ASPECTS OF NITROGEN METABOLISM

IN POLYPORUS TUMULOSUS

A THESIS SUBMITTED FOR THE DEGREE OF

MASTER OF SCIENCE

IN THE UNIVERSITY OF NEW SOUTH WALES

BY

JOHN FRANCIS WILLIAMS

8I0MEOICAL roH ^^UBRARIE^^y

MIN JO * SCHOOL OF BIOLOGICAL SCIENCES

UNIVERSITY OF NEW SOUTH WALES

JANUARY 1957 TO DECEMBER I960 This work was carried out as a part-time study between

January 1957 and December I960, in the School of Biological

Sciences of the University of New South ./ales. The material incorporated in this thesis has not been submitted towards a degree in any other University.

With the exception of the data in Table 19 and the phenolic acid analyses reported in Figure 1$, the results submitted are my own unaided work.

Williams. TABLE OF CONTENTS

Acknowledgements i

Summary ii

1. INTRODUCTION 1

SURVEY OF LITERATURE - NITROGEN METABOLISM IN FUNGI

Introduction 3

t 2. INORGANIC NITROGEN METABOLISM 4

Nitrate Assimilation 4

Nitrite Nitrogen 10

Hyponitrite Reduction 12

Hydroxylamine Reduction 14

Oxime Pathway 15

Ammonia Nitrogen 16

Nitrogen IS

3. THE UTILIZATION OF AMINO ACIDS BY FUNGI 19

The Catabolism of Amino Acids 20

Synthesis and Interconversion of Amino Acids in Fungi 24

(1) The Glutamic Acid Group 27

(2) The Aspartic Acid Group 36

(3) Lysine 42

(4) The Pyruvic Acid Group of Amino Acids 43

(5) Histidine 46

(6) The Serine Group 51

(7) The Aromatic Amino Acids 56

4. URSA AND UREIDES 62

The Occurrence of Urea and its Precursors in Fungi 62 5. THE METABOLISM OF THE NUCLEIC ACIDS AND THEIR

CONSTITUENTS 70 The Degradation of Nucleic Acid Derivatives by Fungi 70

The Uptake, Interconversion and Synthesis of Purines

and Pyrimidines by Fungi 75

6. PROTEINS AND PEPTIDES 55

Peptides of Fungi and Actinomycetes 91

7. AMINES 94

EXPERIMENTAL SECTION

5. PART A - MATERIALS AND METHODS 95

Section (1) The Test Organism 95

(2) The Culture Medium 99

(3) . Cultivation and Harvesting 100

(4) The Mycelium 101 (5) The Culture Liquor 102 (6) The Cell Wall Fraction 102

(7) The Mycelial Extract 103 (5) Acid Hydrolysis of Mycelium and Cell Wall

Fraction 103 (9) Alkaline Hydrolysis of Mycelium and Cell

Wall Fraction 104 (10) Analytical Methods other than Chromatography 104

(13.) Paper Chromatography 105 (a) The Qualitative Identification of

Amino Acids 105 (b) The Sulphur-Containing Amino Acids 115 (c) Urea, Ureides and Guanidine Bases 116

(d) Amino Sugars 116

(e) Visualising Agents for the Guanidino

Group of the ’’Unknown Amino Acid” 117

(12) The Quantitative Analysis of Amino Acids 117

The Estimation of Error and the Statistical

Treatment of Results 124

9. PART B - METABOLIC STUDIES ON POLYPORUS TUMULOSUS 131

Physiological Aspects 132

Measurement of Growth 132

Growth of P. tumulosus on WD5 134

The Effect of Environmental Conditions 146

The Effect of Aeration 147

The Effect of Temperature 143

pH and Growth 146

The Optimal Concentration of Nutrients 149

The Amino Acid Composition of P, tumulosus Fractions 153

The Amino Acid Composition of the Culture Medium 171

The Cell Nall Protein 172

Urea and Ureides 175

The Unknown Amino Acid 173

REFERENCES 131 i

ACKNOWLEDGEMENTS

I wish to express thanks to my Supervisor, Professor B. J. Ralph, for his guidance and assistance during the course of this work, and to Mr. R. Crowden, formerly of this

Department, for many instructive and helpful discussions and for his collaboration in the physiological studies of P. tumulosus.

Miss E. Perry, Mr. F. Wickenden, Mr. P. Brady and

Mr. R. Simson of the Research Laboratory, William Arnott Pty. Limited prepared and photographed the figures in this thesis and have my gratitude•

Dr. S. Ratner (Public Health Research Institute, New York, U.S.A.) and Dr. F. Morrison, Australian National

University kindly donated samples of Argininosuccinic acid and Lombricine.

My thanks are due to Miss Pam Keyes, who typed the final draft of this thesis, and above all, thanks to my wife who, for four years, has shared the life of a part-time student. ii

SUMMARY Principal aspects of the nutrition and metabolism of nitrogen in fungi are discussed.

Physiological studies of the growth of P. tumulosus by still culture on modifications of William Saunders glucose-salts medium were made and have shown that in some circumstances limitation of optimal growth in the organism is the result of unfavourable carbon to nitrogen ratios. The accumulation of phenolic and certain amino acids in culture media and extracts of the organism, as shown by sequential growth studies, is consistent with such C/N imbalance.

The amino acid composition of proteins of the mycelium and cell wall, together with that of the culture medium and mycelial extract, is described. It is established that an unidentified amino acid exists in the mycelial extract of this organism. The sequential appearance of this acid during growth and its chemical and possible biochemical relations to other amino acids are discussed.

Ureidosuccinic acid was found on chromatograms of

P. tumulosus mycelial extracts; this ureide has not previously been reported in fungi. Allantoin, Allantoic Acid and an unidentified ureide (possibly Allantoxanic or Uroxanic Acid) were found in extracts of autolysing cells. 1

INTRODUCTION This investigation was primarily undertaken as part of a comprehensive survey, by this Department, on the biochemistry of the wood-rotting Basidiomycete fungus, Polyporus tumulosus

Cooke, and secondarily to form a supplementary study to a parallel investigation on the phenolic acid metabolism of this organism. Crowden (1) has identified aromatic compounds in

P. tumulosus derived from the Shikimic acid path of biosynthesis and has established that many of these compounds bear a pronounced resemblance to tyrosine. With the provision of this background, research was directed to those aspects of nitrogen metabolism in this organism, which would relate to the nature and incidence of aromatic amino acids. The complementary nature of the metabolic studies on phenolic acids and nitrogen metabolism established the need to standardize culture conditions for the organism.

The definition of the scope of this thesis limited the investigation to the identification of nitrogenous metabolites in the fractions; culture media, mycelia, and mycelial extracts, with a directive to attend in particular to amino acids.

The metabolic studies of the organism cultured on WD5- glycine and WD5-alanine were dictated by an investigation of the pattern of phenolic acid side chain synthesis.

Principally these studies were to serve as an introduction to 2

the role of nitrogen in these transformations and the establishment of the necessary techniques for the more comprehensive investigation of the organism when grov/n with a simple ammonium salts medium (WD5).

A survey of some aspects of the literature on the metabolism and nutrition of nitrogen in fungi has been made.

The recent comprehensive review of Cochrane (2) has provided the chief background against which this survey has been planned, but most of the material reviewed has been obtained from the sections dealing with nitrogen metabolism in the annual review series of Biochemistry, Microbiology and Plant

Physiology. In addition, material not available from these periodicals has been obtained diirectly from various journals,

Proceedings of Symposia, Chemical and Biological Abstracts.

The review deals principally with those aspects of the nitrogen metabolism of fungi which are directly related to the experimental work. While the magnitude of certain aspects of the reported results may not appear to justify such a detailed literature survey, it was deemed necessary for adequate appraisal of these results. ASPECTS OF NITROGEN METABOLISM

IN POLYPORUS TUMUL05US

SURVEY OF LITERATURE

Nitrogen Metabolism in Fungi - 3 -

NITROGEN METABOLISM IN FUNGI

INTRODUCTION

Irrespective of the organism the continuance of life and the synthesis of cytoplasm are dependent on the availability of the same basic materials, namely sources of carbon, nitrogen, water, and mineral salts, together with some mechanism for providing energy in a form that can be utilized in biological systems.

All autotrophs derive their nitrogen from an inorganic source and, depending on the organism, use molecular nitrogen, ammonium ion, nitrate or nitrite. The heterotrophs on the other hand have a specific requirement for one or more substances and are unable to grow in their absence from the culture medium.

The need of a culture for nitrogen is not optimal. It is governed primarily by the supply of an energy yielding carbon compound. Steinberg and Bowling (3) in their studies with Aspergillus niger demonstrated this and established a strict proportionality between carbohydrate supply and nitrogen demand.

Generally nitrogen is taken up from an organic or inorganic medium during the growth phase while amino acids and water soluble nitrogenous compounds, enzymes etc. are found during the latter stages of grov/th and autolysis. It has been found in studies on Scopularopsis brevicaulis that the organism utilizes nitrogen from autolysed cells to - 4 - synthesise new tissue (4).

INORGANIC NITROGEN METABOLISM

Under this heading will be reviewed the role of nitrates, nitrites and ammonium ion as nutrients and metabolites.

Inorganic Nitrate Assimilation

The assimilation of nitrate nitrogen by fungi involves

the reduction of nitrate to ammonia, resulting in an oxidation-reduction change of the nitrogen atom from + 5 to -3, involving a net change of 8 electroins, as shown in Table No. 1

TABLE No. 1.

Oxidation-Reduction States off Nitrogen

and its Compounds

Oxidation-Reduc tion Nitrogen

State of N atom Compounds

+6 n°3, n206

hn03

+4 no2, n204

+3 N203 hno2

+2 NO

+1 N2°- H2N2°2 0 W2 -1 nh2oh

-2 nh2 - NH2

-3 NH3

A number of enzymes, which catalyze the reduction nitrate to ammonia via nitrite and hydroxylamine, have 5

characterised in bacteria and fungi. Reduced pyridine nucleotides act as electron donors and all steps seem to involve metal-dependent flavo-proteins. The assumption is that each enzymic step involves a two electron or proton change.

Nitrate can serve as an excellent source of nitrogen in many fungi (5); however the higher basidiomycetes (6, 7), the Saprolegniaceae ($) and the Blastocladiales (9) fail to utilize it.

The Actinomycetales reduce nitrate (10) although some strains cannot grow with nitrate as the sole nitrogen source

(11, 12, 13). The ability to utilize nitrate can be lost by mutation and it is suggested that the biotic circumstance of an organism, rather than its taxonomic position governs this

(2).

Ammonium nitrate is often used in culture media and experimental evidence for the pattern of utilization is provided by the studies of Morton and McMillan (14) with

S. brevicaulis and Strauss’ work with Neurospora crassa (15).

In S. brevicaulis nitrate utilization does not begin until virtually all the ammonium ion has disappeared from the medium.

It has been established by Morton (14, 16) that nitrate utilization is completely stopped by the addition of ammonium ion (provided a utilizable carbon compound is present to provide an energy source for assimilation), but nitrite - 6 - utilization is not blocked by ammonia; thus it is assumed that in the fungi studied the presence of ammonium ion prevents the reduction of nitrate to nitrite. This is borne out by enzymatic studies which show that nitratase activity declines when ammonium ion is added to the system.

Analytical data on Helminthosporium gramineum (17) shows slow utilization of nitrate, even during rapid assimilation of ammonium ion, while spores of Streptomyces griseus will not grow on nitrate medium, but pregrown mycelium readily uses nitrate (2).

The reduction of nitrate by N. crassa has been intensively studied and has resulted in the isolation of three inducable enzymes from this organism when cultured on nitrate as sole nitrogen source. Nitrate reductase has been characterised as a flavin enzyme with flavin adenine dinucleotide as the prosthetic group.

A flavoprotein enzyme, which catalyzes the reduction of nitrate to nitrite, has been isolated and partially purified from fungi, bacteria and higher plants (18, 19). The electron donors are reduced triphosphopyridine nucleotide (TPNH) for fungi, reduced diphosphopyridine nucleotide (DPNH) for bacteria, and either reduced nucleotide for the higher plants.

Flavin adenine dinucleotide (FAD) is the native flavin.

Stoichiometric experiments showed that for each mole of TPNH oxidised one mole of nitrite was formed according to the following equation. - 7 -

no; + TPNH + H+—► no; + TPN+ + h2o

Thiourea (20), chlorate (21) and flouride (16) inhibit nitrate reductase.

It has been shown (22, 23, 24, 23, 26) that molybdenum is a specific and functional constituent of the enzyme.

Molybdenum has been shown to concentrate in the purified enzyme to the exclusion of other metals. It can be removed from the enzyme by dialysis against cyanide and glutathione.

The dialyzed preparation, washed free from cyanide, failed to reduce nitrate, but when molybdenum as sodium molybdate or molybdenum trioxide was returned to the apoenzyme it restored enzyme activity to about <£>5% of the level in the undialyzed enzyme. No other microinutrient (twenty others were tried) appeared to substitute for molybdenum in this reaction.

A possible sequence of electron transfer in the purified nitrate reductase from N. crassa, Escherichia coli and green plants is suggested by Nicholas and Nason (24) to be as follows:

DPNH+H+ 5 + no:

no:

TPN' +H2° In support of this sequence, it was shown that DPNH can be replaced by reduced flavin (FMN or FAD), or by sodium molybdate reduced with sodium hydrosulphite (NagSgO^K

Thus an external supply of reduced molybdate acts as an effective donor for the enzymic reduction of nitrate. The dialyzed enzyme, which is practically free from Mo mediates

the reduction of FAD by TPNH but not that of nitrate by

FADH2. The most reduced state of Mo in the dithionite

reduced solution of sodium molybdate was shown by paper

chromatography to be Mo (27, 2£). Mo pentachloride, freed from the other valency states of Mo by column

chromatography, acts as an effective electron donor for the

enzymic reduction of nitrate. Other valency states of Mo

have also been prepared. Mo^+ is only stable in strong

ethanol and as it dismutes readily in the presence of a trace 5+ 3 + of water to give Mo and Mo it is most unlikely to be

involved in any physiological process. It is found that Mo-5

prepared from Mo^+ by drastic reduction with zinc dust will,

after removal of Zn, reduce nitrate to nitrite non-enzymically

Mo^+ is oxidised instantaneously by Mo°+, Fe;>+, and Cu +

which are invariably present in fungal growth medium, and

there is insufficient reducing power generated in fungal

cells to effect reduction of Mo^+ to Mo^+. It would appear

then that one of the functions of Mo in the enzyme is to

couple the flavin to nitrate by way of a one electron change,

namely Mo^+ to Mo^+; the latter is then oxidised by the - 9 - nitrate acceptor.

The enzyme has a phosphate requirement that can be replaced completely by tellurate, arsenate or selenate and to a lesser extent by silicate or sulphate (29). It is likely that these complex with the molybdate in the enzyme since the replacement anions have similar atomic radii to

o o

that of phosphate (2.76 A), being within the range 2.4 - 2.8 >

Kinsky and McElroy (30) have shown phosphate to be essential for nitrate reductase action and that it accelerates electron transfer by combining with Mo in the enzyme. Whether or not nitrate reductase is a sing;le or complex protein is still in doubt. Electrophoretic p.atterns of the best preparations still contain at leas t three peaks and they have a weak TPNH oxidase activity. Kin.sky and McElroy (30) have also shown a close parallel betwee:n a TPNH linked cytochrome reductase and nitrate reductase activity in protein fractions of N. crassa. They suggest, however, that probably only one enzyme system is involved in nitrate and cytochrome c reduction. Cytochrome c may be acting here as would a non­ specific dye acceptor, so that not too much significance need be attached to the cytochrome c reductase activity of some isolated enzymes (31).

An active DPNH-cytochrome c reductase which parallels the fractionation of nitrite reductase in N. crassa has been reported (32). Should nitrate reductase be found to be a single protein then it would be similar to other enzymes. 10

e.g. alcohol dehydrogenase, that have several sites on them for the reaction of their various constituents.

Besides the more usually encountered nitrate-nitrite reduction by fungi it has been observed that oxidative synthesis of nitrates, nitrites etc. takes place, thus

Aspergillus flavus growing on glucose-peptone-yeast extract produces nitrite and large amounts of nitrate (33). Norcadia corallina has been reported to oxidise pyruvic oxime and hydroxylamine to nitrite (34, 35).

Nitrite Nitrogen

Nitrite serves as sole nitrogen source for a number of fungi, Phymatotrichum omnivorum (3<6), S. brevicaulis (14), and some of the Aspergilli (37, 38 , 39) use it well, poorly to almost not at all. However the more common experience of workers in this field has been that fungi generally do not grow on nitrite due to the poisonous nature of the unionized acid at low pH (40). This is substantiated by the observation that fungal growth with nitrite is best in alkaline media

(41, 42, 43).

Streptomyces nitrificans produces nitrite when cultured on urea, ammonium carbonate and carbamates. A pyridine nucleotide ammonium dehydrogenase has been suggested to be responsible (44, 45, 46). Nitrate has not been detected in this system.

In 1957 Medina and Nicholas (47) showed that nitrite reductase from N. crassa is a flavoprotein dependent on Fe 11 and Cu for maximal activity. The enzyme is associated with endoplasmic particles and is stabilised in the 10% W/V sucrose which is used to extract it from frozen felts or from acetone powder of the felts (32). Desoxycholate has been used to release the enzyme from particles present in extracts of frozen felts. The fractionation of the enzyme from acetone powders of the felts results in over 100-fold purification. The flavin constituent of the enzyme is FAD identified by paper chromatography, paper electrophoresis and by the D-amino acid oxidase assay. DPNH is a more effective electron donor than TPNH in equivalent amounts. The product of nitrite reduction inhibits the enzyme. The effect of adding hyponitrite and hydroxylamine to the enzyme showed that whereas hyponitrite inhibits the enzyme, hydroxylamine, even at a concentration 16 times greater than hyponitrite, was without effect.

The effect of metal inhibitors on nitrite reductase is reviewed by Nicholas (37). The general metal inhibitors,

KCN, quinoline, Na-EDTA depress enzyme activity as do sodium diethyldithiocarbamate, salicylic acid, thiourea, diquinolyl, -dipyridyl and O-phenanthroline, which are more specific

inhibitors of Fe and Cu. Inhibitors of bound iron, including

Z -heptyl-4-hydroxyquinoline-N-oxide, naphthaquinone and urethane markedly reduce enzyme activity. Uncoupling reagents including 2,4-dinitrophenyl, sodium arsenite, aureomycin and

gramicidin also inhibit the enzyme. Thiol groups have been 12 shown to be essential for enzyme activity.

Silver and McElroy (4$) showed that a nitrite mutant of N. crassa required pyridoxine for growth on nitrite but when ammonia was supplied the vitamin was not required.

Nitrite reductase in this mutant was reduced when pyridoxine was deficient and nitrite accumulated in the medium. The addition of pyridoxine, pyridoxal or pyridoxal phosphate to extracts of pyridoxine-deficient felts reconstituted the enzyme after a short incubation period. Neither pyridoxal nor pyridoxal phosphate was detected in pure fractions of the enzyme. The role of pyridoxine in the enzyme is not known. The purified enzyme has a phosphate requirement that cannot be replaced by other anions and in this respect differs from both nitrate and hydroxylamine reductases.

Hyponitrite Reduction

Intermediates between nitrite and ammonia are still the subject of much speculation.

Early work based on nutrition experiments were often invalid because of the instability of intermediates and the problem of their penetration into fungal tissue and cells.

The growth of Aspergillus niger on nitrohydroxylaminate seems to be the only instance in which an inorganic compound intermediate between nitrite and ammonia has been found to serve as a nitrogen source for growth (49, 50). Even here there is doubt about the stability of the compound in the culture solutions used. - 13 -

In the reduction sequence a compound with a reduction

charge of - 1 on the nitrogen atom, namely, nitrous oxide or

hyponitrite or nitramide, would be expected to be an

immediate product of nitrite reduction. Hyponitrite

decomposes rapidly to ^0 and H2O in acid solution but is

stable for several hours at neutral or alkaline pH. Nitramide behaves in the reverse way with regard to pH but yields

similar products. Hyponitrite has been reported in growing

cultures of nitrifying organisms (51). McNall and Atkinson

(52) showed that E, coli can utilize hyponitrite and hydroxylamine as a nitrogen source.

Hyponitrite reductase has been found in N. crassa grown

on nitrate nitrogen but was absent in felts grown on ammonia

nitrogen (47, 53). The enzyme appears to be a flavoprotein requiring DPNH as the electron donor. Its action is inhibited

by metal chelating agents and by - SH blocking agents, the latter effects being reversed by glutathione. It is likely that nitrite and hyponitrite reductases are distinct enzymes

since the reduction product of nitrite reductase inhibits

the enzyme whereas that of hyponitrite does not; a purified nitrite reductase does not reduce hyponitrite and a deficiency

of Zn depresses nitrite reductase only. Hydroxylamine is

the immediate reduction product of hyponitrite reductase

since it accumulates as an oxime in media, and to a lesser

extent in felts, of Mn deficient cultures, because of restricted formation of hydroxylamine reductase. - 14 -

Hydroxylamine Reduction Zucker and Nason (54) identified a DPNH-dependent hydroxylamine reductase in N, crassa. Stoichiometric data show that the enzyme mediates the following irreversible reaction:

NH2OH + DPNH + H+-----> NH3 + DPN+ + HgO

This enzyme is a flavoprotein inhibited by metal­ chelating agents, and is formed only in the presence of nitrate or nitrite.

The enzyme is much reduced in extracts of Mn-deficient felts and under these conditions oximes (from keto acids) accumulate in both media and felts. A deficiency of Mn reduces hydroxylamine reductase activity and also depresses nitrite and hyponitrite reductase, presumably because they are on the same route. Recent work has shown that a deficiency of Mg also markedly reduces hydroxylamine reductase activity in extracts of N, crassa (32). The enzyme was found to be more stable when extracted in 10$ W/V sucrose (pH 7.5). Although Mn and Mg are essential for enzyme activity, neither metal accumulated in purified fractions of the enzyme nor stimulated purified fractions of the enzyme when added to it. It would thus appear that the two metals are required for enzyme formation rather than for its action,

Hydroxylamine is reduced to ammonia by hydrogen in cells of Clostridium perfringens (55). -15-

Qxime Pathway

A possible alternative scheme for utilization of hydroxylamine would involve its chemical reaction with

/X-keto acids to form oximes which would then be reduced to amino acids. This would not involve the formation of ammonia.

Silver and McElroy (48) showed that cell free extracts of

N. crassa mutants, unable to grow on nitrate or nitrite, contained enzymes which could reduce nitrite through hydroxylamine to ammonia. They also found that some mutants required pyridoxine before they could grow on nitrite but when ammonia was the sole nitrogen source the vitamin was not required. They propose the following scheme to explain their results.

pyridoxal oxime pyridoxal phosphate phosphat* amino acids

pyridoxamine phosphate — keto acids

In the absence of pyridoxine there is a marked accumulation of ammonia and keto acids in the felts and in the medium, presumably because of the reduction in pyridoxine- dependent transamination processes. This seems to indicate that ammonia is an obligatory intermediate in the reductive

sequence in the organism. - 16 -

Nicholas (32) prepared pyruvic oxime, (X -ketoglutarate oxime and pyridoxal oxime in pure form. All of these

compounds hydrolyse at physiological pH (6 - 8) to yield

hydroxylamine. Oximase activity was not detected in N. crassa

grown on a variety of nitrogen sources.

Although a variety of electron donors and cofactors

were tried, no convincing evidence was obtained to show

that the route proposed by Silver and McElroy is operative

in N. crassa. It seems clear that the main function of

oximes in the reductive pathway is the detoxication of

hydroxylamine and that they are not reduced direct to amino acids. The use of -labelled oximes has confirmed that

oximes are not utilized in N. crassa since no amino acids

contained the tracer; the C*^ was all recovered in the corresponding keto acid after release of hydroxylamine which was reduced to ammonia via hydroxylamine reductase (32).

Ammonia Nitrogen The inorganic pathway of nitrogen in fungi, as in most

organisms, is postulated by Van Kluvyer as follows. 2N0~ —► 2N0T H2N202 —2NH20H —>- 2NH ^ The reduction of nitrate by fungi is assimilatory in

contrast to the bacteria where assimilation is not

necessarily part of the pattern of nitrate reduction.

The most readily utilizable form of nitrogen for fungi,

therefore, is ammonia, from which the organism is able to - 17 - build up, by means of the requisite energy-donating mechanisms, all the complex nitrogenous substances necessary for growth and reproduction. Fungi will also assimilate amino acids, amines, amides, and even complex nitrogenous substances, such as polypeptides and proteins, but the fate of the majority of these materials, upon assimilation, lies in their degradation and conversion into ammonia which is then incorporated into the cell substance.

With the exception of some members of the lower

Phycomycetes (56, 57, 53) it is the general rule that fungi as a group can grow with ammonium ion as the sole nitrogen source. The utilization of ammonia requires oxygen and a carbon compound. Ammonia utilization increases with increase in pH and has no definite pH optimum (14). Ammonia enters and leaves the cell by passive diffusion of the undissociated NH^ molecule. As with microorganisms generally,fungi assimilate ammonia with the formation of glutamic acid via glutamic dehydrogenase. Other amino acids can be formed from glutamate by transamination. The assimilation of ammonium ion from sulphate, nitrate and chloride invariably results in a marked lowering of pH in the culture medium, due to the production of free acid upon the removal of cation. This is a very general reaction and Cochrane (2) lists seventeen fungi in which this phenomenon was observed. The drop in pH due to acid formation - id -

can be overcome by using ammonium salts of acids whose dissociation is small e.g. ammonium carbonate, phosphate (59) or the ammonium salts of utilizable organic acids (14).

Alternatively, buffering of the culture medium corrects this effect of acid pH on growth rate (60).

Nitrogen

In spite of repeated claims to the contrary there does not exist good corroborated evidence that fungi fix nitrogen.

However, Ingram (61) reports that yeasts can exchange isotopic nitrogen with the atmosphere, indicating that atmospheric nitrogen can find its way into the nitrogen compounds of the yeast. There is presumably no net increase in the total nitrogen content of the cell population under these conditions and hence no net assimilation of nitrogen. - 19 -

THE UTILIZATION OF AMINO ACIDS BY FUNGI

In the fungi as in other organisms it is generally- agreed that the amino acids are assimilated as such and that conversion to ammonia before assimilation of the nitrogen is at best a secondary pathway. The amino acids are assimilated at varying rates from the medium, depending on the organism, the period of incubation, the nature of the basal medium, and the concentration of the amino acids used. Typically, the amino acid content of the growth medium drops as the acids are taken up and rises again as they are liberated during autolysis.

Although numerous studies have been made on the utilization of different amino acids as sole nitrogen source with the one organism, it is frequently found that an amino acid allows excellent growth of one organism and very little of another. With fungi it is not possible to say at this stage whether this reflects permeability, enzymatic capacities, or merely such secondary problems as acidity changes consequent upon utilization. It is, however, generally conceded that glycine, asparagine, glutamic and aspartic acids are most likely to support good growth. On the other hand, the literature records a number of examples not consistent with the above generalization, i.e., failure to grow well with asparagine (62, 63, 64), aspartate (17, 65) and glutamate (65, 7). Other examples of this can be found, sufficient to establish the point that although generalizations may 20 be made which fit most fungi, they will not necessarily apply to any one fungus which happens to be of interest.

Although uncommon in nature, specific requirements appear with reasonable frequency as a result of induced mutations in fungi. As examples: Mycena rubromarginata responds in growth to phenylalanine and tyrosine but can grow in the absence of both of these aromatic amino acids (66)

Eremothecium ashbyii requires several amino acids, but only for growth in acid medium (67); absolute requirements in

Trichophyton spp. for histidine (6£) and T. tonsurans for arginine (69) have been reported; Labryinthula minuta var. atlantica requires leucine (70); Lentinus omphalodes requires tryptophan (71).

In regard to the transport of amino acids into fungal cells, it is known that a free amino acid pool exists, and that transport across the membrane occurs in both directions.

In bacteria, yeast, and animal cells there is good evidence that some or all amino acids enter the cell by an active transport mechanism requiring an expenditure of metabolic energy, but different organisms cannot at present be fitted into any single pattern (72, 73, 74). The Catabolism of Amino Acids

Amino acid breakdown is considered under two headings:

(1) The metabolism of the amino group, and

(2) The metabolism of the carbon chain.

In fungi the enzymes and metabolites associated with these 21

changes are imperfectly described, and although much can be inferred from relationships established in other organisms, the evidence presented for fungi is largely incomplete and circumstantial.

A logical approach to this problem is to investigate what compounds are either oxidised or deaminated by resting cells. Wolf (75) studied the oxidation of twenty three amino acids by Penicillium chrysogenum and found that under the experimental conditions this organism rapidly oxidised glutamic acid, alanine and proline, other amino acids slowly, and cystine not at all.

Gottleib and Ciferri (76) working with washed cells of

Streptomyces venezuelae were able to show liberation of ammonia from glutamate, arginine, proline, and other amino acids, but not from tyrosine, tryptophan, leucine or cysteine.

It was also shown that only those amino acids which could be deaminated by this organism served as sole nitrogen source for growth. Leptomitus lacteus both oxidises and deaminates alanine, glycine and leucine (58).

Specific enzymes associated with the metabolism of the amino group and the carbon chain of amino acids in fungi have been studied. D-amino acid oxidase activity has been reported in Neurospora crassa (77)> Aspergillus niger,

P. chrysogenum, P. roqueforti, P. notatum (78), but was not found in mycelial extracts of P. sanguineum (78).

A number of microorganisms possess L-amino acid oxidase 22

activity. These include the N. crassa oxidase, whose formation is doubtfully suggested as being biotin dependent.

Indirect evidence supports the assumption that the prosthetic group of this enzyme is flavin adenine dinucleotide (79). Some strains of N, crassa may produce both D- and L-amino acid oxidases while others produce only a D- or L-specific oxidase (79, 30, 81). L-amino acid oxidase has been found in strains of Penicillium spp. and A. niger (32).

The formation of (X -keto acids by growing or resting cells supplied with the corresponding L-amino acid is often observed and is taken as presumptive evidence for an L-amino acid oxidase (83). It should be stressed though that chemical attack on an L-amino acid does not, by itself, prove that an L-amino acid oxidase is present. A combination of trans­ amination and glutamic dehydrogenase activities could bring about oxidative deamination without the participation of an oxidase. Glutamic dehydrogenase is widely distributed in nature and has been found in mycelial extracts of N. crassa (84). It is highly specific enzyme for L-glutamic acid, and in fungi requires the participation of triphosphopyridine nucleotide

(TPN). The reversible deamination of glutamic acid with this enzyme is a reaction of considerable metabolic significance, being one of the major mechanisms for the interconversion of the L-amino group nitrogen and ammonia. - 23 -

A serine dehydrase, from N. crassa (85, 86) effects the non-oxidative deamination of L-serine and L-threonine, pyridoxal phosphate being the coenzyme.

Aspartase activity has been reported in N. crassa (87) and P, notatum (88).

The fragmentary evidence for the catabolism of the carbon chain of amino acids by fungi prevents any attempt at a systematic treatment of the topic. Nevertheless, some of the data available is consistent with the break-down pathways known in other organisms.

Tyrosinase is present in some fungi and actinomycetes (89)

In N. crassa (89), tyrosine is oxidised via 2,4-dihydroxy- phenylalanine , and it is usually assumed that dark insoluble pigments in fungi are products of tyrosinase action (90).

In liver, tyrosine is oxidised to acetoacetate and fumaric acid as follows (91):

Tyrosine-- p-Hydroxyphenylpyruvic acid ------^

2,5-Dehydroxyphenylpyruvic acid ---

Homogentisic acid---^ Acetoacetate + Fumarate

Homogentisic acid has been isolated as a product of the tyrosine metabolism in A. niger and some Penicillium spp.

(92, 93) while p-hydroxyphenylpyruvate is implicated in the tyrosine metabolism of N. crassa (94)• There is good circumstantial evidence for the break-down of homogentisic acid to T.C.A. cycle intermediates but the reactions are not known in fungi. - 24 -

Tyramine has been isolated from A. niger (95) which is

suggestive of the tyrosine decarboxylase action described

by Gale et al. (96) with Streptococcus faecalis.

The catabolism of tryptophan in N. crassa has been

studied principally in relation to the formation of nicotinic acid. Bonner and Yanofsky (97) have reviewed this work.

An anaerobic deaminase, aspartase, provides a mechanism

for nitrogen fixation similar to that induced by glutamic

acid dehydrogenase, but in this case producing aspartic

acid. This enzyme has been reported in N. crassa and

P. notatum (£8).

In the reaction catalyzed by this enzyme a molecule of ammonia is removed from aspartic acid to yield fumaric acid,

the equilibrium favours the formation of aspartic acid, thus providing another means of incorporating inorganic nitrogen.

It is not yet known whether this reaction makes a significant contribution to amino acid synthesis in fungi.

SYNTHESI5 AND INTERCONVERSION OF AMINO ACIDS IN FUNGI

With few exceptions fungi can be considered to synthesize

all their amino acids from certain inorganic sources of nitrogen, the foremost of these being ammonia. Among the most important mechanisms for incorporation of this nitrogen

into organic structures are those provided by the enzyme

systems, glutamic dehydrogenase and aspartase. Of these two

systems, present evidence indicates the former to be by far - 25 -

the more important. The formation of glutamic and aspartic acids in this way occurs at the expense of citric acid cycle. It is known that the cells of fungus mycelium contain a considerable pool of free amino acids (98,

99j 100, 101) among which glutamic acid, aspartic acid and

alanine are predominant. These amino acids have keto acid precursors which are intermediates in citric acid cycle; thus

a dynamic state may be visualised in which the citric acid

cycle generates the eC-keto acids which in turn combine with

ammonia, or undergo transamination. As a result, when amino acids are being synthesised in the cell, carbon is being

drawn from the citric acid cycle, energy is being produced

and protein synthesis is enhanced largely via glutamic acid.

The occurrence of the citric acid cycle in animal cells is well established and there is good evidence that it is at

least potentially functional in fungi, yeast, and bacteria. However, a real question exists whether its major metabolic importance is in supplying the carbon skeleton of the amino

acids (102, 103), and/or the additional role of providing

energy to the cell (103, 104). With the probable exception of the oxidative decarboxylation of o(-ketoglutarate, the

phosphorylations associated with the cycle are at the co­

factor level. It is not certain whether the citric acid

cycle is the only terminal respiratory system in fungi but

there can be no doubt that it plays a major role. - 26 -

Much of the present knowledge on the general biosynthesis of amino acids in microorganisms has been obtained using an experimental technique which involves supplying the organisms, which are in a state of actively synthesising amino acids and proteins, with sources of radioactive carbon isotopes (C ^ or C ^), isolating the protein, and degrading it to its constituent amino acids. The amino acids are broken down in turn to afford a picture of the extent of isotopic labeling of individual carbon atoms or groups of carbon atoms and in this way certain conclusions can be drawn as to the mechanism of their synthesis. In general the amino acids constituting the proteins of fungi or other microorganisms after growth on radioactive carbon source, fall into several distinct groups according to their degree of labeling with the isotope (106, 107, 108).

These comprise (109) : (1) The Glutamic Acid group, consisting of glutamic acid

proline

arginine; (2) The Aspartic Acid group containing aspartic acid

methionine

threonine

isoleucine;

(3) Lysine; - 27 -

(4) The Pyruvic Acid group including

alanine

valine

leucine;

(5) Histidine (110, 111);

(6) The Serine group,

serine

glycine

cysteine;

(7) The aromatic amino acids,

phenylalanine

tyrosine

tryptophan.

• • Ehrensvard (112), and Abelson and Vogel (106) found that this classification applied to Neurospora crassa, Torulopsis utilis and Escherichia coli, with the sole exception that lysine is derived in the latter from aspartic acid via (X - £-diamino pimelic acid (113), an amino acid which has not been detected at all in fungi (114).

Abelson and Vogel found that the above classification is consistent with the existence of citric acid cycle in both

N. crassa and T. utilis.

(1) The Glutamic Acid group

The relative activities of the amino acids isolated from

T. utilis after growth in the presence of various tracer compounds labeled with is shown in Table No. 2 (106). TABLE No. 2.

Incorporation of C~^ from Tracer Compounds into Amino Acids of T. utilis Protein

Tracer Compound

Glutamic Arginine Proline Aspartic Threonine Isoleucine Methionine Lysine Acid Acid

C1^ C02 100 500 95 230 230 240 220 0

Acetic Acid 100 250 129 33 30 30 35 200 1-CU

C1^ Aspartic 100 100 105 167 160 170 170 70 Acid

C1^ Glutamic 100 SO 67 26 25 24 22 40 Acid

Glutamic Acid 100 90 70 0 0 0 0 0 29 -

This shows the division of these particular amino acids

into three of the seven groups mentioned. Allowance should

be made for the fact that the relative activity of arginine

is increased by direct incorporation of from carbon

dioxide or acetate into the amidine moiety.

The data are consistent with the view that the five-carbon

skeletons of proline and arginine are derived from glutamic

acid. Evidence for the biosynthetic sequence of the compounds

is yielded by isotopic competition experiments in which unlabeled compounds, which are presumed intermediates in their

synthesis, are allowed to compete with the added labeled

compounds. Any reduction in the radioactivity of the products

as compared with that of products of the control experiments may be taken as evidence for the participation of the

nonradioactive competitors in the biosynthesis and hence

that these compounds are natural intermediates. The results

of such experiments are illustrated in Table No. 3.

It can be seen that ornithine, citrulline, and arginine,

compete effectively as sources of arginine and proline,

while added proline suppresses the radioactivity of the

proline of T. utilis protein, but not that of glutamic acid

or arginine.

Glutamic acid semialdehyde contributes to proline, and

to some extent to glutamic acid and arginine. On the basis

of both these results and parallel studies with mutants of

N. crassa and T. utilis, Ableson and Vogel proposed the - 30 -

TABLE No, 3.

Effect of Competitors on the Radioactivity of the Amino Acids

of the Glutamic Acid group

in Torulopsis utilis

Competitor Amino Acid

Glutamic Acid Arginine Proline

None 100 100 100

Ornithine 75 12 4

Citrulline 95 25 25

Arginine 95 15 15

Glutamic Acid 65 70 6 Semi Aldehyde

Proline 100 100 0 - 31 -

following sequence of reactions for the biosynthesis of the members of the Glutamic Acid group.

Glutamic acid A’-Pyrroline-S------* Proline semialdehyde carboxylicruuxync acidaciu II2C------CII2 II2C------CH2 CIIOCII2CH2CH NII2 C02H i i i i 11CV ^CHCOjH HaC^ ^CHCOjII A X 1 H Exogci ornithine Glutamic acid W \\ C02HCH2CH2CHNH2C'02H W \N w w w Glutamic acid -* Ornithine ----- *■ semialdehyde ----- ► Citrulline ------Arginine

chxh2co2h chxh2co2h CHXH2C02H

ch2 ch2 ch2 1 QH2 ch2 1 ch2 ch2 CH-, 1 1 nh2 NH XH 1 CO 1 C=XH XII2 xh2

This scheme is not consistent with the earlier

postulation by Fincham (115), that proline plays a major role as a precursor of ornithine and of arginine in N. crassa The contribution of the semialdehyde to arginine indicates

that it is an ornithine precursor, a biosynthetic sequence

which was proposed earlier for this organism (116); but in

view of its conversion to glutamic acid (Table No. 3) it

appears that part of the exogenous material does not form

ornithine directly. The two biosynthetic pathways leading, - 32 -

on the one hand, to proline, and on the other hand to arginine, may well be physically separated in the cell.

Thus, when only traces of highly radioactive ornithine were used in the growth medium, the specific activities of the recovered proline and arginine were in the ratio of 100:6, but when the added ornithine was diluted with carrier this ratio was 100:80, indicating that exogenous ornithine as a source of proline does not equilibrate with endogenous ornithine acting as a source of arginine. Added ornithine also appears to function as a source of the arnidine carbon atom of arginine, possibly by decarboxylation to carbon dioxide which is readily incorporated. The added ornithine also contributes to glutamic acid, probably via the semialdehyde, and presumably mediates the contribution of citrulline and arginine to.proline by way of the ornithine cycle (117) and glutamic acid semialdehyde (118). This relationship between glutamic acid, proline, and ornithine in M. crassa and

T. utilis differs from that in S. coli, in which ornithine is formed via a series of acetylated intermediates (119, 120, 121)

These latter intermediates appear to play no part in fungal metabolism, since N- & -acetyl ornithine, a precursor of ornithine in E. coli, did not reduce the radioactivity of arginine in isotopic competition experiments with T. utilis.

It is noteworthy that an enzyme catalyzing the reduction of

^-pyrroline-5-carboxylic acid to proline has been observed in N. crassa and in rat liver (122). This enzyme requires 33 -

DPNH to effect the reduction.

Vogel (121) has drawn attention to a parallelism, of interest from the point of view of comparative biochemistry, between the inter-relationship of glutamic acid, proline and ornithine in N, crassa or T. utilis, and that in mammals as found by btetten (123). The two fungi resemble mammals more than the bacterium, E. coli, and Vogel also points out the similar observation by Yanofsky (124) that the relationship between tryptophan and nicotinic acid in N. crassa resembles that in mammals, but differs from that in bacteria.

In the case of ornithine formation it has been shown that the transamination of glutamic acid semialdehyde is a reversible reaction favouring the semialdehyde, presumably because of the cyclization of the latter. This seems to indicate that in the fungi, and possibly in mammals, a mechanism has been evolved, namely the above-mentioned physical separation of intermediates, which permits an adequate rate of synthesis of ornithine in spite of the unfavourable equilibrium. In E. coli, however, the goal of ornithine synthesis is achieved by the use of N-acetylation which prevents the cyclization of the semialdehyde and hence maintains the equilibrium.

Srb and Horowitz (117) showed the participation of ornithine in urea cycle in N. crassa, and its conversion into arginine via citrulline, although the detail of the mechanisms are not as complete as those described for animal systems. - 34 -

There is evidence for all of the reactions of the cycle in

N. crassa, these being consistent with the analagous mechanisms described for mammalian liver.

Ratner (125) has shown that these latter mechanisms, which form part of the Krebs-Henseleit (126) cycle, involve several stages. The first is the addition of carbon dioxide and ammonia to ornithine in an endergonic reaction for which

ATP furnishes the driving force, and in which carbamylglutamic acid or other acyl glutamic acids function catalytically (127).

In mammals the utilization of ATP results in the formation of an unstable intermediate called compound TtXt! (127,

128, 129). Compound ,1X,f is a derivative of carbamyl phosphate, which is the active donor of the carbamyl group to ornithine

(129, 130, 131).

Hall et al. (132), have recently isolated and characterised N-acetylglutamic acid in yeast, this is the naturally occurring co-factor for carbamyl phosphate biosynthesis in both liver and yeast.

The union of carbamyl phosphate and ornithine forms citrulline with liberation of carbamylglutamic acid and inorganic phosphate. These reactions may be summarised as follows:

Carbamylglutamic Acid + ATP + NH0) + CO2 >

Compound ,TX” + ADP.

Compound TTX1T + Ornithine ^ Citrulline + Carbamyl Glutamic

Acid + P0y+. - 35 -

The citrulline so formed is then converted into arginine in two stages which involve formally the addition of ammonia and the removal of water. Only aspartic acid can donate the ammonia, and again the energy from ATP is required to drive the condensation reaction.

Citrulline + Aspartic Acid + ATP Mg ^++ Argininosuccinic Acid

+ ADP + H3 PO^.

The phosphate bond energy is thereby incorporated into the amidine moiety of argininosuccinic acid, which is readily cleaved to form arginine and fumaric acid. Two enzymes are required to effect the condensation reaction yielding argininosuccinic acid (125). Both of these enzymes and argininosuccinase, causing cleavage of argininosuccinic acid, are present in N. crassa, yeast, and a number of bacteria which utilise citrulline (118, 133).

It will be seen that the action of argininosuccinase is analogous to that of aspartase in yielding fumaric acid and a base, but in the mammalian systems studied, aspartase activity has not been evidenced in any enzyme system associated with this cycle. It is conceivable, then, that in fungi, as in mammalian liver, the ammonia required for arginine synthesis is fixed by CC -ketoglutaric acid to yield glutamic acid via glutamic dehydrogenase. The glutamic acid transaminates with oxaloacetic acid to form aspartic acid, which in turn donates ammonia to citrulline with the formation of arginine. - 36 -

Arginine can be degraded to ornithine in a cyclic process which, in mammals, can be diverted to urea formation.

In N. crassa urea has not been reported although the organism forms urease, it being supposed that the cycle serves primarily to supply arginine for protein synthesis.

It is of interest that both fungi and bacteria contain the enzyme, arginine desimidase, which bring about the reversal of the above-described formation of arginine from citrulline (134, 135). In the further degradation of citrulline, ATP is generated:

Citrulline + H^PO^ + ADP - > Ornithine + CO- + NH^ + ATP.

This sequence is of importance since it enables the cell to use arginine for the production of energy in the absence of carbohydrate. In bacteria, because carbamylglutamic acid and its analogues are not involved in the sequence, it is not simply a reversal of the formation of citrulline, as in mammals.

(2) The Aspartic Acid group The close relationship which exists between members of this group is well brought out in Table No. 2, in which it may be seen that the relative radioactivities observed in aspartic acid, threonine, isoleucine, and methionine are almost identical in T. utilis when grown on a variety of labeled substrates.

The function of aspartic acid as a precursor of threonine was demonstrated for N. crassa and T. utilis by 37 -

Abelson and Vogel. Using yeast, Black and Wright (136, 137) demonstrated the following sequence of reactions and

discovered the intermediates -aspartyl phosphate and

aspartic acid--semialdehyde.

Aspartic Acid + ATP -Aspartyl Phosphate

(^-aspartokinase)

fi-Aspartyl Phosphate —Aspartic Acid p-Semialdehyde

+ TPNH +H+ + TPN+ + Hq PO. 3 4 {A- semialdehyde dehydrogenase)

Aspartic AcidA-Semialdehyde Homoserine

+ DPNH + H+ + DPN+

(homoserine dehydrogenase)

The formation of threonine from homoserine has been demonstrated in N. crassa and E. coli as well as in yeast, and would appear to be a general sequence in microorganisms

(13^, 139, 140, 141). The further fate of threonine in both N. crassa and T. utilis was elucidated by Abelson and Vogel (106) by isotopic competition studies analogous to those described for glutamic acid. The results establish the following sequences:

Aspartic Acid--- >. Homoserine --->- Threonine --->-

0( -Ke to butyric Acid >. fl(-Ke to-^-Methyl ---Isoleucine Valeric Acid

Adelberg (142) has proposed a mechanism for the

biosynthesis of isoleucine in N. crassa based on isotopic - 33 - competition experiments. It was found that threonine suppressed the incorporation of radioactivity into carbon atom positions, 1, 2, 4, and 5 of isoleucine when uniformly labeled acetic acid was used as the carbon source for the organism. In addition, threonine - 1, 2 - yielded the dihydroxy acid - 1, 2 - showing that the and of isoleucine came from C^, and C, of threonine. j 4 Adelberg proposed that the primary product derived from ketobutyric acid and acetaldehyde is produced by an aldol condensation followed by hydration and that this trihydroxy acid then undergoes a pinacol rearrangement to the ketohydroxy acid:

CH, ILC OH CH, 1 I 3 CH, CH, + ii2o 1 1 1 + 1 -> HOC—CHCO,H CO CO CHO iX 1 CILCHOH ch3chchohco2h co2h (VIII) (IX)

The ketohydroxy acid is then supposed to be hydrated and reduced in turn to give the dihydroxy acid and finally isoleucine. Though the isotopic data are consistent with the proposed pathway, the actual reactions and intermediates involved must still be elucidated. Nevertheless, the proposed shifts of the methyl and ethyl groups in these formulations represent novel reactions, at least for - 39 - biological systems, and demonstration of such phenomena at

the enzymatic level would be of interest.

This type of synthesis of isoleucine is paralleled by

that of valine in which pyruvic acid condenses with acetaldehyde to form acetolactic acid (143) which then rearranges in the above manner to give a keto hydroxy acid

(144, 145, 146). A biochemical relationship between valine and isoleucine was originally indicated by Bonner et al. (147), who found a single mutant of N, crassa which required both amino acids for growth. These investigations revealed that N. crassa mutants accumulated the two X,^ -hydroxy acids, having the carbon chains of valine and isoleucine (14$, 149, 150) and the above schemes provide a ready explanation for this,

since the formation of these hydroxy acids only requires that the ketohydroxy acid precursors be reduced. The generality of the schemes proposed for the formation of threonine and isoleucine in various microorganisms is borne out by observations on other isoleucineless mutants of N. crassa, which will grow on the four-carbon-atom acids, o(-ketobutyric acid, x-aminobutyric acid, and threonine (150,

151, 152). This is also observed with E. coli, in which these four carbon acids suppress the radioactivity of isoleucine formed from C^-glucose (113). In the latter organism Umburger (153) has shown that L-threonine is an obligatory precursor of isoleucine. - 40 -

The fact that the formation of methionine in N. crassa

(154) proceeds via homoserine, suggests that it occurs by a mechanism similar to that found in mammals, i.e., by the condensation of homoserine with cysteine to yield

cystathione, which in turn gives rise to homocysteine and methionine. Cystathione has been shown to be a highly effective competitor in the synthesis of radioactive methionine in N. crassa and T, utilis (107) and is thus implicated as a methionine precursor. Methionine can take part in transmethylation reactions so that it is formed from homocysteine by the transfer of a methyl group from an energy rich ’’onium” compound such as betaine or the the tins

(the reactions do not involve ATP or other energy sources), or it can form homocysteine by the transfer of its methyl group to acceptor molecules (155). Vdnether methionine always functions as a methyl donor as such, or is present as an activated complex, is uncertain. Such an activated compound has been found in certain microorganisms, including fungi, in the form of S-adenosylmethionine (156, 157, 15&, 159, 160), which is formed from methionine and ATP. Its

structure has been unequivocally established by synthesis (161) By donating its methyl group to an acceptor molecule

it presumably forms S-adenosylhomocysteine and this is possibly

the active form of homocysteine, which in fungi, actually accepts methyl groups to form methionine (162).

S-adenosylhomocysteine has been found in animal tissues, - 41 -

and its structure proved by complete synthesis (163). It is possible that the methylation of homocysteine occurs by © the intermediate addition of formaldehyde from the Mone carbon pool” to yield S-hydroxy-methylhomocysteine, a compound which has been found in pigeon liver homogenates

(164).

McRorie et al. (165) have observed, however, that certain mutants of N, crassa yield compounds, intermediate between homocysteine and methionine, which have chemical and biological properties different to those of the

5-hydroxymethyl compound and to the alternative m-thiazane-

4-carboxylic acid. The position is still open,and as regards fungi, little is known at the moment about this aspect of cell metabolism.

It is noteworthy that 5-adenosylhomocysteine has been shown to be formed from adenosine and L-homocysteine by direct condensation rather than via methionine. This reaction has been demonstrated in animal tissues (166). De La Haba and Cantoni (167) recently described an enzyme from yeast which specifically condensed adenosine and L-homocysteine to yield S-adenosyl-L-homocysteine. In bacteria methionine is also formed from homocysteine and 5-methyl methionine (166).

In addition to transferring methyl groups, the active form of methionine can give up its 5-methyl grouping intact, e.g., a mutant strain of Aerobacter has been found in which

S-adenosylmethionine transfers its 5-methyl group to - 42 -

X-aminobutyric acid, a direct reversal of one path of

methionine degradation in fungi. It has also been shown

that methyl mercaptan and ethyl mercaptan (169, 170) can

give rise to methylthioadenosine and ethylthioadenosine in

yeast, presumably via adenosylmethionine and adenosylethionine.

bince both of these mercaptans may be formed in normal cell

metabolism these reactions may well be of importance in

the general economy of the sulphur compounds.

(3) Lysine

The biosynthesis of lysine has been studied in N. crassa,

yeast, and bacteria. Two synthetic pathways have been

discovered, and one of these appears to involve intermediates

which are also formed in the degradation of lysine in higher

animals.

Using isotope labeling experiments (171, 172, 173) the

distribution of radioactivity in carbon atoms 3, 4, 5 and 6

of lysine isolated from T. utilis after growth with acetate

labeled carboxyl and methyl groups, accords well with that

expected in the succinic acid moiety of o(-ketoglutarate,

while the radioactivity in carbon atoms 6 and 2 of lysine

suggest that these carbons find their origin in an intact

acetate radical (174, 175, 176).

Using this evidence Stassman and Weinhouse (171, 172)

postulate that the acetate radical condenses with the ketonic

grouping of 0t-ketoglutaric acid to yield homocitric acid as

the first product of biosynthesis. This reaction is analagous - 43 - to the citric acid-forming reaction by condensation of oxaloacetic acid with acetic acid. The further reactions postulated are again analogous to those of the citric acid cycle yielding oC-ke to adipic acid (the homologue of

X-ketoglutaric acid) which is then supposed to yield X-amino adipic acid presumably by transamination. This amino acid is known to be the precursor of lysine in N. crassa (177) and various other microorganisms (173,

179) and is presumed to be converted into lysine by a series of reactions resembling that involved in transamination of glutamic acid to ornithine previously described.

Another finding of possible significance is that X-amino £ -hydroxycaproic acid can serve as a lysine precursor

(179, 130). It is probable that both X-aminoadipic acid and X-amino- £-hydroxycaproic acid are converted to o(-aminoadipic acid - £-semi aldehyde, this being the precursor of A*-piperidine-2-carboxylic acid. It has been shown that N. crassa (1$1) can convert X-keto-^ -aminocaproic acid (which is in equilibrium with the cyclic form of ^-piperidine-2-carboxylic acid) to pipecolic acid or to lysine. (4) The Pyruvic Acid group of Amino Acids The incorporation of radioactive tracers into a further series of amino acids of T. utilis (106), is shown in Table No. 4. - 44 -

TABLE No. 4. Incorporation of Radioactive Tracers into the Amino Acids

of the Protein of Torulopsis utilis Tracer Protein Amino Acid

Alanine Valine Leucine Serine Glycine Cysteine

Alanine 100 40 60 5 5 Serine 0 0 0 100 100 100 i ir\ —

Glycine 0 0 0 15 100 I

It is apparent that alanine functions as a precursor of both valine and leucine and this presumably occurs via pyruvic acid as an intermediate, since isotopic competition experiments reveal that unlabeled pyruvic acid depressed the incorporation of radioactivity into alanine, valine and leucine. similar experiments with -ketoisovaleric acid show

the suppression of the radioactivity of valine and leucine while oC-ketoisocaproic acid suppresses only the activity of leucine. Isotopic competition experiments show that added non-radioactive valine affects both the incorporated radio­ valine and radio-leucine of T. utilis protein while added non-radio-leucine affects the radio-leucine.

The role of pyruvic acid as a precursor of valine in

T. utilis was established by Strassman et al (144) and in

E, coli by Abelson (113) while the function of oi -ketoiso­ valeric acid as a forerunner of valine in N. crassa was - 45 -

revealed by Bonner et al (147). Furthermore, the mediation of (X -ketoisocaproic acid in the biosynthesis of leucine in other organisms is exemplified in N. crassa (106) and E. coli

(106). The conversion of (X -ketoisovaleric acid to

0(-ketoisocaproic appears to involve the loss of a fragment containing a single carbon atom, and the replacement of this by a fragment containing two carbon atoms. In view of the findings of Ehrensv&rd et al. (174, 175) that the carboxyl group of leucine in both T. utilis and E. coli appears to arise in the carboxyl group of acetate it seemed probable that the two carbon fragment was closely related to acetic acid.

This probability was realized when Reiss and Bloch

(162) showed that and of leucine in Saccharomyces cerevisiae, grown on labeled glucose or acetate, are derived directly from an acetate unit. A similar conclusion was reached by Strassman et al (163) from an examination of the radioactivity of each carbon atom of leucine derived from

T. utilis after growth on glucose in the presence of tracer amounts of variously labeled acetic and lactic acids. These workers also found that the labeling of the isobutyl group of leucine matched that of the corresponding group of valine thus confirming that the carbon chain of valine arises from the same source as that of leucine as shown by Abelson’s isotopic competition experiments. By analogy with the reactions of the citric acid cycle, btrassman et al.(163) - 46 - propose a condensation between (X-ke to iso valeric acid and

the methyl carbon atom of acetyl CoA to give o(-hydroxy- (X-isopropylsuccinic acid, which is converted (in analogy with the conversion of citric acid to Q(-ketoglutaric acid)

to (X-ketoisocaproic acid. Transamination of the latter compound would yield leucine.

Alanine has received almost no attention in studies on fungi. The studies of Wang et al. (1&4) on the extent of labeling of alanine from yeast grown on pyruvic acid, suggest

that alanine is formed directly from pyruvic acid by trans­ amination and consequently administration of pyruvic acid-

2-C^^ resulted in heavy labeling of C2 of the alanine. However the C-^ and positions were also labeled to some extent. This labeling probably resulted from equilibration with oxaloacetic acid, since the ratio of the activities in these carbon atoms was very similar to that observed for

the corresponding carbon atoms of aspartic acid.

(5 ) Histidine Isolation of histidine from the cellular protein of

T. utilis, 3, cerevisiae, and Pseudomonas fluorescens, grown in the presence of formic acid-C^^, and followed by degradation, shows that Gg of the imidazole ring is derived exclusively from formic acid (1&5, 1$6). Neither glycine-1-

nor bicarbonate-C^^ was incorporated into histidine and

since fungi incorporate the former directly into the

imidazole ring of purines it is apparent that the imidazole - 47 -

ring in the latter compounds is synthesized by a route different to that for histidine. The observation of Broquist and Snell (187) that Lactobacillus casei shows an increased requirement for purines when grown in the absence of histidine is not to be interpreted therefore to mean that the purines serve as histidine precursors. More probably these purines serve as sources of formic acid or single carbon intermediates in the formation of the C2 of the imidazole ring of histidine. Studies on the breakdown of histidine in either micro­ organisms or liver revealed that one product consisted of a compound which yielded formic acid and glutamic acid on hydrolysis (186, 189, 190, 191, 192). This substance proved to be formarnidinoglutamic acid and consequently the formic acid produced by degradation originated in C2 of the imidazole ring (193, 194, 195, 196, 197, 196, 199). This fact suggested that the synthesis of histidine might occur by the reversal of the degradation reactions, possibly from glutamic acid. Investigation of this hypothesis by Levy and Coon (110) showed it to be untenable for fungi. On the other hand when yeast was grown on glucose-l-C^\ the specific activity of the histidine equalled that of the glucose, but C2 of the ring only contained 9% of the total radioactivity of the molecule (110), showing that the five carbon chain is derived more or less directly from glucose.

The probability that a sugar functions as the source - 48 - of the carbon chain of histidine is enhanced by the findings that mutants of N. crassa (111) and £. coli (200) accumulate

compounds which are related to histidine, but which contain

one to three hydroxyl groups in the side chain. Compounds

of this type are formed in the non-enzymic reaction of

D-arabinose or D-ribose with ammonia and formaldehyde in the

presence of copper, thus:

HC—NH * Cu++ ^ \ CHO 2NH3 + HCHO-----> CH I /y CHOH N I + CHOH CHOH I CHOH CHOH I CHtOH CHsOH i>-AY///Arotrihydroxy- propylimidazole

and it seems probable that an enzymic reaction of a similar

nature might occur in N. crassa (201). Working with various mutants of this organism, Ames and Mitchell (202) isolated

three phosphate esters which proved to be derived from

hydroxy compounds of the above type, and on the basis of this

evidence were able to propose the following reactions for

the later stages in the biosynthesis of histidine: - 49 -

H II H HC —X IIC—N HC—N \ \ /H /CH i > C—X C—N C—X 1 1 1 1 I nTT —Pc —AUun * ^ t CH, CII2 1 I H-C-OH c=o CIIXII2 I ch2opo3h2 ch2opo3h2 ch2opo3h2 Imidazoleglycerol Imidazoleacetol L-Histidinol phosphate phosphate phosphate asz

r H

1 HC> H(D—N n

C 1 C n> CH2 —-—» ch2 chxh2 chnh2 1 CH2OH COOH L-Histidinol L-Histidine

Enzymes which catalyse the first two reactions have been isolated from N. crassa; that which catalyzes the transformation of imidazole acetol phosphate to L-histidinol phosphate being known as imidazole acetol phosphate transaminase, and that which brings about the conversion of imidazole glycerol phosphate to imidazole acetol phosphate being called imidazole glycerol phosphate dehydrase. The former enzyme, in common with most transaminases, requires pyridoxal phosphate as a co-enzyme (203). Both enzymes fail to act on the dephosphorylated compounds, which are also accumulated by the histidineless mutants examined. 50 -

Using N, crass a mutants, Ames (204) has shown that

L-histidinol phosphate is converted into L-histidinol by a phosphatase in the normal biosynthetic sequence of reactions.

Histidinol itself has also been shown to function as a precursor of histidine in E. coli by Uestley and Ceithame

(205). These latter workers also emphasize that the five- carbon chain of histidine is formed prior to the formation of the imidazole ring.

Little is known regarding the source of the nitrogen atoms for the imidazole ring of histidine in microorganisms, although investigations by Neidle and 7/aelsch (2 0 6) using

E. coli have shown that of the imidazole ring (that yielding the amino group of glutamic acid on degradation) is derived from the amide group of glutamine.

A detailed description of studies relating to the origin of the imidazole ring in histidine has recently appeared

(207). The amide-N of glutamine was directly incorporated into the L-^ position of the imidazole ring by S. coli, and the and atoms of purines were incorporated as a unit into the and C2 positions of histidine. Conversely it has been found that histidine donated not only a formyl group, but also a nitrogen atom, in the formation of purine precursors (208).

These findings are consistent with the fact that, in

Lactobacillus casei (209), the N-atom of aspartic acid labels both of guanine and of histidine and that C2 °T guanine 51

yields C2 of histidine.

(6) The Serine group

This group deals principally with the reactions of serine and glycine. An examination of the biochemical literature of the filamentous fungi reveals very little study of these

amino acids, and it will be necessary to quote data on yeast

and other microorganisms in order to present a background for

the possible intermediary metabolisms of these amino acids.

An examination of Table No. 4 shows that the addition of radioactive serine to T. utilis results in labeling of

the serine, glycine, and cysteine of the protein of this organism at equal levels of specific activity. The use of radioactive glycine as a substrate gave rise to labeling of

serine and cysteine at only 15% of the initial specific activity - a finding in agreement with the earlier observations of Ehrensv&rd et al. (210). It was concluded

that serine is a major precursor of glycine and cysteine

in T. utilis, resembling N. crassa and E. coli in this

respect (113).

In more detailed investigations, in which the labeling of the individual carbon atoms of glycine and serine derived

from 0. cerevisiae protein was determined, Wang et al. (1$4)

found that the isotopic patterns in these amino acids are

similar, each having the same extent of labeling in C-^ and

These authors concluded that the origin of this series of amino acids does not lay directly in the conversion of pyruvic 52 -

acid to serine, as postulated for rats (211), since the labeling patterns were dissimilar to that of alanine.

The origin of the glycine skeleton itself is to be

sought in more complex metabolites, and indeed Meltzer and

Sprinson (212) proposed that 0C>£ -cleavage of threonine would yield glycine. This hypothesis is not compatible with

the results of Wang et al., since the isotope distribution

in glycine did not match that of the corresponding moiety of threonine and, furthermore, Ehrensv&rd et al. (174)

showed that the proposed cleavage of threonine would yield

two molecules of acetic acid-2-C^. Wang et al. therefore postulate that glycine is formed from pyruvic acid-2-C^ via

Krebs cycle as shown in Scheme A.

CH, Clif 0 C02H

|C02 C - C - c1/f - c

| Randomization

C - C1^ - C1^ - C —_____ ^ c1^ - c + clk - c I Fumaric Acid Krebs I or Scheme C Cycle f Succinic Acid co2h c1^ 0 c14 h2 ch2 co2 h

0(-Ketoglutaric Acid

Scheme A 53

Conversion of c(-ketoglutaric acid to the four carbon dicarboxylic acids followed by (X,£ -fission would then produce the isotopic pattern experimentally found for

glycine. Similar operation of the cycle with acetic acid-

1_C^ would yield glycine-l-C^ in accordance with the

finding of Ehrensv&rd (Scheme B).

Krebs CHj C1^ 02H —C1^ - C - C - C1Zf --*.

C - c1^ --► ch2 nh2 c1^ o2h

Scheme B

This scheme is also consistent with the observation by

Shemin (213) that C-^ of glycine may arise from of glutamate

A further metabolic pathway involving direct interconversion of the four carbon acids formed from pyruvic acid-2-C^ would result in the preferential labeling of 0>2 of both

glycine and serine (Scheme C). The slightly higher isotope

level in this carbon atom observed by Wang et al. (184) was

considered to reflect a limited contribution of this nature.

It is apparent that the relationship between pyruvic

acid, glycine, and serine is still somewhat obscure and it

is possible that the net balance between glycine and serine may vary according to the conditions. Reactions, other than

those noted above, which may well contribute to the formation

of serine are:

(a) The reverse of the catabolic deamination of serine to 54 -

pyruvic acid, although no evidence for this has as yet been obtained, and,

(b) The transamination of ft -hydroxypyruvic acid which has been established in other organisms (214). If the formation of -hydroxypyruvic acid from D-glyceric acid can be established, the connection of serine with pyruvic acid would be evidence, but it is perhaps significant that the reverse of this reaction has been noted (215).

The work of Wang et al. makes it probable that formylation of glycine can yield serine, and an enzyme, isocitritase, derived from certain bacteria and S. cerevisiae

(216) has been reported, which splits isocitric acid to form succinic acid and glyoxylic acid. Transamination of glyoxylic acid will yield glycine. Although this enzyme is present in fungi, recent isotopic (21?) studies have shown that the formation of glycine from citrate is not a path of major importance.

The various routes which might contribute to the formation of glycine and serine have been reviewed by

Ehrensv&rd (112) and presented in a cyclic form associated with Krebs cycle. The formation of glycine via Krebs cycle in this manner would lead to the labeling pattern of glycine and serine observed by Wang et al. (184) and is presumably therefore, the major pathway.

However, the mechanism by which glycine is converted into serine has not yet been elucidated but, if it resembles 55 -

that in other organisms, it may well be mediated by folic acid as has been found to be the case for a large number of transfers of single carbon units, bprinson has suggested that these transfers can take place according to the following scheme (2l£, 219, 220).

Histidine

Tryptophan h' N —^ N-CHO ^ purines Formic Acid / R? * Glycine " r

Formyltetrahydrofolic acid -b-CH^ Methionine oerine CH20H group - ■ NN-CH20H -v ^ -N-CH^ Choline

R2 -C-CH^ Thymine

Hydroxymethyltetrahydrofolic acid

Osborne and Talbert (221) have isolated an enzyme (from beef, or pigeon liver acetone powders) which catalyzes the conversion of hydroxymethyltetrahydrofolic acid into - formyltetrahydrofolic acid.

Formyl groups can be transferred from histidine (see above) via formamidinoglutaric acid (222), tryptophan, etc., to tetrahydrofolic acid and in turn to purines, while hydroxymethyl groups can similarly be transferred to folic acid, and hence to acceptor compounds, such as homocysteine by a reductive mechanism or, in the present instance, to an activated glycine molecule to form serine. - 56 -

An enzyme system,recently isolated from extracts of

Clostridium cylindrosporum, has been found to catalyze the conversion of serine into glycine and formic acid: This enzyme system requires DPN, managanous ion, pyridoxal phosphate, and orthophosphate for its activity and, although citrovorum factor and tetrahydrofolic acid function as co­ factors, very much more active natural coenzymes have been isolated (223, 224). These natural coenzymes are probably pteridine polyglutamates, the monoglutamate being inactive

(225, 226).

(7) The Aromatic Amino Acids

Certain Neurospora mutants accumulate in their culture fluid large amounts of the substrate of genetically blocked reactions, while other mutants will grow on later intermediates in the biosynthetic pathway involving the blocked reaction. Hence, by observing cross-feeding between two strains which have a requirement for one amino acid (the end product of the biosynthesis being studied), information on this biosynthesis is obtained, one strain providing quantities of an intermediate previously unavailable, the other strain aiding in the recognition of the intermediate by furnishing a method for biological testing. By utilizing a wide variety of such strains of Neurospora and other organisms requiring a mixture of tyrosine, phenylalanine, tryptophan, p-amino benzoic acid, and p-hydroxybenzoic acid,

Davis (109, 227, 228) and co-workers have established a 57 -

scheme of reaction sequences as follows:

^Cll2 —C—COOH-----* Phenylalanine HO COOH COOH

rS „oO

HO COOH COOH COOH

jf) — — jfj ---->"Z7 Anthranilic acid Indole •Tryptophan OY OH O’Y'OH HO^YHO' Y' 0H OH OH 5-Deh y droquin ic 5>Dehydroehikimic Shikimic acid acid acid

This scheme is now widely thought to apply in its major respects to the synthesis of aromatic compounds in

a large number of organisms and in higher plants. The pathway from 5-dehydroquinic acid through 5-dehydro-

shikimic acid, shikimic acid compound ,TZ1M is not certain, but Gilvarg, in an unpublished communication to Davis has

shown that it is an acid labile conjugate of shikimic acid

with pyruvic acid, the latter being liberated on hydrolysis.

Hence, it is suggested that the pyruvic acid is attached to

the ring by an enol-ether linkage, subsequent rearrangement

yielding prephenic acid, which aromatizes extremely readily, indeed spontaneously at pH values greater than 7, losing the elements of water and carbon dioxide to form phenylpyruvic - 56 -

acid (229, 230). The compound HVtT, accumulated by mutants blocked before 5-dehydroquinic acid (231) was shown by Davis (226) to be an open chain phosphorylated keto acid. At the same time a study of the incorporation of radioactive glucose into shikimic acid revealed that C-p Cg, and the carboxyl group of this acid originated from a glycolytic fragment, the remainder of the molecule being derived from a four- \ carbon compound containing to of the glucose. Further experiments with cell extracts demonstrated that dehydro- shikimic acid can be synthesized from compound "V" and also in small yield from various hexose phosphates (232). This acid was obtained in large yield from sedoheptulose-1,

7-diphosphate (233), carbon atoms 4 - 7 of the heptose being converted quantitatively and exclusively into to of the product, the remaining three carbon atoms of the heptose being inverted prior to recombination with the four carbon fragment and cyclization. Inhibition by flouride ions of the enzyme catalyzing the conversion of triose phosphate to phosphoenolpyruvic acid completely prevents the synthesis of dehydroshikimic acid from sedoheptulose diphosphate, but the synthesis proceeds when phosphoenolpyruvic acid is added together with fluoride. Furthermore, the addition of D-erythrose-4- phosphate and phosphoenolpyruvic acid to an enzyme extract leads to the production of dehydroshikimic acid, indicating 59 - that sedoheptulose is not an obligatory intermediate in the production of aromatic compounds. The formation of the ring compounds may therefore be visualised as occurring by the following sequence:

Phosphoenolpyruvic Acid + D-Erythrose-4-phosphate I 2 Keto-3-deoxy-7-phospho-D-glucoheptonic acid i Dehydroquinic acid

The intermediate, 2-keto-3-deoxy-7-phosphoheptonic acid has been synthesised and shown in E. coli at least to be readily utilized in producing 5-dehydroshikimic acid

(234). It therefore appears that the complete path of biosynthesis has been outlined in this organism. With regard to the biosynthesis of tryptophan, most evidence for the individual steps involved has been adduced from the study of mutants of N. crassa. As a result Haskins and Mitchell (235) have been able to demonstrate the series of reactions summarised on the following page. The final step in the formation of reactions consists of the condensation of serine with indole by means of an enzyme, tryptophan synthetase, which requires pyridoxal phosphate as the co-enzyme. Suskind and Kurek (236) prepared an active tryptophan synthetase from N. crassa. Indole originates from anthranilic acid by an unidentified compound. The nature of this compound X was uncertain until Yanofsky (237, - 60

Ser.ne Tryptophan ch2oh Enzyme-Pyridoxal phosphate CH:CHNH2C0?H

C02H

Indole Kynurenme 3Hydroxykynurenme COCH2CHNH2COzH ^vn.C0CH2CHNH2C0zH

Phenylalanine •— Compound X Anthranilic acid 3-Hydroxyanthranilic acid Tyrosine ^ CO,H CO.H fj Ammobenzoic acid Cj(V' MU NH..

Nicotinic acid ^Niacin) „ CO. H

23a, 239) found that, in E. coli, a.nthranilic acid reacts with 5-phosphoribosyl-l-pyrophospha te (derived mainly from the reaction of fructose-6-phosphat^e with 3-ph°sphoglycer- aldehyde and partly via the shunt rmechanism) to yield indole- 3-glycerol phosphate. This compound is split by a separate enzyme to yield indole and triose phosphate and its isolation suggests the possibility that tryptophan may be synthesised

in some organisms by a mechanism other than the coupling of

serine with indole, since the analogous compound, imioa^ole

glycerol phosphate, yields histidine directly. Yanofsky (240) has recently shown that the tryptophan

synthetase of E. coli participates in the conversion cf indoleglycerol phosphate to indole, while Doy and Gibson (241 - 61 -

have shown that mutant strains of Aerobacter aero genes and

E. coli, which require tryptophan or indole for growth accumulate a new compound, 1-(O-carboxyphenylamino)-1- deoxyribulose, which may be the immediate precursor of indolylglycerol phosphate. - 62 -

UREA AND UREIDES

THE OCCURRENCE OF UREA AND ITS PRECURSORS IN FUNGI

Urea was first isolated from fungi in 1903 (242) followed by the discovery of urease in 1904 (243). The

French workers Brunei (244), Fosse (245) and Brunel-Capelle (246) isolated the ureides, allantoin and allantoic acid, from several fungi, mainly basidiomycetes, and the same authors demonstrated the presence of allantoinase and allantoicase in fungal extracts.

The ureido group is found in three compounds of known occurrence in fungi, allantoin, allantoic acid and citrulline.

In animal and bacterial biochemistry interest has lately arisen in four other ureido compounds:

(a) ureidosuccinic acid (orotic acid metabolism). (b) ureidoglutaric acid (citrulline synthesis).

(c) /S-ureidopropionic acid (uracil metabolism), (d) 0-ureidoisobutyric acid (thymine metabolism). Evidence may eventuate to show the involvement of these ureides in fungal biochemistry.

Urea is a utilizable source of nitrogen for fungal growth and experiments show utilization by Candida albicans

(247) , Aspergillus niger (248), Streptomyces griseus (11),

Venturia inaequalis (249), Coccidioides immitis (250) and several other species of Mucor (251). Urea is formed in the fruiting bodies of several basidiomycetes (252, 253). Ivanov (254) considers that many - 63 -

fungi store nitrogen in the form of urea when they are growing in a medium rich in nitrogen while excess of carbohydrate in the medium tends to suppress urea storage.

When grown on a peptone medium A. niger secretes urea into the medium, but if carbohydrate is then supplied, the urea disappears.

In pure culture urea is formed by many saprophytic fungi and some actinomycetes (255, 256, 257) while oxygen and exogenous ammonia increase urea formation by sporophores

(258, 259).

The biochemical origin of urea in fungi has not been proved, but there are several possible sources:

(1) The breakdown of arginine in ornithine cycle.

Ivanov (260) noted that exogenous arginine increases the accumulation of urea in a variety of fungi and arginase activity has been demonstrated in cell free preparations from basidiomycete sporophores (259).

(2) Urea appears to be formed by the action of one or more enzymes which act on guanidine and some of its derivatives to yield urea and ammonia; this activity is reported in Streptomyces griseus (261) and in several saprophytic fungi (262, 263).

(3) Lamaire and Brunei (264) report that cell free preparations of A. niger decompose calcium cyanamide with formation of urea.

(4) The breakdown of purines in fungi to yield urea has - 64 -

been suggested (2), and the following path of degradation

proposed.

Guanine

Hypoxanthine - Xanthine -- Uric acid ------►

Adenine

Hydroxyacetylene dlureide carboxylic acid --- ►

Allantoin«*Allantoic acid ---- »•

Urea and

Glyoxylic Acid

Although the role of the above path has not been demonstrated in any single organism, the following evidence

is consistent with the proposition.

(a) Xanthine oxidase and uricase occur in several

fungi (265), and uricase activity has been induced in

Torulopsis utilis (266).

(b) 5umi isolated and identified uric acid in the

spores of A. oryzae (267)*

(c) Allantoinase, an enzyme which catalyzes the hydro­

lytic cleavage of the ring of the allantoin molecule to form

allantoic acid, is found in cell free extracts of

basidiomycete sporophores (263) and in A. niger (269).

(d) Fosse and Brunei (245) discovered significant

amounts of allantoic acid in basidiomycetes.

(e) Brunei (270) discovered allantoicase, an enzyme which hydrolyses allantoic acid to urea and glyoxylic acid, - 65 - in both A. niger and A. oryzae.

The above pathway could thus serve to convert purine nitrogen into urea nitrogen, which might then become generally available to the organism through the action of urease.

The yeast Saccharomyces cerevisiae (271) can use allantoin, urea or ammonia as sources of nitrogen but its biotin requirement is different for each compound. With allantoin the biotin requirement is four times, and with urea twice that required for growth with either an ammonium salt or arginine. These decreasing quantities of biotin necessary to produce comparable growth tend to support the idea that in this organism allantoin is degraded to urea and then ammonia, and also indicate that urea is probably not an intermediate in the utilization of arginine. If, however, ureides in fungi do not all result from the degradation of uric acid, then they must arise from other substances, or by synthesis from simpler compounds. An obvious suggestion is that the ureides might arise by combination of urea with glyoxylic acid. Brunei and Brunei-

Capelle (272) report that these two substances are condensed to form allantoin in cell free extracts of basidiomycetes.

Urease, the enzyme hydrolysing urea, is of general occurrence among fungi and has been reported in many species of Aspergilli (269, 273, 274, 275), in Penicillium spp. (276), and some basidiomycetes (252, 277). The urease of A. niger - 66 - occurs in the growth medium during autolysis (273) and in basidiomycetes it is found to occur most abundantly (like the ureides) in the hymenial layers (252). Although urease occurs in Neurospora crassa (11?) urea is not formed in this organism. The arginine which is formed via ornithine cycle is presumably used for protein synthesis.

In bacteria, fungi, and the higher plants, urease appears to be a constitutive enzyme, being present in organisms grown in media without urea (278), but in some species, for example Pseudomonas aeruginosa (279) it is formed only if urea is present in the medium.

Citrulline, the other ureide of consequence in fungi, has been shown by Srb and Horowitz (117) to partake in ornithine cycle. A more detailed treatment of its role in this cycle as a urea precursor will be discussed with that of arginine in the section on amino acids. Canavanine, the guanidinoxyamino acid structurally related to arginine, has not been detected as a urea precursor in fungi, but metabolic studies show it can inhibit growth of N. crassa (280) by forming canavininosuccinic (281) acid which can serve as a substrate for argininosuccinase. This reaction yields urea and canaline as its end products and thus serves to inhibit arginine formation.

Canavanine is converted to guanidine and homoserine by Streptococcus faecalis (282). Such a conversion would - 67 - explain why canavanine replaces threonine in some mutants of

N. crassa (2$3). The origin and fate of the ureides in fungi is at present not clear. These compounds appear often in specific fungal tissues, particularly during periods of intense cell activity. Uric acid, allantoin, urea, citrulline and its metabolically related amino acids, arginine and ornithine, can act as sole sources of nitrogen for fungi, and the nitrogen of these compounds seems to be readily available for growth. However it is not yet possible to formulate a mechanism of utilization.

The following Tables (Nos. 5, 6, and 7) show growth and utilization data for several species of fungi cultured on urea, ureides and associated compounds. TABLE No, 5.

Comparative Growth of Fungi with Nitrogen Supplied as Nitrate-N, Ammonium-N or Urea-N

(Growth was determined as dry weight of mycelium unless otherwise stated.)

SPECIES NITRATE-N AMM0NIUM-N UREA--N AUTHORITY

Phvmatotrichum 100 14 104 Ezekiel, Taubenhaus & Fudge omnivorum (1934) (284). Aspergillus niger 100 101 90 Steinberg (1937) (149).

Actinomyces 100 100 478 Cochrane & Conn (1947) (285). coelicolor Memnoniella 100 120 125 Perlman (1948) (286). echinata

Colletotrichum ^ 100 124 124 Mathur, Barnett & Lilly 1 indemut’nianumK (1950) (287).

Phytophthora 100 - 38 Newton (1957) (288). parasitica

A Growth measured as colony diameter.

TABLE No. 6.

Species Which can use Urea, Ornithine, Citrulline or Arginine as Sole Nitrogen Source

(U = urea, 0 = ornithine, C = citrulline, A = arginine)

FUNGI Ability to grow on ol SPECIES U ol A AUTHORITY

Candida spp. + • • • Wickerham (1946) (247)

Aspergillus niger • 4* • + Steinberg (1942) (248)

Streptomyces griseus + • O + Delaney (1948) (11)

Venturia inaequalis + • • + Leben & Keitt (194&) (249) w /

TABLE No. 7,

Species which can use Uric Acid or Allantoin as Sole Nitrogen Source

FUNGI Ability to use as sole nitrogen source

SPECIES Uric acid Allantoin •

Sterigmatocystis • + Brunei (1939) (269) nigra

3. phoenicis • 4- Brunei (1939) (269)

Piricularia oryzae + • Leaver, Leal & Brewer (1947) (289)

Saccharomyces • + Di Carlo, Schultz & McManus cerevisiae (1951) (290) Torulopsis utilis + + Di Carlo, Schultz & McManus (1951) (290) - 70 -

THE METABOLISM OF THE NUCLEIC ACIDS AND THEIR CONSTITUENTS

THE DEGRADATION OF NUCLEIC ACID DERIVATIVES BY FUNGI

The nucleic acid of microorganisms is the subject of many early classical researches on nucleic acids generally, and on their degradation by various enzymes, in particular ribonuclease (291).

On comparative grounds it may be supposed that fungi, like bacteria, attack nucleic acids by nucleodepolymerase action (ribonuclease) yielding primarily nucleotides. Table

No. 8 shows such activity associated with some fungi.

Ribonuclease, which has been obtained in crystalline form, hydrolyses the bond linking CT-5 of one nucleotide residue in nucleic acid with the phosphate group attached to

CT-3 of an adjacent pyrimidine nucleotide. The corresponding phosphate links between pyrimidine nucleotides and purine nucleotides or between different purine nucleotides are resistant to the action of ribonuclease, which can therefore be regarded as a specific phosphodiesterase (301). As a result of its mode of attack on nucleic acid, the major proportion of the mononucleotides produced consist of the pyrimidine derivatives, uridylic and cytidylic acids, while the remaining products are polynucleotides derived principally from the purines, adenine and guanine (302, 303).

The nucleotides themselves are attacked by non-specific phosphomonoesterases to yield the nucleosides as follows: - 71 -

* H3P04

Sadasivan (304) has demonstrated the hydrolysis of

AMP to base and phosphate with an alkaline phosphatase from acetone powders of Penicillium chrysogenum. Similarly, using aqueous extracts of the mycelium of Aspergillus niger and P. chrysogenum, Krishnan (305), and Krishnan and Bajay

(306) hydrolysed ATP to orthophosphate and base, the enzyme

system being shown to have pyrophosphatase, metaphosphatase and depolymerising phosphatase activity.

Dialyzed preparations (free of phosphate donors) from

an acetone powder of yeast, bring about the hydrolysis of adenosine-3!-phosphate or adenosine-5T-phosphate to yield adenosine and phosphoric acid (307, 308). l/Vhen yeast is allowed to autolyze, the nucleic acid is converted into adenosine in 60% yield within 20 hours.

This second step in the classical pathway of nucleic

acid degradation is often, and perhaps usually, carried out by non-specific phosphatases.

The dephosphorylation of riboflavin monophosphate by extracts of P. chrysogenum (309) is presumably, although

not necessarily, effected by such phosphatases. A phosphatase TABLE No. 6.

Enzymes Acting on Nucleic Acids, Nucleotides and Nucleosides

Substrate Type of Action Occurrence

Ribonucleic Acid Depolymerisation Streptomyces spp. (292, 293)

Aspergillus oryzae (294)

Deoxyribonucleic Acid Depolymerisation Neurospora crassa (295)

Streptomyces sp. (293)

Nucleotides Deamination Aspergillus spp. (294, 296, 297, 293) Nucleosides Deamination Aspergillus oryzae (294, 293, 299) Neurospora crassa (300)

Nucleosides Hydrolysis Aspergillus oryzae (294) of Streptomyces sp. acts both on glycerophosphate and on nucleotides (293).

Diphosphopyridine nucleotide is attacked by the specific enzyme, diphosphopyridine nucleotidase, which splits off nicotinamide from the dinucleotide, leaving adenosine diphosphate ribose. The enzyme is formed by Neurospora crassa (311) and its high concentration in the conidia suggest; that it is formed only during sporulation (312). The concentration of enzyme in the mycelium is increased by deficiencies of zinc, nitrogen, or growth factors (313, 314).

The enzyme of N. crassa, unlike the corresponding animal enzyme, is not inhibited by nicotinamide except at very high concentration and does not catalyse an exchange reaction between the dinucleotide and free nicotinamide (315).

Degradation of nucleosides is brought about by enzymes known as the nucleosidases to give ribose (or deoxyribose from deoxyribosides) together with appropriate purine or pyrimidine

Such an enzyme is the specific uridine nucleosidase isolated from yeast by Carter (316) which brings about the hydrolysis of uridine to uracil and ribose. This enzyme will not catalyze the degradation of any other nucleoside tested and cannot split uridylic acid. It has optimal activity at pH

7.0. A series of similar enzymes capable of hydrolysing various nucleosides was detected by Heppel and Hilmoe (317) but as yet no evidence has been obtained for the existence in fungi of nucleoside phosphorylases analogous to those - 74 -

found in bacteria (313, 319, 320). Deamination of nucleotides and nucleosides is usually effected by specific enzymes, but an adenyl deaminase of A. oryzae acts on both types of compound, deaminating adenosine, adenosine-5*-phosphate, adenosine-3’-phosphate, adenosine diphosphate and diphosphopyridine nucleotide (321).

Chargaff and Kream (321) found a cytosine deaminase and

Wange et al. (322) detected a specific cytidine deaminase

(converting cytidine to uridine) in both yeast and Escherichia coli. Di Carlo et al. (290) detected guanine deaminase in

Qaccharomyces cerevisi^ . The information on deaminases is extremely limited, since few studies on isolated systems have been made and those carried out have been concerned with a limited number of organisms. In fact, the enzyme from Torulopsis utilis, referred to as an adenase by Roush

(323), has been partially purified (324) and shown to be an adenine transaminase which brings about the transfer of the

6-amino group of adenine to an X-keto acid in the presence of pyridoxal phosphate and cupric ions.

In Nocardia corallina the pyrimidines, thymine and uracil, are metabolised by way of barbituric acid (325). Presumably the overall process is also reponsible for the catabolism of cytosine and 5-methylcytosine. In bacteria this has been observed to lead to urea and malonic acid (326). The above data are too scattered and incomplete for a coherent picture of nucleic acid breakdown in fungi to be proposed, but they - 75 -

do indicate that catabolism of the purine ribonucleotides in fungi is similar to that in other organisms. It would thus seem likely that the free purines, adenine, hypoxanthine, and guanine, are converted to xanthine, which is broken down by xanthine oxidase to uric acid (265).

THE UPTAKE, INTERCQNVERSION AND SYNTHESIS OF

PURINES AND PYRIMIDINES BY FUNGI

Some basidiomycetes have been reported as having a partial requirement for adenine (7)> and adenine administered to these organisms does accelerate growth but does not influence final yield (327). Eypoxanthine, partially replaced by guanine, accelerates both spore germination and early growth of Phycomyces blakesleeanus (326, 329, 330, 331,

332, 333).

Purine and pyrimidine-requiring mutants in fungi have been obtained (334, 335, 336) and adapted for bioassay (337).

For both Neurospora sitophilia and Fusarium oxysporum f. nicotianae, the purines are generally adequate sources of nitrogen, the pyrimidines poor sources, and the methyl

purines, caffein and theophylline, unutilizable (336). A

variety of unrelated fungi are able to grow well with xanthine

as the sole nitrogen source (339) and N. corallina can obtain

both carbon and nitrogen from uracil (340).

Kerr (341) showed that T. utilis incorporated the adenine

skeleton into both the adenylic acid and guanylic acid moieties of ribonucleic acid, while guanine gave rise to - 76 - guanylic acid only. This particular yeast was found to utilize all the naturally occurring purines and pyrimidines except thymine (290). However the purine nucleosides are assimilated poorly, and the parent bases and corresponding nucleotides are assimilated even less readily, while the pyrimidine nucleosides and nucleotides ADP, ATP, ribonucleic acid and deoxyribonucleic acid did not serve as sources of nitrogen at all (290). S. cerevisiae is able to utilize allantoin nitrogen fully. Di Carlo et al. (271) succeeded in preparing an active allantoinase from this yeast.

As a result of experiments involving the growth of yeast

(T. utilis) on labeled purines such as adenine-3-C^, guanine

8-C*^ and 2,6-diamino purine-2-C^, Kerr and Chernigoy (342) came to the following conclusions: (a) In the biosynthesis of RNA, preformed adenine is utilized even more extensively than formic acid.

(b) Out of each three molecules of adenine incorporated into RNA, two molecules give adenylic acid while one yields guanylic acid.

(c) Guanine, and to some extent diaminopurine, suppress the utilization of adenine for the synthesis of guanylic acid. (d) Diaminopurine serves readily as a precursor of guanine but not of adenine and it suppresses the utilization of formic acid for guanine synthesis to about the same extent as adenine, but less effectively than guanine.

(e) The increased utilization of formic acid for adenylic - 77 -

acid synthesis which occurs in the presence of guanine or diaminopurine results from the sparing of formic acid from the synthesis of guanylic acid. In these experiments about

50% of the total purine supplied was utilized for nucleotide synthesis.

The failure of excess of diaminopurine to suppress markedly the use of guanine for the formation of guanylic acid is relevant to the hypothesis of Bendich et al. (343) that diaminopurine is an intermediate in the transformation of adenine to guanylic acid (in rats). If the diaminopurine were an obligatory intermediate, excess of unlabeled compound should sharply reduce the activity of any guanylic acid synthesised from labeled adenine in comparison with a control.

That this was not found to be the case suggested that adenine may be converted into adenosine and hence into diaminopurine riboside before finally giving rise to guanosine.

The finding that glycosides of the purines are involved in these conversions parallels the observation that their earlier biosynthesis de novo involves the glycosides rather than the free bases. It is generally recognised that yeasts and fungi resemble mammals and fi. coli in being able to

synthesize purines and pyrimidines from simple precursors,

i.e., carbon dioxide, formic acid, ammonia and glycine. In the synthesis of guanine, is derived from carbon dioxide,

and Cg from formic acid or substances which give, rise to formic acid (serine, methionine etc.) C^, C^, and N« from the 7£

carboxyl, methylene, and amino groups of glycine (344, 345,

346) and the remaining nitrogen atoms from ammonia thus:

:^C02 ~ NH NH-- fC ^ V" ' uOoH /

HCOpK

The methylene carbon atom of glycine is not used for the

synthesis of uracil or cytosine and hence the pyrimidines

are synthesized by a route independent of that for purines.

The synthesis of purines and pyrimidines in RNA and DNA has been reviewed by Siminovitch and Graham (347) who carried out

a systematic survey of the precursors of the nucleic acids

in S. coli by means of isotopic competition methods similar to those described for amino acids. In line with the observations made by other workers (see above) these authors conclude that there is no interconversion between the precursors of the purines and pyrimidines. In accord with this conception, orotic acid-6-C^ (labeled uracil-4- carboxylic acid) is utilized for the synthesis of uracil in fungal RNA as in mammals,'but is not incorporated into guanine. Lactic acid-2,3-C"^ is also used, presumably for the formation of the "non.ureido" carbon atoms via oxaloacetic acid and aminofuramide (or the related ureidosuccinic acid or carbamylaspartic acid) as suggested for N. crassa by

Mitchell and Houlahan (343) and for E. coli by Weed and

Wilson (349).

Of some interest in this connection is the finding of

Hermann and Fairley (350) that (X -aminobutyric acid supports - 79 -

the growth of pyrimidineless mutants of N. crassa and that this amino acid is incorporated in pyrimidines. Mitchell and Houlahan (348) are of the opinion that aminofuramide or its pentoside, oc-N-pentosylamino-fumaric acid diamide, is converted into a pyrimidine nucleoside through two inter­ mediates related to orotic acid riboside which has been isolated from the mycelium of a uridine requiring mutant of

N, crassa (351). The observation that certain pyrimidine auxotrophs grow better on cytidine or uridine than on the free bases again suggests that the latter are not the natural intermediates in nucleoside synthesis. Davis (109) has recently (1955) suggested that ureidosuccinic acid

(carbamylaspartic acid) formed by donation of a carbamyl group from carbamyl phosphate to aspartic acid, gives rise to orotic acid and finally pyrimidines.

A complete sequence for the formation of uridine triphosphate (UTP) and cytidine triphosphate (CTP) in N. crassa and coli has been postulated by Liebermann (352) to consist of the following reactions:

L-Aspartic Acid + L-Ureidosuccinate L-Dihydroorotate Carbamyl Phosphate

Qrotate 0ritidine-5T -P0^ >- Uridine-5f-P0^ + CC^

UDP UTP CTP

Enzymes bringing about several of these transformations have been obtained (353); those concerned with the final formation of CTP are specific for UTP, and ammonia is the $0 - source of the nitrogen atom added during the reaction.

In experiments on the incorporation of into the purines and pyrimidines of T. utilis (342) the adenine of the nucleotide fraction was labeled far more extensively than the adenine in nucleic acid, a fact indicating a greater turnover rate in this fraction. This is possibly related to the existence in this organism of a pool of free nucleotides similar to the pool of free amino acids. The intermediates involved in the formation of the purines in fungi have not yet been established with certainty

However Stetten and Fox (354) found in the culture filtrates of bacteria which had been inhibited with sulphonamides, an amine which was identified by Shive et al. (355) as 5-amino-

4-imidazolecarboxamide. In Qphiostoma multiannulatum there is evidence for the role of this base in purine biogenesis (356). Purine reversal of sulphonamide toxicity to

Eremothecium ashbyii (357) is consistent with the finding. This base accumulates because the sulphonamides inhibit a subsequent step in the biosynthesis of the purines which requires p-aminobenzoic acid as a co-operator.

A similar, and possibly identical base was isolated by

Chamberlain et al. (35$) from culture filtrate of yeast grown on a medium deficient in biotin and containing excess methionine. An important observation was that this base was able to support the growth of strains of

Schizosaccharomyces octosporus which require adenine and 61 -

hence it was concluded that it is a true intermediate in purine synthesis. Similar conclusions were reached by

Williams (357), who showed that authentic 5-amino-4- imidazolecarboxamide enables fungi to synthesise adenine and guanine at a rate comparable with that attained with glycine or formic acid, and by Bergmann et al. (360), who found that the amine is utilized by purine-requiring mutants of S, coli.

Bergmann et al. have shown that this compound can serve as a purine precursor in yeast, 0. multiannulaturn, E. coli, lactobacilli, and pigeon liver homogenates. An amine closely related to 5-amino-4-imidazolecarboxamide, namely amino- imidazole, has been shown to accumulate in biotin deficient yeasts (361, 362), and a role for biotin has been suggested in the conversion of this amine to the carboxamide. The amine may well be that observed earlier by Chamberlain et al.

(356), instead of the carboxamide mentioned above. Its accumulation is inhibited by purines- and by aspartic acid, the latter fact accounting for the growth stimulatory effect of the amino acid under conditions of biotin deficiency.

A soluble enzyme system isolated from S. cerevisiae by

Williams and Buchanan (363) catalyzed the incorporation into inosinic acid of the imidazole carboxamide, labeled in the carboxamide group with C"^. The authors report that this enzyme system is rather unstable but that their studies suggest that it resembles the more stable system from pigeon liver which is described below and which has given substantial 82 - information on the steps in purine biosynthesis. 5-amino-4-imidazolecarboxamide requires only one carbon atom to complete the purine ring, namely at C^, and Vitamin B-^2 is able to increase the incorporation of the base into the purines (360). Vitamin B-^ is known to play a vital role in the transfer of one carbon fragment in many organisms and it appears that this function is involved in the completion of the pyrimidine ring, possibly by way of formylamine. In

E. coli it was in fact found that the N-formyl compound was utilised to a greater extent than the free base and in this case vitamin B-^ had no effect. Recently, important work on the intermediates for purine biosynthesis de novo have been carried out with pigeon liver extracts and the following sequence of reactions demonstrated. See Fig. 1 (364, 365, 366, 367, 368, 369, 370). All the intermediates have been isolated and it is assumed that the free base aminoimidazole amide ribotide acts sluggishly as a purine precursor in some instances because it must first be converted to the ribose phosphate derivative thus recalling the behaviour of 2,6-diaminopurine in its conversion to guanylic acid. Guanosine (or guanylic acid) synthesis in 0. multiannulatum has been suggested to proceed from adenosine (or adenylic acid) i.e. by further step or steps added to the following equation (371)*

"X" ---Inosine ------Adenosine ------>-

Unknown Intermediates -- Guanosine FIGURE No. 1.

Path of Purine Biosynthesis, photphati 2 9

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°°N V ill ql < H 6 y<, , *-v/

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J w - 84 -

2,6-diaminopurine replaces guanine for some mutants of this organism, the corresponding riboside does not (372).

It is proposed that carbon dioxide is fixed by aminoimidazole ribotide. In this connection it is relevant to record that an intermediate in the formation of 5-amino-4-imidazole- carboxamide from 5-aminoimidazole ribotide in avian liver has been shown by Lukens and Buchanan (373) to be 5-ainino-

4-imidazole-(N-succinylocarboxamide)-ribotide.

This compound accumulates when ATP, aspartic acid, and bicarbonate are incubated with 5-aminoimidazole ribotide and an enzyme concentrate. When incubated with a second enzyme system, this intermediate yields the carboxamide ribotide and fumaric acid, recalling the fission of argininosuccinic acid. A related compound, adenylosuccinic acid (374, 375) has been isolated from yeast autolysates (376) and shown to arise from the reaction of fumaric acid with adenosine-5!- phosphate.

Adenylosuccinic acid has been proposed as a precursor of adenylic acid (377); an adenine-requiring mutant of

N. crassa accumulates both this compound and its riboside

(376). At the present time the reaction appears to be limited to fumaric acid, adenosine-5T-phosphate, and the corresponding deoxyribosyl derivative of adenine. This compound is con­ ceivably an intermediate in the incorporation of the 6-amino group into the purine nucleotides. Borne support for this - 85 -

view is supplied by the findings of Liebermann (379, 380) that a purified enzyme from E. coli yields adenylosuccinic acid from inosine acid, L-aspartic acid, and guanosine triphosphate (GTP). Since there is evidence that inosine or inosinic acid is the first purine to be synthesised naturally (381) , such a reaction, when coupled to the hydrolytic reaction described for yeast and fungi, would provide a mechanism for the formation of adenine. The enzyme isolated was highly specific for aspartic acid and for GTP.

Abrams and Bentley (381) described a similar enzyme system in bone marrow extracts. In this system one enzyme known as inosinic acid dehydrogenase catalyzes the oxidation of inosinic acid (IMP) to xanthosine-5’-phosphate (IMP) by

DPN. IMP is then aminated to guanosine-5T-phosphate (GMP) in the presence of L-glutamic acid or L-glutamine, ATP, and magnesium ions. The third reaction in this system is identical with the above-described conversion of IMP to AMP.

It seems probable that the intermediate in GMP production from XMP is a guanyloglutamic acid analogous to the adenylosuccinic acid mentioned previously. It may be significant that extracts of a guanine-requiring mutant of

Aerobacter aerogenes, which excretes xanthosine, is unable to aminate XMP, suggesting that the above amination reactions are the essential steps in the biosynthesis of the guanine of nucleic acid in this organism (382).

The link between IMP and the amino imidazole carboxamide - 36 -

ribotide is the reaction catalyzed by the enzyme inosinic acid transformylase (333), which involves the reaction of serine with the carboxamide to form glycine and IMP, an example of the one carbon transfer reactions. This reaction

< has been shown to reoxuire anhydroleucovorin .as a co-factor, together with glutamine the role of which has not been elucidated as yet (334). In fungi and other organisms (335,

336) the various nucleotides can also undergo interconversion by exchange reactions, for example, nucleoside triphosphates transfer phosphate groups to nucleoside diphosphate, thus

ATP + IDP-- >- ITP + ADP (nucleoside diphosphate kinase)

and to nucleoside monophosphates, ITP + AMP --ADP + IDP

ATP + AMP ---2ADP (adenylate kinase)

It was observed by Gabriel and Hoffman-Ostenhof (337) on the other hand, that yeast extracts do not effect the transfer of phosphate from ATP to guanosine, cytidine or uridine, but could bring about the transfer to adenosine.

The precise role played by such reactions in the overall metabolism of the cell is still a matter for speculation although the function of a number of the compounds formed is beginning to be understood, e.g., UTP (uridine triphosphate) is involved in the synthesis of UPDG (uridine diphospho- glucose), which functions as a co-enzyme for galactowaldenase

(333), and GTP functions in several reactions involving the conversion of 0{ -ketoglutaric acid to succinic acid in the - &v7 -

Krebs cycle (3$9, 390) and the above described amination reactions.

It is thought that the nucleoside triphosphates are concerned in the synthesis of nucleic acids (391) and, in this connection, it is pertinent to observe that AMP has been shown to be as good a precursor for RNA in pigeon liver as adenine itself, and that it is incorporated with the ribose phosphate bond intact (392, 393, 394), a finding supported for all nucleotides by that of Reichard (395) working with rats. Reichard also found that the specific activity of pyrimidine deoxyriboside isolated from nucleic acid was identical with that of incorporated, labeled pyrimidine riboside, and concluded that the deoxyribosides are formed directly from the ribosides. Grunberg-Manago et al. (396) have recently described an enzyme which brings about the condensation of purine and pyrimidine nucleoside diphosphates:

nX:R.PP (X.R.P.)n + nP

X = purine or pyrimidine, R = ribosyl and P = phosphate This enzyme, polynucleotide phosphorylase, has been isolated

from a number of sources including fungi. Ochoa et al. (397) have shown that the RNA synthesized by this enzyme in systems

isolated from Azotobacter vinelandii is identical in all

respects with the natural RNA. It is noteworthy that in rat

liver, for example, the incorporation of labeled phosphorus

into the di- and tri-phosphates corresponding to all four

ribose nucleotides occurs at the same rate and that these - 8?a-

nucleotides are in equilibrium with the ATP-ADP system (39$).

The presence of all these compounds in fungi, coupled with the knowledge of the above phosphate exchange reactions, suggests that the enzyme system described by Grunberg-

Manago et al. (396) and Ochoa and co-workers (397) may be responsible for elaborating fungal ribonucleic acid. It appears likely, on the other hand, that the synthesis of

DNA occurs from the appropriate nucleoside triphosphates, as

Bessman et al. (399) have isolated an enzyme from S. coli which brings about the condensation of these compounds derived from adenine (A), guanine (G), thymidine (T), and cytidine (C), according to the following equation: n(Tp32pp + Qp32pp + Cp32pp + Ap32pp) + DNA y ++,

DNA - (TP32 + GP32 + CP32 + AP32) n + 4nPP

In this system, which has been enriched one thousand-fold,

DNA is required as a primer and RNA is inactive, a situation quite unlike that in the above reaction with polynucleotide phosphorylase in which preformed RNA is not needed (400). In the above reaction TDP cannot replace TPP.

A further role of purines (401) lies in the synthesis of riboflavin. It was found that the pyrimidine ring of adenine is incorporated intact into the isoalloxazine moiety of the riboflavin of Eremothecium ashbyii, a finding in agreement with the earlier observation that biochemical pre­ cursors of the purines, e.g., carbon dioxide, formate, and glycine, are also precursors of riboflavin and that purines increase the yield of riboflavin. - 66 -

PROTEINS AND PEPTIDES The fungi are known to decompose proteins (402, 403) in soil and other materials. Most of the work which has been done centres attention on the enzymic breakdown of protein, rather than the use of proteins for growth studies. It is known however, that in pure culture, proteins such as gelatin, casein, and egg albumin can serve as a source of nitrogen for some fungi, but it is doubtful whether fungi can utilize highly purified proteins in the complete absence of any other nitrogen source.

Simple linear peptides, such as di-and tri-peptides, support the growth of Aspergillus spp. (404) and Ustiliago spp. (405, 406), this is undoubtably due to the ubiquitous nature of enzyme systems which hydrolyse peptides to amino acids.

Waksman and Starkey (407) in their study of the utilization of proteins by fungi and found that Streptomyces violaceus-ru.ber and Rhizopus sp. utilize casein, zein, gliadin, fibrin, gelatin and egg albumin. It has also been shown that proteins are used more rapidly if there is no other carbon source. Evidence has accumulated to show that generally fungi hydrolyse fibrin (406, 409) and casein (409, 410, 411, 412,

413). Preparations hydrolysing keratin have been obtained from

Streptomyces sp. (414) and it is assumed that keratin breakdown by the dermatophytic fungi is carried out by an - 69 -

extracellular enzyme (415, 416). So far there has been a failure to identify the fungal endopeptidases with the known extracellular enzymes of animal origin. It is known, that the endopeptidases studied in crude and purified form have pH optima which are on the alkaline side of neutral (406,

417, 416, 419) but the fractionation of A. oryzae surface culture filtrates (274) yields proteases having pH optima of 4.5. That fungal endopeptidases are of the trypsin type is evidenced from substrate and inhibition studies on

Streptomyces sp. (420), Trichophyton gypseum (406), and other fungi (419). Pepsin substrates are attacked by

Streptomyces proteolyticus (421) but at pH values far removed from pepsin optima. As with endopeptidases from bacterial sources, the crude and highly purified preparations from the fungi consist of mixtures of different enzymes (419, 423, 424, 425).

Because of its commercial importance to the baking industry, the endopeptidases of A. oryzae have been extensively studied and three different enzymes acting on proteins have been isolated (274, 422, 426). The major component of this mixture of three has been crystallised (427) and found to act on most proteins, including those which lack aromatic amino acids.

Evidence strongly suggestive of the presence of a lysozyme-like enzyme in culture filtrates of different species of Streptomyces has been recorded. This enzyme has been - 90 - identified as a mucopolysaccharase (273, 426)crasa result of the combined action of ribonuclease and a protease (429). The formation of exocellular protein-hydrolysing enzymes by some fungi and actinomycetes proceeds in synthetic media with an inorganic nitrogen source (40S, 430, 431). For other species a more complex medium may be required. There is no reliable evidence of enzyme induction by substrate, although use of protein medium may increase yields (432).

Culture conditions for endopeptidase formation by A. oryzae show optima for carbon source, salts, iron and temperature (431)* Culture filtrates or mycelial extracts of fungi commonly, perhaps universally, hydrolyse peptides, although the specific enzymes carrying out these hydrolyses are unidentified; A. parasiticus forms four exopeptidases including an amino peptidase which is activated by zinc and reducing agents (433)

These same activators affect the aminopeptidase of Penicillium spp. (434). Many of the fungi (435) form dipeptidase, aminopeptidase, a carboxypeptidase, and an aminotripeptidase, assayable with triglycine (436). It would appear that fungi produce a battery of enzymes attacking peptides of low molecular weight, but no detailed classification exists, though the availability of pure test substrates makes the development of such a classification a possible enterprise. - 91 -

Peptides of Fungi and Actinomycetes

Because of the importance of the peptide antibiotics from fungal sources, considerable interest has been centred on the biological screening, isolation, and constitution of such materials.

Glutathione (an ubiquitous peptide) although possessing no known antibiotic properties, is most important because of its role as a reducing prosthetic group in triose phosphate dehydrogenase, and its possible role in protein synthesis

(437)* This peptide has been detected in Neurospora sitophilia (437) but because of the widespread occurrence of triose phosphate dehydrogenase, it is probably common in fungi generally. One species of the genus Glomere11a (438) requires glutathione for growth because it is unable to synthesise the glutathione component dipeptide glutamyl- cysteine . The peptide antibiotics produced by Streptomyces spp. (439) and I-Iicromonospora sp. (440) were first isolated by

Salesman and Woodruff (441). This group of antibiotics is known as the actinomycine; they are all heteromeric peptides containing the same chromophore.

The differences in the peptide chain distinguish the individual actinomycins, many of which have been described

(439, 442). A difficult problem in classifying antibiotic - 92 - products is the production of different types of antibiotic during different stages of life cycle.

Viomycin, streptothricin and streptolin (437) are basic peptides which have been isolated from various actinomycetes such as Streptomyces lavindulae. Levomycin has been isolated in pure form from an unknown streptomycete but the structural chemistry of this antibiotic has not been determined although its hydrolysis products have been identified. It is tentatively described as an antibiotic belonging to the class containing an aromatic group linked to a peptide moiety. Two peptide antibiotics, causing wilt in plants, have been isolated from fungi. Lycomarasmine is produced by Fusarium lycopersici and is the pathogenic agent in the infectious wilting of tomatoes. Enniatin, also a wilting toxin (443) is a dipeptide isolated from Fusarium lateritium (444, 445, 446). Lycomarasmine is the more important of the two and has been reviewed by G&umann (444). It is a heteromeric peptide with the structure N-( OL ( oC-hydroxy-propionic acid))- glycyl-asparagine. The complex which it forms with iron is responsible for the toxic effect on certain plants. The amanita peptide toxins of mushrooms have been identified by Block et al. (447). The toxins are found in the closely related Amanita phalloides, A. verna, and A. verosa and are responsible for at least ninety percent of the deaths caused by mushroom poisoning. Three closely related peptides have been isolated from the crude toxin amanitine and have - 93 -

been called

The European form of A. phalloides contains all three compounds, amounting in total to about 0*005% of its fresh weight (448). Other Amanita spp. have only one or two peptides, but most have more.

On hydrolysis, (X-amanitine (C 2^14^10^ ^ yields aspartic acid, glycine, hydroxyproline, cysteine, and an unidentified amino acid. It is believed that c(-amanitine is the amide of ft -amanitine. A thirty gram specimen of

A. phalloides was found to contain about one to two mgs. of the amanitines and zero to five mgs. of phalloidine. To kill a 25 gram mouse, 2.5, 8-10 and 40-50 micrograms of -amanitine,

fl -amanitine and phalloidine, respectively were required.

Phalloidine may be of less importance in mushroom poisoning than the amanitines, not only because of its lower toxicity, but also because of its lesser stability to heat.

The enzymatic basis of peptide synthesis in fungi is at present unknown. Extracts of Neurospora crassa mycelium show a ^-glutamyl transferase activity, and conceivably this system could function in the synthesis of glutamic acid peptides (450). In Bacillus subtilis glutamyl di - and tri-peptides are synthesised by transfer reactions of this general type (451). - 94

AMINES

Amines, in fungi, can occur as metabolic products by the decarboxylation of a(-amino-dicarboxylic acids. Only one specific enzyme, glutamic decarboxylase, yielding

y'-aminobutyric acid is known to occur in fungi (452, 453) •

However a large number of amines have been isolated from fungi, presumably originating by decarboxylation of known amino acids. Histamine, tyramine, isoamylamine, agmatine, putreseine and cadaverine (454, 455, 456) have been isolated from sclerotia of Claviceps purpurea. From

the fruiting bodies of basidiomycetes, putrescine, phenyl- ethylamine, isoamylamine and cadaverine have been reported

(457, 456, 459, 460).

It has been found that amines are generally poor, or unavailable, sources of nitrogen for fungi (461), although

several fungi act on histamine (462), and a mutant of

Aspergillus nidulans requires putrescine for growth. This

requirement is not met by L-ornithine or by other amines (463)

The toxin of Amanita muscaria is a substituted amine and

has been extensively studied by Kdgl et al. (464). The

structure has been established and confirmed by synthesis as

S thy1-5-dirne thy1aminoe thy1-2-methy1-furan-3-carboxylate.

Choline is ubiquitous in fungi (455, 460), but doubt

still exists as to whether it is present in the free state or v in phospholipids. Cyclic choline sulphate has been isolated

from fungi (251, 465, 466). - 95 -

Barley which has been scabbed by Fusarium gr amine arum and other Fusarium spp. contains a water soluble material identified as choline by Christensen et al. (467). This finding has been refuted and shown to be in error by

Schroeter (466). Mutants of Neurospora crassa (469) require choline and a mutant can be used for the bioassay of choline

(470, 471). Horowitz has shown that N. crassa synthesises choline by stepwise methylation of aminoethanol.

Aminoethanol —Monomethylaminoethanol —■»-

Dimethylaminoethanol Choline Trimethy 1 amine , presumably arising fror: choline , (472), has been isolated from numerous basidiomycetes e*g. Tilletia levis, Phallus impudicus, Russula aurata (459, 460, 473), and

Claviceps purpurea (455). Ergothionine, which has the structure of the trimethyl betaine of thiolhistidine, has been isolated in sclerotia and in cultivated mycelium of C. purpurea (455, 474) and in the mycelium of several common fungi (475). Ergothionine is synthesised on the same pathway as histidine (476), but histamine is not a precursor of either of these components

(477). Although betaine (476) has been isolated from fungi it is possible that it does not occur in the free state, but is isolated as a degradation product of ergothionine.

The fruiting bodies of Amanita mappa contain bufotenine

(5-hydroxy-N-dimethyl tryptamine) (479) which suggests a biosynthetic relationship with 5-hydroxytryptophan, an amino - 96 - acid which up to this time has not been observed in fungi, but is known in other organisms (479, 480). - 97 -

ASPECTS OF NITROGEN METABOLISM

IN

POLYPORUS TUMULOSUS

EXPERIMENTAL SECTION

Materials and Methods

Resuits

Discussion - 9S -

MATERIALS AND METHODS

PART A.

Section (1)

The Test Organism The organism used throughout these investigations was the basidiomycete fungus, Polyporus tumulosus Cooke. The original Stock - Culture of this organism (accession number

119) was obtained from decaying heartwood of the Zest

Australian Jarrah Eucalyptus maginata.

Soon after these present studies had commenced, however,

Stock - Culture 119 underwent a gross physiological change, resulting in markedly decreased yields of metabolites. The cause and precise nature of the change are not understood. Consequently a new stock culture (accession number 553) was obtained and all observations reported in this thesis have been made with 553 as the experimental material. The pattern of gross physiological change accompanying growth have been faithfully followed by 553 , but with only minor variation in calendar age due to somewhat modified culture conditions.

As far as can be ascertained, the two cultures 119 and 553 each represent authentic specimens of P. tumulosus.

Throughout these investigations the Stock - Culture was maintained on 2% malt agar slopes with six-monthly sub­

culturing.

The Stock - Culture 553 was a gift from the laboratories of the C.S.I.R.C., Division of Forest Products, Melbourne, 99 - through the courtesy of Mr. N. Walters. Section (2)

The Culture Medium - WD5 (modified William Saunders,

5% dextrose The fungus was cultivated routinely on a chemically defined liquid medium of the following composition, affording a carbon to nitrogen ratio of 31:1.

Glucose 50 gm./litre

(nh4)2so4 3.0 gm./litre

CaCl2.6H20 0.5 gm./litre MgS0^.7H20 0.25 gm./litre Thiamine HC1 2.0 mgs./litre

h3bo3 1.0 mgs./litre MnCl2.4H20 1.0 mgs./litre

T1N03 1.0 mgs./litre ZnS0^.7H20 1.0 mgs./litre

FeClv6H20 0.5 mgs./litre

CuS0^.5H20 0.1 mgs./litre KI 0.1 mgs./litre

made to 1 litre with distilled water. Sterilization of the medium was achieved by steaming for one hour in the presence of 0.3 mis. of chloroform per litre. The liquor after sterilization had a pH of approximately 4.5. In particular experiments the composition of the medium was altered as required. The initial investigations with this organism involved growth on WD5 100 medium with the addition of a single amino acid complementing the nitrogen level of the culture liquor, i.e.

(1) Glycine 2.0 gm./L designated WD5-Glycine and

(2) Alanine 2.0 gm./L designated WD5-Alanine.

Lection (3)

Cultivation and Harvesting

Mycelial slips of F. tumulosus (approximately 1 cm . - obtained from agar slopes) were inoculated into 100 ml.

conical flasks, each containing 50 mis. of culture liquor.

Care was taken during inoculation to ensure that the slip

inoculum did not sink, as this always caused a noticeable

delay in the initiation of growth. Any such cultures were

rejected. The cultures were set aside to grow for an

appropriate growth period (usually 7 weeks) in a room held at 25°C. It was found in preliminary studies, that there was a

great deal of heterogeneity between samples treated in the same way. Accordingly, a sufficient number of flasks (usually

six) were prepared to allow an adequate number of replications

of each sample to be made. In certain instances, however,

when rare or expensive chemicals were involved, only three

replications were usually made.

The harvest time was dependent on the nature of the

particular investigation to be undertaken. In most cases

it was possible to carry out periodic harvests, throughout all phases of growth and autolysis. When this was not 101

practicable, the time taken for the mycelium to completely cover the surface of the liquor was noted, as this represented the attainment of a comparable extent of growth, and the cultures were harvested when it was adjudged that growth had concluded, i.e. when autolysis had just commenced. The onset of autolysis was marked by the appearance of brown liquid droplets on the surface of the mycelium and a tendency for the mat to break away from the walls of the culture flask.

In a few instances cultures were harvested after a pre­ determined time interval (Table 16).

At harvesting the mycelial pelts were separated from the liquor by filtration on a Buchner funnel, and pressed ,Tdry,T between layers of filter paper. The liquors, and mycelia, respectively, of all replicate samples were pooled and subjected to various analytical procedures.

Section (4)

The Mycelium

The mycelium, after being pressed T,dryn, was weighed

(recorded as wet weight), and a sample (about 5«0 grams) dried

to constant weight in a vacuum desiccator over (recorded

as dry weight). The dry weight of mycelium was taken as a measure of the extent of growth of the organism and was used

to determine the economic coefficient, E.C.(481).

m r _ Mycelium (gnus, dry weight) ,, 100 "J*°' “ Carbohydrate consumed A 1

In some instances the lipid content was determined by 102

exhaustively extracting dry, ground mycelium in a Soxhlet apparatus successively with diethyl ether, chloroform, and acetone, the extracts then being weighed.

Section (5)

The Culture Liquor

The volume of the combined filtered liquors was measured and the pH determined. The volume was then readjusted to the original volume with distilled water (corresponding to 50 ml. for each flask harvested). The level of residual carbohydrate, total nitrogen, and free ammonia, were determined. In order to chromatographically investigate the composition of the culture liquor it was necessary to concentrate it twenty-fold under reduced pressure.

Section (6)

The Cell Wall Fraction

The cell wall fraction of P. tumulosus was prepared by

Leis and Ralph (4S2). These workers used a "grinding device" especially developed for the isolation of specific fractions of filamentous fungi. The aim of their degradative procedure, in this instance, was the liberation of the cytoplasm, the nucleus, endosplasmic reticulum, cell wall membrane and ultimate isolation of the free cell wall of hyphal cells,

^rotein-containing samples of the cell wall material of

P. tumulosus were kindly presented to the author for amino acid analysis. - 103 -

Section (7)

The Mycelial Extract

From one to two grams of dry mycelium (accurately weighed) were ground with 10 ml. portions of 7Oyo V/V boiling ethanol in a mortar, total grinding time about 10 minutes.

The suspension was centrifuged at high speed and the clear supernatant liquid collected. The residue was re-extracted

(as above) three more times and the total volume of super­ natant liquid concentrated to dryness under vacuum at 45°C.

This residue was taken up in 10$ V/V isopropanol solution and made to a volume of 10 ml.

All mycelium harvested during sequential studies of

P. tumulosus were treated in the same manner and the respective extracts used for analyses.

Section (6)

Acid Hydrolysis of Mycelium and Cell Wall Fraction

The residue left after extraction with 70$ ethanol was dried to constant weight. The dry material was ground to a fine, pale grey-coloured powder, which was Soxhlet extracted.

0.6 gm. of ground, dry lipid-free mycelium or cell wall fraction was hydrolysed with 20 ml. 6N A.R. hydrochloric acid under an open reflux condenser for 24 hours (temperature of reflux 112°C). The hydrolysate was freeze-dried and stored in a vacuum desiccator over K0H. The residue was taken up in 10$ V/V isopropanol and the resulting solution made to a volume of 5 ml* This solution was used for the amino - 104 - acid analyses of the mycelium and cell wall proteins,

Section (9) Alkaline Hydrolysis of Mycelium and Cell Wall Fraction

0,25 gm. of ground, dried, lipid-free material was hydrolysed with 1,4 gms. of barium hydroxide in 10 ml. of distilled water. The hydrolysis was conducted by auto­ claving the reactants, in a sealed tube, at 15 p.s.i.g. pressure for 24 hours. Barium ion was removed from the hydrolysate as barium sulphate and the filtrate concentrated by freeze-drying. The residue was dissolved in water

(adjusted to pH 8.0) to give a final volume of 10 ml. Aliquots of this solution were used for the determination of tryptophan.

Section (10) Analytical Methods other than Chromatography

(a) pH was determined with a glass electrode pH meter (Radiometer Type PHM 22p). (b) U.V. spectra were read on a Unicam quartz spectrophotometer type SP.500. (c) Reducing sugar, as glucose, was estimated by the Somogyi and Nelson colorimetric procedure, after the method of King (4^3)• (d) Total nitrogen was determined by the semi-micro

Kjeldahl method (484). (e) Ammonia was determined by distillation using the

Markham Still (485).

(f) Urea was determined using the microdiffusion - 105 - apparatus and method of Conway (436). (g) Amide Nitrogen - by the method of Borsook and

Dubnoff as modified by Conway (487).

(h) Citrulline was estimated using the diacetyl monoxime method of Knivett (488). 1.0 ml. aliquots of mycelial extracts were sampled for analyses.

(i) Ornithine was estimated in mycelial extracts by the method of Chinard (489). 1*0 ml. aliquots of mycelial extract fraction were sampled.

(j) Hexosamines, as glucosamine, were estimated by a modified Slson and Morgan method (490). Section (11)

Paper Chromatography (a) The Qualitative Identification of Amino Acids Three two-dimensional solvent systems were used to resolve and identify the amino acids present in the various fractions described in Sections 5, 7, 8 and 9* These are, respectively, the solvent systems of (1) Hardy et al. (491), (2) Levy and Chung (492) and (3) Stepka (493)•

As it was found essential to have complete familiarity with the regions of the chromatogram holding the resolved material, maps and photographs of the two dimensional chrom­ atograms were prepared,(see Figs^ No. 2, 3, 4, 5, 6)and used as a reference guide for identification. It was originally intended to tabulate Rp values but experience showed this to be pointless and in complete FIGURE No. 2. Map of an amino acid chromatogram

developed with solvents N/Q of

Hardy et al. (491).

In this and subsequent Figures refer to "Index to Amino Acids", page 110. SECONDARY SOLVENT , , G-THIONE FRONT OF Q

ARG UNKNOWN ACID FIGURE No. 3. Map of an amino acid chromatogram

developed with solvents of

Levy & Chung (492). is?

Vo 0 5 O 0 I 0 o ACH

ACETIC

-

dUTANOL

8 CD 108 FIGURE No. 4-.

Map of an amino acid chromatogram

using the solvents of

Stepka (493)- STEPKA butanol- acetic ACD- WATER PRO

«X-AL

ORN

STEPKA PHENOL - WATER 109 FIGURE No. 5>.

Photograph showing separation of amino acids

with solvents N/Q of Hardy et al• (491).

FIGURE Ho. 6.

Photograph showing separation of amino acids

with solvents of Etepka (493).

110

INDEX TP AMINO ACIDS

0C-AL (X-ALANINE ISO ISOLEUCINE

-AL 0-ALANINE LEU LEUCINE

ARG ARGININE LYS LYSINE ARG SUCC-A ARGININOSUCCINIC ACID PIET METHIONINE

a-nh2 | NOR NORLEUCINE ASPARAGINE ASP-NH2 J N-VAL NORVALINE

AA ASPARTIC ACID ORN ORNITHINE

CIT CITRULLINE ^ -PHE fl -PHENYLALANINE CYSTEINE PHE CIS CYSTINE PR PROLINS GLUC GLUCOSAMINE PRO

GA GLUTAMIC ACID SER SERINE g-nh2 GLU TAMINE THR THREONINE

G-THIONE GLUTATHIONE TRY TRYPTOPHAN

GLI GLYCINE TYR TYROSINE

HIS HISTIDINE VAL VALINE

OH-PR ) HYDROXYPROLINE OH-PRO f Ill

agreement with Proom and Woiwood (494) who ’’have not found the Rp value of any great use in identification, and although it is often quoted with an astonishing degree of precision, it is at best only a rough guide and should never be used as the sole means of identifying an unknown substance”.

Whatman No. 1 paper, (chromatographically washed with a 0.8 gm./L Versene solution for phenol solvents) was used exclusively for both qualitative and quantitative chrom­ atography, both ascending and descending techniques being employed. Equilibration was dispensed with, and all chromatograms were run in a temperature controlled room at

70°F.

With the technique of Hardy et al. (491) their solvent combination, designated N/Q, was used; this is the reverse of their suggested Q/N, but this extension of their technique achieved sharper resolution and provided the means of detecting the ’’unknown” amino acid isolated from mycelial extracts of this organism. Details of the procedure adopted are as follows.

Paper. Whatman No. 1., 9” x 9" squares, development by the ascending technique.

Loading. 5 micrograms of each amino acid. kobile solvents . N. n-Propanol :Butanone ^2*3 : Dicyclohexyl amine,

10:10:5:2.

Q. Ethanol :n-Butanol J^O .-Piperidine ,

10:10:5:2. 112

Visualisation. The dry chromatogram was sprayed with a 5% V/V solution of dicyclohexylamine in absolute ethanol, air dried and dipped into a 0.25% solution of ninhydrin in

acidified acetone (7% V/V with acetic acid). The chromatogram was then heated in an air oven at 75°C to reveal the spots.

An examination of the map (Fig. 2) and photograph (Fig.

5) shows that many of the amino acids are closely associated,

but the colours imparted by dicyclohexylamine (Table No. 9)

served as markers and greatly facilitated identification. TABLE No. 9.

Colour* with

Amino Acid Dicyclohtexylamine Aspartic Acid Turquoise Cystine C armine

Alanine Blue-purple Histidine Grey

Serine Grey-purple Phenylalanine Grey-brown Proline Yellow

Hydroxyproline Yellow Threonine Grey

Tyrosine Grey-brown Glycine Wine red Other amino acids Purple

A All colour stable for 1-2 hours. - 113 -

The solvent system N/Q failed to satisfactorily resolve

the following groups of amino acids,

(1) Glutamine, Glutamic acid, Citrulline.

(2) Asparagine, Aspartic Acid.

(3) Lysine, Ornithine.

(4) Leucine, Isoleucine, Norleucine.

(5) Cystine, Cysteine.

(6) Methionine, Valine.

Any ninhydrin-positive spot within these doubtful regions

of the chromatogram had to be resolved with a second or

third two dimensional system. Glucosamine is ,Tlost,? in

solvent Q.

With the method of Levy and Chung, lysine and ornithine

were not separated, otherwise these solvents afforded

reasonable separation of most amino acids. Cystine did not

show particularly well and cysteine not at all. Arginine

tended to streak and argininosuccinic acid appeared in a

crowded region of the chromatogram making identification

difficult. Glucosamine also occurred in a crowded region

rendering the identification uncertain by ninhydrin alone.

It was found that in general the combination of a

phenolic solvent system on paper scoured of cations with

Versene showed high sensitivity.

Details of this procedure are as follows

Paper. Whatman No. 1, lStf x 18", Versene-washed and pinked

along two edges at right angles; development was by the - 114 -

descending technique.

Loading. 200 micromoles of each standard amino acid were dissolved in 10 ml. of 10$ isopropanol, pH 2.0 - 2.5. The standard amino acid solution was thus 0.02 M, or Ijdl £ 0.02ztM of the amino acid. Loadings of 1 - 4*1 were applied to chromatograms.

Mobile Solvents. Two solvents were used.

(1) n-Butanol-Acetic Acid-Water. Levy and Chung used a solvent mixture of 4:1:5 but better resolution was obtained with these solvents in the proportions 4:1:1 (495).

(2) Phenol-Cresol-Buffer. This solvent consisted of

15 gm. Phenol A.R., 30 gm. m-Cresol, Chromatography Grade, and 7.5 ml. Borate Buffer pH 8.3. The buffer is made by mixing 300 ml. 0.1 M sodium borate and 60 ml. 0.1 N sodium hydroxide.

Visualisation. The chromatogram was sprayed with a mixture of 50 ml. 0.1$ ninhydrin in ethanol, 2 ml. collidine and

15 ml. glacial acetic acid and heated in an air oven at 75°C for 5 minutes. The colours imparted to the amino acids by the Collidine Reagent are shown in Table No. 10.

The technique of Stepka was used to resolve the amino acids, lysine and ornithine. In addition this method afforded a markedly different chromatographic pattern, useful in confirming the results of previous techniques.

The details of this system of chromatography are as follows: - 115

TABLE No. 10. Colour^ with

Amino Acid Collidine reagent

Histidine ^ Turquoise

Tyrosine r Blue Phenylalanine J Cystine Brown

Asparagine Brownish-orange

Tryptophan Yellow-brown

Threonine ] Gre;y Methionine ) Aspartic Acid Bri.ght Blue Glycine Red dish-purple

Serine Muddy Yellow Remaining Acids Blue-purple

Isoleucine is more reddish than leucine immediately after chromatograms are visualised.

Paper. Whatman No. 1, 18" x l$Tt, Versene-washed and pinked

along two edges at right angles. Descending solvent flow

for 24 hours with each solvent.

Loading. 3-5 Ag. of each amino acid. Mobile solvents. (1) n-Butanol .‘Acetic Acid:Water, 100:22:50

(parts by volume). (2) Phenol:Water, 100:39 (W/V).

Visualisation. The reagent described for Levy and Chung.

(b) The Sulphur-Containing Amino Acids

Cysteine, cystine, methionine, homocysteine and - 116 -

homocystine were resolved by the technique of Strack et al.

(496) and visualised by the sodium nitroprusside reagent of

Toennies (497). Loadings of 5 - 20 ja± of fractions

(Lections 5, 7, 9) were applied to chromatograms.

(c) Urea, Ureides and Guanidine Bases

Separation of the above compounds was achieved using the phenol reagent of Williams (49&v). The Levy and Chung

(492) technique applied equally well to urea and ureides.

The p-dimethylaminobenzaldehyde reagent of Marini-Bettolo and Frabacchi (499) was used to visualise urea and ureides and the nitroprusside spray reagent of Kirby-Berry et al.

(500) for guanidine bases.

(d ) Amino Sugars

The amino sugars were identified by chromatography using:

(1) The solvents of Levy and Chung (492), and,

(2) An ascending unidimensional technique using the solvent mixture n-Butanol .‘Acetone :Water in the ratio 4:1:1.

Chromatograms were visualised by any of three following reagents:

(1) The ammoniacal silver nitrate reagent of Partridge

(501) , or,

(2) The acetylacetone-dimethylaminobenzaldehyde reagen- of Partridge (502), or,

(3) The ninhydrin reagent (section 11). - 117 -

5-10 in 1 loadings of fractions provided sufficient material for resolution by the above solvents.

(e) Visualising Agents for the Guanidino Group of the

,TUnknown Amino Acid”.

(1) Sakaguchi reagent.

(2) Pentacyanoaquoferriate reagent.

(3) Diacetyl reagent.

All three of the above are described by Smith (503) and were used on the amino acid chroma to grams described in

Section 11 (a). Visualisation was achieved by "smoking” the reagent onto the chromatogram with a Ue Vilbiss glass nebulizer No. 40. This precaution was necessary to prevent diffusion of the spot.

Section (12)

The Quantitative Analysis of Amino Acids

The method of amino acid analysis was developed for these investigations. The principle of the method depends on the quantitative separation of amino acids on buffered filter paper and their direct estimation on paper by spectrophotometry. The solvents used to achieve the separations are modifications of those described by McFarran and Mills (504). Table No. 11 shows the five solvent systems used and the amino acids which may be determined with each system. These solvents were used in conjunction with the buffer solutions at pH 6.2, 8.4, 9*0 and 12.0 recommended by

McFarran (505). The systems were prepared by shaking the TABLE No. 11.

SOLVENT SYSTEMS AND CONDITIONS FOR THE QUANTITATIVE DETERMINATION OF AMINO-ACIDS solvent Composition Millimolarity of Amino .Running Time Visualising Agent Amino Acid Determined of the Solvent Acid Standard Solution In Hours

A 95 ml. phenol distilled 1.0, 2.0, 3.0, 4.0, 40 Ninhydrin Reagent Aspartic Acid 5 ml. 2-butanol 5.0, 6.0 No. 1. Glutamic Acid

100 ml. buffer (pH 12.0) Serine Glycine

, Threonine Alanine

B 95 ml. m-cresol distilled 1.0, 2.0, 3.0, 4.0, 60 Ninhydrin Reagent Tyrosine

5 ml. 2-butanol 5.0, 6.0 No. 2. Histidine

100 ml. buffer (pH 6.4) Valine Methionine

C 99ml. 2,4/2,5-lutidine 1.0, 2.0, 3.0, 4.0, 40 Ninhydrin Reagent Arginine distilled 5.0, 6.0 No. 2. Lysine

1 ml. phenol distilled Proline developed Proline

100 ml. buffer (pH 6.2) with Isatin Reagent Valine Phenylalanine

Tyrosine

D 95 ml. 2,4,6-collidine 7.0, 8.0, 9.0, 10.0, 60 Ninhydrin Reagent Tryptophan 5 ml. phenol distilled 11.0 No. 1.

100 ml. buffer (pH 9.0) • E 50 ml. 2-methyl-2-butanol 1.0, 2.0, 3.0, 4.0, 40 Ninhydrin Reagent Leucine 50 ml. methyl ethyl ketone . 5.0, 6.0 No. 1. Isoleucine

0.5 ml. 90% formic acid 10 ml, water - 119 -

organic solvents in a separatory funnel with the appropriate buffer or with water, setting the mixture aside overnight, and then separating off the aqueous layer. (There was no separation of layers in system E).

Buffered Filter Papers.

21” x 24” sheets of Whatman No. 1. filter paper were dipped into a buffer solution of the same pH as the solvent in which they were to be used. They were then dried and stored in a clean, dry place. standard Amino Acid Solutions.

Stock solutions of each amino acid (with the exception of tryptophan) were prepared, at a concentration 8mM with respect to each amino acid in 10% V/V isopropanol solution.

Measured aliquots of the stock solution were diluted with

10% isopropanol to yield 1.0, 2.0, 3.0, 4.0, 3.0 and 6.0 mil. standard solutions. Tryptophan standard solutions were prepared at 7.0, 8.0, 9.0, 10.0 and 11.0 mM.

Visualising Agents.

Ninhydrin Reagent No. 1. 1000 ml. of n-butanol and

500 ml. of water were shaken in a separatory funnel and the two layers separated. 100 ml. of n-butanol, 5 gm. of ninhydrin, and 50 ml. of glacial acetic acid were then dissolved in the water-saturated butanol.

Ninhydrin Reagent No. 2. The same procedure as for

Ho. 1 was followed, except that 25 ml. of glacial acetic acid were used. 120

Isatin Reagent. 4 gm. of isatin were dissolved in

1000 ml. of 95fo ethanol containing 40 ml. of glacial acetic acid

Procedure

The buffered papers were marked by drawing a line approximately 4" from the 21" side of the paper marking 1" spacing points on this line. To these were applied 5 fb 1 aliquots of the standard amino acids. All aliquots of

standards were applied in duplicate, i.e., there were two

separate spots for each standard. Depending upon the concentration of amino acid, 2-5 ytt'l aliquots of fractions to be analysed were applied in triplicate spots. Two separate chromatograms, similarly loaded, were run simultaneously.

The drip end of the chromatogram was pinked to facilitate even flow of solvent through the paper.

The chromatograms were developed in the appropriate

solvent by the descending technique for the specified time

(see Table 11). Developed chromatograms were air-dried in darkness and prepared for spectrophotometry. This preparation

involved visualising, the first step of which was carried out

by mounting the chromatograms on a frame, stretching them

taut and spraying until just wet (as judged by change in

opacity) with the designated visualising agent. Sprayed

chromatograms were heated in an air oven at 60°C for 15 minutes, except in the case of chromatograms sprayed with

isatin reagent, which were heated at 100°C for 5 minutes. 121

Fig. No. 7 shows the selective separation of amino acids resolved in solvent A.

Final preparation for the measurement of O.D. involved the removal of a rectangular section (3.5 cm x 1.5 cm) of the chromatogram holding the visualised spot due to the amino acid being determined, and the positioning of this section on the cell carrier in such a way that the light beam and vertical axis through the region of highest O.D. were coincident. Where the colour yield of a given spot was too low for accurate visual location of the region of highest

O.D., location was achieved geometrically.

Typical standard curves obtained by using this method are shown in Fig. 8. Table No. 12 shows an amino acid analysis of hydrolysed casein using this technique.

Calculation

The concentration of amino acid in gm./lOO gm. mycelium is calculated using the following formula.

/, rr, C x M.W. x 10“3 x V gm. a.a./lOO gm. =______s Vy x M.

C = millirnolarity of amino acid being estimated as read from

the cone./O.D. standard curve.

M.W. = molecular weight of the amino acid being determined.

V = volume in microlitres of standard amino acid solutions s applied to the chromatogram (usually 5).

Vy= volume in microlitres of extracts or hydrolysates applied

to the chromatogram.

FIGURE No. 7. Photograph of a chromatogram showing

the separation of amino acids resolved in solvent A (Table No. 11)

123 - 124 -

M = mass of mycelium taken for extraction or hydrolysis (gm.)

The Estimation of Error and the Statistical Treatment of Results The magnitude of error involved in the amino acid analyses was determined by fitting the line of least squares to the results used to plot the standard curves (506).

If in a set of observations, y = f(x), the general equation describing a straight line relationship between x and y may be written

y = mx + b

m = slope of the line, and

b = the intecept on the y axis.

It is required to consider a bivariate distribution in which one set of results can be represented by (x^, y^) x^ being the controlled variable, viz. concentration. This is not absolutely controlled, it should be noted, as it is subject to small experimental fluctuations, regarded as remaining constant for the work being here statistically considered. Such fluctuations are acceptable if all that is required from the resulting least squares line is the expected O.D. of a specimen of known concentration. However it is wished to ascertain the concentrations corresponding to a known O.D. while keeping in mind that the experimental method leading up to the measurement of this O.D. introduces further errors.

Sets of results, such as those under consideration, - 125 -

TABLE No . 12. The Amino Acid Analysis of Casein The following analysis was performed on acid hydrolysed pure casein (Nutritional Biochemicals Corporation. Hammers1,en quality nitrogen (13 . 9-%) . Reported figures Steele et al~ Gordon et al~ a . a . a .a . N. a .a . ~ - Alanine 3, 72 0 , 58 3.31 2 ,69 Argi nine 4 , 27 1 , 37 3.72 3.65 Aspartic Acid 7 . 30 0 , 77 6 ,95 6 , 30 Glutami·c Acid 20. 30 1 , 93 20 .80 20 .00 Glycine 1 ,94 0 , 36 1 .97 2.40 Hi stidine 3 ,60 0 . 97 .3 .00 2. 76 I sol eucine 5, 55 0 . 59 5.91 5,40 Leucine 10.40 1 .11 9 . 39 8. 20 Lysine 7,05 1 . 35 7 .68 7, 30 llethionine 3, 18 0 . 30 2 .94 2 . 50 Phenylalanine 5 .05 0 .43 4 .82 4 . 55 Proline 9 .50 1 .16 10.40 10.10 Serine 4 , 55 0 ,61 5, 67 5,60 Threonine 4, 30 0 . 51 4 , 26 4 .36 Tryptophan 1 , 26 0 . 17 1 .24 1 .10 Tyrosine 5,80 0 ,45 5.62 5, 60 Valine 6, 70 -0 .80- 6 , 79 6 .40 13.46 a .a . = Grams of amino acid/100 grams of casein. (All results are expressed on a 14. 3~ nitrogen basis) . a . a .N. = Grams of amino acid nitrogen/100 grams of casein. REFEREKCES 1 . Steele , B. E. , Sauberlich, H. E . , Reynolds , r.; , S . and Baumann , C. A. , J . Biol . Chem . , (1949) , ill, 533

2 . Gordon, \'I. G. , Semmett , W. F . , Cable , R. :; . and !-'!orris , r.: ., J . Am . Chen- . Soc . , {1949) , 71 , 3293 . - 126 - often present considerable difficulty in handling, even to an experienced statistician, and in such cases the variability of the final concentration result is conveniently neglected (507). The method of least squares is to fit a line y = rnx + b to the results (x„. , y^) such that the sum of the squares of the deviations of each y^ from this line is least.

>. (y^ - (mxi + b)' is least. The least squares line for a given series may be obtained by the use of a set of ’’normal equations’’. These ’’normal equations” are derived mathematically (50S) and from them the expressions for m and b are described as follows;

m= 2xiyi- Zxi gyj ______n_____ - < Zxd2 n

-m 2xi n and the errors in y> m, and b are calculated by the following var = Sd2 (y) ^ “ ^calc“ ^obs^ (n - 2) var (m) = var y. 1______= var y.______1____ ri 2 £ Xi - nx 2 2 ^Xi ” var (b) = var y. ______o = var (y) ^ Xi_____ o n y xi Hx±^ * v (xi - - 127 -

Confidence limits for usual scientific observations are chosen so as to encompass at least 90% of the observations i.e. it is expected that no more than 10% of observations lie outside these limits if errors are due solely to chance fluctuations. i 90L/o confidence limits are + ((var (m, b, y))2

The following are typical details of calculations performed in the compilation of statistical data in Table No.

15* See table No. 14 for numerical data on arginine.

var (y) = l£43 x 10~° = 460.75 x 10“6 4 S (y) = 4.293 x 10~2 = 2.146 x 10~2 = 0.021 2 i S = standard deviation = (var)2

4y90^ = + 0.035

J y'' = 90% confidence limit

At mid scale 4y ^ 6°/o

TABLE No. 13.

X X - X (x - x)

1.0 1.25 1.5630

1.5 0.75 0.5625 2.0 0.25 0.0625 2.5 0.25 0.0625

3.0 0.75 0.5625

3.5 1.25 1.5630 2.25 4.3760 - 12a -

var (m) = 460.75 x 10-6 x 1 4.3760 105.4 x 10"6 s (m) = 10.27 x lO-3 0.0103 + 0.0169 8.09/o var (b) = 460.75 x 10” x 34.75 6 x 4.376 610 x 10"6 S (b) = 0.0247 4 b90^ = 23%. Consider the concentration corresponding to an O.D. of

0.500 (mid scale). From an error no greater than _+ 0.035 is expected. x = 2.25 i.e. £x n m = 7.9&0 - 7.076 = 0.904 = 0.207 34.75 - 30.39 k7R- b = 3.145 - 2.a00 = 0.345 = 0.0575 6 6 y = 0.207x + 0.05^ The max. error in (y - b) is + ( + 4b^)* = + (0.0352 + 0.02472)® = +_ 0. 043 Max. °/o error in (y - b) = + 0.043 x 100 = + 9.3$ 0.442 1 Max. fo error in m = + a.09% Max. % error in (y - b) = (9.S' + a.09^)^ = + 12.7%

It is seen that the estimated maximum possible error in arginine concentration at mid scale is 13%.

The least squares line is that line which would be TABLE No. 14

Least Squares Line for Arginine

x = concentration y = optical density

2 X Z 2£Z X y calc. Ax V2 X 10

1.0 0.260 0.260 1.00 0.265 0.005 25

1.5 0.400 0.600 2.25 0.368 0.032 1024

2.0 0.450 0.900 4.00 0.472 0.022 484

2.5 0.560 1.400 6.25 0.575 0.015 225

3.0 0.685 2.055 9.00 0.679 0.006 36

3.5 0.790 2.765 12.25 0.783 0.007 47

13.5 3.145 7.9SO 34.75 1S43 - 130 -

expected if the experimental technique were perfect. From the values for y so obtained, it is possible to calculate the standard error of the observed points from those predicted, and use 90% confidence limits of + 1,64 S.E.

For example, for arginine, the standard error in y is

0.021 and the 90yo confidence limits are + 0.035. Thus, if an O.D. of any sample of arginine is measured from the same chromatogram, there may be assigned to it an error of

_+ 0,035. The concentration corresponding to this O.D. is then determined from the least squares line (see Table 15).

Discussion of Method

The addition of 2-butanol (509) to the phenol solvent of McFarran and Mills (504) improved the separation of glutamic acid, aspartic acid, serine, glycine, threonine and alanine and led to the formation of compact round spots.

The addition of 2-butanol to the m-cresol solvent made possible the separation of tyrosine and histidine, which were not separable when only buffered cresol was employed.

The lutidine solvent recommended by McFarran (505) separated lysine, arginine, proline and valine but failed to separate phenylalanine from tyrosine. A solvent system made up of lutidine, buffer solution, and a small amount of phenol, gave a good separation of lysine, arginine, proline, valine, phenylalanine and tyrosine. Leucine and isoleucine formed a complex in the benzyl-alcohol-butanol solvent. However, it was found that a solvent system made up of 2-methyl-2-butanol, - 131 -

ethyl methyl ketone, formic acid, and water gave a satisfactory separation of these two amino acids, and the spots formed were round and compact. Tryptophan was not completely separated from tyrosine in buffered collidine, but the addition of phenol increased the R^ value of tryptophan, and decreased that of tyrosine, so that they could be completely separated. The amino acids, valine and tyrosine, may be determined equally well with solvent systems

B, or C.

Preliminary experiments showed that the use of five standards in the preparation of the standard curves greatly improved the reproducibility of the results. Only those values for optical density of the hydrolysate spots that fell within the concentration range of the standards were taken for calculation of the results.

PART B.

Metabolic studies on Polyporus tumulosus

The study of the metabolism in P. tumulosus presents a considerable problem in technique, a problem which is a direct consequence of the slow growth, and inherent metabolic inactivity, of the organism. For example, attention may be drawn to the fact that a culture of this organism requires some 35 days to consume 5 gm. of glucose and elaborate 4.5 gm. dry weight of mycelium (see Figs. 9, 11, 13). As a result, many of the conventional experimental approaches of biochemistry are difficult to apply. - 132 -

It has generally been found that cell free extracts show exceptional lability and this has made the task of concentration and purification of enzymes extremely difficult. In order to observe the time-consuming transformations of substrates, it has been necessary, for the most part, to maintain the integrity of the whole organism.

This study, therefore, is essentially a physiological one although it is possible to present a reasonable bio­ chemical interpretation of certain observed phenomena.

Physiological Aspects Factors pertaining to the methods of cultivating fungi, and to the multitudinous effects of varied environment are dealt with in detail in the texts of Cochrane (2 ), and Foster (510). Measurement of Growth

As with other organisms, the precise definition of growth in fungi depends on the method of measurement used. Increase in dry weight, increase in protein or some other standard material, increase in cell number, are all criteria which have been used by various authors to describe growth.

The ultimate choice of method is usually the one which allows the greatest experimental convenience for the individual investigator.

With the filamentous fungi, it is probably most common to employ dry weight of mycelium to describe the extent - 133 -

of growth. The purely vegetative growth which follows inoculation into liquid media is typically variable both in gross extent and in weight.

Crowden ( 1 ) reports the degree of heterogeneity of

F. tumulosus and this is clearly contrasted in Table No. 16 which shows the extent of growth over a 21 day period on

WD.5 medium.

TABLE No. 16

Variation in Extent of Growth of 10 Cultures

of P, tumulosus

Experimental Data of Crowden ( 1 )

Growth (mycelium dry weight)

0.157 gm. 0.182 gm. 0.195 gm. 0.207 gm. 0.245 gm. 0.269 gm. 0.274 gm. 0.282 gm. 0.296 gm. Mean Dry Weight = 0.238 gm. Standard Error of the Mean = 0.014 gm. Standard Deviation = 0.044 gm. 95$ fiducial limits = + 32 mgs.

Each of the ten inocula used were obtained from a single agar slope, extreme care being taken to ensure uniformity of size of the mycelial slips. In addition, the culture flasks were especially selected for uniformity of size and shape. - 134 -

Similar heterogeneity was reported by other workers in this department (511,512) for shaken cultures, where measured amounts of homogenised mycelium were used to inoculate culture flasks.

These data clearly emphasize the necessity of making as large a number of replications as possible, particularly if quantitative interpretation is required.

Growth of P. tumulosus on WD5

Within two to three days of inoculation mycelial hyphae radiated from the surface of the inoculum and extented over the surface of the liquor. There was progressive compaction of the young hyphae into a mat as the culture increased in area. The upper surface was usually well covered with a

"fluffy" white mass of aerial hyphae whilst the under-surface took on a smooth and "leathery" texture. There was little subsurface growth of the mycelium.

In the culture flasks used for these experiments the mat attained sufficient size to fully cover the surface of p the liquor (about 20 cm .) in approximately 17 days, and undoubtedly, the attainment of full surface cover represented a distinct physiological stage in the growth of the organism.

Carbohydrate was fully utilized and autolysis commenced in about 34 days. At about 40 days, droplets of a brown liquor appeared on the surface of the mycelium. These increased in number as autolysis proceeded. Eventually the whole mat became dark and fragile. - 135

At 20 days the liquor had a pale yellow colouration and Crowden ( 1 ) reports that the material responsible for the colour could be completely removed by continuous diethyl ether extraction over 6 hours. As growth progressed this colour intensified through orange-yellow to a final orange- brown shade. The liquor from the autolysing cultures showed little decrease in intensity of colour even after exhaustive ether extraction of up to AS hours.

During the period of active growth the pH of the liquor fell from 4.5 to 1.6, and remained low until considerable autolysis had taken place. Quantitative data describing the growth of the organism is classified in Table 17 and

Figs. 9, 10. It can readily be seen from the figures that during active growth, the fall in pH, the utilization of carbohydrate, and the production of mycelium, are closely correlated. The most efficient rate of utilization of carbohydrate occurred during the period immediately prior to the attainment of full surface cover. The pattern of utilization and distribution of nitrogen in the culture liquor is shown in Figs. No. 11,14 together with a presentation of changes in pH, wet weighed mass of mycelium and glucose assimilation. These data are presented for growth of the organism on WD5-Glycine, WD5-Alanine. In the case of media containing glycine or alanine there is a preferential utilization of inorganic nitrogen, a greater FIGURE No. 9. Graph Showing Sequential Change in Glucose

Utilization and Yield of Mycelium

of Polyporus tumulosus Cultured on WD5. P.TUM ULOSUS STILL-CULTURED ON WD 5.

2 4- DAYS OF CULTURE GLUCOSE O 5 0 13 3 63 20 2-2 27 0-9 34 0-13 GLUCOSE UTILISATION 42 O 4« O 56 O

20 30 4 DAYS OF CULTURE

DAYS OF MYCELIUM CULTURE WET WT. O O MYCELIUM PRODUCTION 13 07 20 3 4 27 7-1 34 a 9 41 7-6 49 7 5 56 4-5

DAYS OF CULTURE FIGURE No. 10. Polyporus tumulosus cultured or WD5.

Graph Showing Sequential Change in Free Ammonia Total Nitrogen, Organic Nitrogen, and pH of Culture Medium.

FIGURE No, 11. Polyporus tumulosus cultured on WD5-Glycine

(1) Glucose Utilization

(2) Mycelium pH (3) Culture Fluid pH GLUCOSE UTILISATION P. TUM. STILL CULTURED ON WPS GLYONE.

pH CHANGES WITH TIME Of P. TUM,

Doys o* mycelium Colt ore PH.

O 4 7-49

IS *44 21 29 4 74 MYCELIUM pH- >6 4-20 42 *•1 4t ISO

FIG. 2

Doys of Culture

Doys •» Culture CFpH

O 4-0 4 M4 It 2*24 CULTURE FLUID pH »l I4t »f 2I» 24 l«7 42 2HI 4t 2 40

FIG. 3 10 40 - 14C - 141 - FIGURE No. 13. Graph Showing Sequential Change in Glucose Utilization

and pH of Mycelium and Culture Fluid of Polyporus tumulosus Cultured on WD5-Alanine P TUMULOSUS STiLL-CULTURED ON WD5 ALANINE

DAYS OF CULTURE GLUCOSE O 50 8 5-0 14 420 19 279 25 145 12 077 GLUCOSE UTILISATION 19 095 44 057

20 30 DAYS OF CULTURE

DAYS OF MYCELIUM CULTURE dH O _ 8 14 580 19 5-65 MYCELIUM pH 2» 4-RO 32 380 39 >90 48 3 60

DAYS OF CULTURE

DAYS OF C F pH CULTURE O 4-0 8 4-27 14 >55 CULTURE FLUID pH 19 250 25 200 12 170 39 1-70 46 170

DAYS OF CULTURE

FIGURE No, 14. Polyporus tumulosus Cultured on WD5-Alanine. Graphs Showing Sequential Changes in Free Ammonia

Total Nitrogen, Organic Nitrogen and Mass of Mycelium. WD5 ALANINE DAYS OF FREE DAYS OF .TOTAL CULTURE. AMMONIA. CULTURE. NITROGEN FREE AMMONIA TOTAL NITROGEN O 3-0 IN O IN • 2-91 9 1-130 14 2 63 CULTURE FLUID. 14 1-013 CULTURE FLUID. 19 1-90 19 0-813 25 WO 25 0-628 12 096 32 0-560 39 062 39 0493 46 I-2B 46 0568

DAYS OF CULTURE DAYS OF CULTURE

DAYS OF ORGANIC DAYS OF WET WEIGHT. CULTURE NITROGE9 ORGANIC NITROGEN CULTURE. CHANGE IN MASS O IN O O OF 8 O 75 8 0381 CULTURE FLUID MYCELIUM(WET 14 0-336 14 71 19 0324 19 IX WEIGHED) H 0293 25 132 32 0-312 32 144 39 39 IX 46 08? 46 ii-o

30 4 DAYS OF CULTURE DAYS OF CULTURE - 143 FIGURE No. 15.

Graph Showing Growth of P. tumulosus in media

with Amino Acids as the Source of Nitrogen. :gss:::::s;s5s:ss:Ss 11

r

n 0 8 z 1 ■ l « I1 u si O ^

o 2? s < o - £ 2 2 C u 0 c -2 1 & 2 1

T rr.dZIXEir

* z ^ a * y ! * s I*i t.j *! 1111 i | ? >- UJ */) cc ac a > > < 2 fc m h D u »- u 5 I u ‘ I 13 144 FIGURE No. 16. Graph Showing Sequential Change in pH of Culture Medium of Polyporus tumulosus Cultured

on Different Ammonium Salts. \ 1 J[T? 5 ? £ g s ii « o j 9 « | c n <* w m _ 5a s 1 • D ° 8 ¥ 8 ? ? s U. > X a ♦ « * * O i i! ! ; 2 ? ? ? ; l * + 1 * «• J_ t s •« 2 5 5! & i £s222°?" i 512 ujE s * ? s : v - * * i 2 2 « s 2 z i i o g ? t 5 1 r s i SALT. n’uMr i i 0 1 h

U

s 1 i i l lnc«bati«ft

0*y* - 145 rate of assimilation of ammonia, and a greater yield of mycelium than if the organism is cultivated on WD5 alone.

Fig. No. 15 shows the comparative growth of P. tumulosus with the amino acid featured, as sole nitrogen source.

Maximum yield was achieved with aspartic acid (2.25 gm. wet weight) which compared poorly with the yield (8 gm. wet weight) of mycelium from WD5.

In Fig. No. 16 growth and accompanying pH changes of

P. tumulosus on different ammonium salts are shown. These data are in agreement with the findings of Morton and

MacMillan (14) that nitrogen is assimilated by utilization of ammonium ion (probably via glutamic dehydrogenase) with liberation of the anion of the ammonium salt into the medium as acid. Maximum growth of this organism was consistently associated with acid pH. During autolysis there was a liberation of inorganic

l nitrogen back into the culture medium.

It is evident from data shown in Figs. No. 9, 12 and 14 that maximum growth of this organism, within the limits of these experiments was achieved with WD5~Glycine or WD5- Alanine (C/N = 20/l). This is considered to be due to the more favourable carbon to nitrogen ratio of these media.

Consideration of P. tumulosus cultivated on glycine, alanine or other amino acids of the series examined (Fig. 15) at

C/N = 3l/l show that these amino acids as sole nitrogen source failed to stimulate growth to that engendered by WD5 - 146 -

(C/N = 3l/l)• These data, then, indicate that growth on the glucose-salts medium of WD5 was dependent on a favourable

C/N ratio with the presence of nitrogen in the preferred inorganic form. When the data on WD5-Glycine is examined

(Fig. No. 11, 12) it is found, that during the period corresponding to complete utilization of carbohydrate some

70% of the total nitrogen supplied (including 8OJ0 of inorganic nitrogen, and less than 50/^ of organic nitrogen) had been utilized. It thus seems probable that nitrogen is limiting in WD5 cultures, and if culture conditions were to correct this limitation, provision for the synthesis of enzymes, responsible for regulating the rate of assimilation of metabolites to the requirement of optimum growth, would be possible.

The Effect of Environmental Conditions

It is seldom possible, or for that matter desirable, to precisely duplicate the natural ecology of any organism in the laboratory; consequently it is doubtful if one ever observes an ideally controlled experiment. It is usually possible to vary one or two parameters at a time, and to maintain an environmental constancy not normally available to the organism in nature.

Metabolism in general, and hence growth, can be greatly affected by culture conditions, such as variations in the carbon/nitrogen balance, pH, temperature, oxygen tension, and the availability of trace nutritional requirements. - 147 -

It is advantageous to determine the extent to which environment can modify metabolic activity.

The Effect of Aeration

Fungi usually colonize environments in which the oxygen supply is limited, e.g. relatively stagnant aquatic media, decaying vegetable matter, cores of trees, etc. They are, however, generally considered to be obligate aerobes, although the quantitative relation of growth and oxygen supply vary considerably among the different forms.

In some methods of culturing microorganisms, varying degrees of aeration are readily obtainable within the one culture medium. As a result when grown as a surface (still) culture, the cells of the maturing mat are, in many cases, of visibly different types. The aerial hyphae are remote from the nutrient solution, and are exposed to the atmosphere of the culture flask, while the cells on the underside of the mat are in close contact with the nutrients and soluble metabolic products in the culture fluid, but may suffer from pronounced anaerobiosis. It is probable that in most conditions of still culture on a liquid medium, the culture as a whole is deficient in oxygen.

Improved oxygenation can be obtained by mechanical shaking or stirring, or by forced aeration of the culture liquor. Whilst such treatment ensures that the cells inhabit a more uniform environment, such aeration may in many instances subject the organism to unnatural oxygen tensions. - 148 -

Undoubtedly the level of oxygenation will affect the metabolic behaviour of the organisms. P. tumulosus was shown to accumulate aromatic compounds, only under the comparatively anaerobic conditions of still culture. There has not, however, been any detailed quantitative investig­ ation of the effect of oxygen tension on the metabolic character of this organism.

The Effect of Temperature

A temperature of 25°C was found to be most favourable for the vegetative growth of P. tumulosus. This is shown in Table No. 18 where the growth (diameter of colony) over the surface of agar plates during one week was measured.

TABLE No, 18.

Temperature °C 5 15 20 25 30 37

Growth (cm) 0.9 4.9 5.3 5.5 1.9 _

The effect of temperature on the assimilation or production of nitrogenous metabolites with this organism was not made. pH and Growth

In contrast to the true bacteria and the actinomycetes, fungi show a propensity to invade acid environments, moreover, the ability of fungi to raise or lower the pH of an initially unfavourable medium seems to be well developed.

Many are able to grow quite well, at a pH which does not permit the initiation of growth, although the larger basidiomycetes, in general, are unable to grow at a pH - 149 -

above 7 (513).

Usually the culture solutions provided for fungal growth are poorly buffered, and in consequence, when many of the higher fungi are cultured on a synthetic medium the pH of the culture liquor may fall to very acid values.

The pH is affected during growth by metabolic activity.

It is raised by the absorption of anions or by the production of nitrogenous compounds, and lowered by the formation of organic acids, or by absorption of cations.

One can expect a change in pH to exert its effect in several ways, e.g. by altering the solubility of trace metal ions, or by changing the ionization state of organic mater­ ials in the liquor, and hence the entry of these materials into the cell.

The Optimal Concentration of Nutrients

Most media provided for fungal growth contain considerable quantities of soluble carbohydrates (usually glucose) as well as a readily available supply of nitrogen (mostly ammonium salts). There may, however, be a considerable imbalance in the supplied C/N ratio. Skinner et al. (514) point out that the C/N ratio of the Czaapek-Dox medium commonly supplied to fungal cultures may reach as high as

40/1.

In the case of the wood-rotting fungi, the level of carbon available will most likely be governed by the activity of their extracellular secretions (cellulases in particular). - 150 -

Moreover, one would expect a consistent shortage of nitrogen, unless these fungi have some ability to fix atmospheric nitrogen. Little, however, is known about this facility in fungi.

To study the growth response of F. tumulosus to steadily increasing C/N ratio, the fungus was cultured on the basic glucose-salts medium, in which the carbohydrate concentration was varied from 1 - 6%. The results of this investigation are classified in Table No. 19. It can be seen that the extent, and rate of growth, and the economic coefficient are clearly dependent on the initial level of carbohydrate, and that lower carbohydrate concentrations permit more efficient growth.

The rate and efficiency of growth both increased to maximal values as growth proceeded, but the attainment of these maxima were delayed as the initial carbohydrate level was increased. The early phase of the process was not obvious

at C/N ratios of 6.3 and 12.0, but it could probably be assumed to have taken place before 14 days.

Although relatively more sugar was utilized at higher

initial levels of carbohydrate, the dry weight of mycelium produced was not increased proportionately. Evidently carbohydrate was converted to alternative metabolic products. TABLS No. 19. The Effect of Initial Carbohydrate Concentration on Growth of P, tumulosus - Experimental data of Crowden ' 1

Initial glucose cone, gm/100 ml. 1 2 3 4 5 6

C/N ratio 6.3/1 12.6/1 18.9/1 25.2/1 31.5/1 37.8/1 Days to harvest a 0.200 0.212 0.308 0.378 0.254 0.244 14 b 0.705 0.710 1.350 1.587 1.900 1.800 c 28.3 29.8 22.8 23.2 13.4 13.5 a 0.258 0.432 0.706 0.568 0.464 0.394 21 b 0.990 1.912 2.260 2.180 2.450 2.350 c 26.0 22.1 31.2 26.0 19.0 16.7 a 0.244 0.362 . 0.664 0.736 0.908 0.683 25 b 1.000 1.991 2.930 3.432 3.810 3.420 c 24.4 18.2 22.6 21.5 23.8 20.0 a 0.494 0.796 0.986 1.056 35 b 2.993 3.972 4.853 5.945 c 16.5 20.00 20.3 17.7 a 0.720 1.040 42 b 4.910 5.985 c 14.7 17.4 a 0.560 0.832 49 b 4.995 5.994 c 11.2 13.8 a 0.780 56 b 6.000 c 13.0

a = mycelium production (gm. dry weight/100 ml. liquor).

b = rate of carbohydrate utilization (gm. glucose used/100 ml. liquor).

nmn r* r’Aciffi ant

FIGURE No. 17.

>hikimic Acid Pathway for the Biosynthesis of

Aromatic Amino Acids

- 153 -

THE AMINO ACID COMPOSITION OF P, TUMULOSUo FRACTIONS Results and Discussion

A study was made of the composition, sequence of

production, and qualitative nature of amino acids in

P. tumulosus. Tables No. 20-24 present this data when the

organism was cultured on WD5-Glycine and WD5-Alanine. As

outlined in the introduction to this thesis, the organism was

cultured on these media as part of an investigation of phenolic

acid metabolism and this study was directed by the need to

elucidate the contribution of nitrogen metabolism to the production of aromatic intermediates.

The presence of aromatic amino acids in the mycelial

extracts (Tables 22 & 24) during the first 15 days of growth, coupled with their trace appearance in older cultures, is consistent with the suggestion that nitrogen is limiting at

certain phases of growth. Crowden ( 1 ) reports an increase in the accumulation of phenolic acid metabolites during the period of decline in aromatic amino acids. This accumulation

could be the result of an overflow or shunt of normal tyrosine metabolism, or a limitation of p-hydroxyphenylpyruvic

transaminase i.e. insufficient enzyme or nitrogen (See Fig.

No. 17). Further evidence in agreement with this suggestion

is provided by quantitative studies yet to be described.

Further detailed description of, or comment on, Tables No. 20-

24 will not be made since these data do not differ

significantly from those provided by the quantitative study, - 154 -

which lend themselves more readily to discussion: - 155

TABLE No. 2Q.

mino Acid Composition of the Mycelium of Polvporus tumulosus

Alanine + + Lysine +

Arginine + Methionine +

Aspartic Acid + Phe ny1alanine +

Cystine T Pro line +

Glutamic Acid ++ Serine +

Glucosamine + Threonine +

Glycine + Tryptophan +

Isoleucine + Tyrosine +

T = trace

+ = present

+ + = very prominent. - 156 -

TABLh Ho. 21.

WD5-GLYCINE Amino Acids in the Culture Medium of Polyporus tumulosus of days of incubation: 9 15 21 29 36 42 49

Alanine - T T - T T -

Arginine - - - 4- + 4-4-

Citrulline TT - - - - -

Glutamic Acid - TT - 4- T -

Glycine + 4- ++ ++ ++ 4-4- 4-4- 4-

Isoleucine T ------

Lysine - -- T T 4- 4-

Ornithine -- - T 4- 4- 4-

Methionine T ------= absent + ss present T = trace + + = very prominent 157

TABLE No. 22.

WD5-GLYCINE The Amino Acid Composition of the 70$ Ethanol Extract of

the Mycelium of Polyporous tumulosus

of days of culture: 9 15 21 29 36 42 49

' (X -Alanine + 4- T + + 4" T

-Alanine T ------

Arginine + + + T ++ 4-4- 4-4-

Aspartic Acid T + 4* - - - -

Citrulline - T + 4“ 4- 4- 4* 4* 4-

Cystine T ------

Glutamic Acid ++ + 4* + 4-4- + 4- -

Glycine ++ + + ++ 4- 4-4- 4-

Isoleucine + T - TT - -

Lysine + - T T 4- 4- 4-

Methionine + 4~ - T T - -

Ornithine - T + T 4- 4- 4-

Phenylalanine + + - - - --

Serine + + - ++ 4- 4-4- -

Threonine - - - TT - -

Tryptophan T T - - - - -

Tyrosine ++ + + - - - 4-

= absent + = present

T = trace + + s very prominent

Unknown Amino Acid L + + 4-4- 4- 4- - 15$ -

TABLE No, 23.

WD 5-ALANINE

The Amino Acid Composition of the Culture Medium

of Polyporus tumulosus

No. of days of culture: 8 14 19 25 32 21 46

Alanine 44 ++ ++ +•+• ++ - + +

Arginine - - - T - T T

Glutamic Acid - - - T - + -

Glycine T T T T - - -

Lysine -- - T - T -

Ornithine - - - T - T -

- - absent + = present T = trace ++ = very prominent - 159 -

TABLE Mo. 24.

WD5-ALANINE

The Amino Acid Composition of the 70yo Ethanol Extract of

the Mycelium of Polyporous tumulosus

Mo. of days of culture: 6 14 19 25 32 39 46

X-Alanine ++ ++ 4* 4* 44 4-4- 4- 4-4-

-Alanine “ - - - 4 4- 4-

Arginine T ++ - 4- - 4 “f*

Aspartic Acid -- “ - T - “

Citrulline + + 4- 4-4- 4-4- 4-4- -

Glutamic Acid - 4 4 4- 4-4- 4-4- 4-4-

Glycine - T T - T TT

Isoleucine TT T “ “ “ -

Lysine - T + 4 4- 4- 4-

Methionine - T T “ T “ -

Ornithine - T T T 4- 4- 4-

Phenylalanine + 4- “ -“ - 4-

Serine - - T - TT -

Threonine - “ T - -- “

Tryptophan T T - - -- -

Tyrosine + 4- - “ - - 4

Unknown Amino Acid _. 4 4-4- 4-4- 4-4- 44

absent 4 - present

trace 44 = very prominent TABLE No. 25.

Amino Acid Analysis of the 10% Ethanol Extract of the Mycelium of Polyporous tumulosus (All results are expressed on an anhydrous basis)

1st sample 13 days 2nd sample 20 days 3rd sample 27 days Ath sample 3A days 5th sample LI days 6th sample L9 days 7th sample 56 days

Amino Acid a.a. a.a.N 9* • • a.a.N a.a. a.a.N %N a.a.N %N Q. • S • a.a.N %N a. a. a.a.N %N cl« cl * a.a.N m X- Alanine trace trace - 0.231 0.0360 6.05 0.343 0.0540 8.18 0.137 0.0215 3.16 0.164 0.0255 3.13 0.083 0.0130 1.79 0.071 0.011 1.52 Alanine Arginine 0.718 0.231 16.86 0.101 0.0325 5.46 0.333 0.1070 16.21 0.974 0.313 46.02 0.974 0.3135 38.46 0.915 0.294 40.55 0.781 0.251 34.62 Unknown Acid very prominent present

Aspartic Acid trace trace 0.007 0.0005 0.08 trace trace 0.003 - 0.002 — 0.003 — 0.006 0.0005 0.07 Citrulline 0.031 0.0075 0.55 0.037 0.0090 1.51 0.028 0.0070 1.06 O.O46 0.011 1.62 trace trace 0.022 0.005 0.69 0.044 0.0105 1.45 Cystine trace trace trace trace trace trace trace trace trace trace trace trace trace trace trace trace trace trace trace trace trace Glutamic Acid 0.080 0.0075 0.55 0.088 0.0085 1.43 0.131 0.0125 1.89 0.073 0.007 1.03 0.076 0.0070 0.86 0.066 0.006 0.83 0.052 0.005 0.69 Glucosamine 0.043 O.OO35 0.27 0.034 0.0025 0.42 0.013 0.0010 0.15 0.014 0.0010 0.15 0.010 0.0005 0.06 0.016 0.0015 0.21 0.025 0.0020 0.27 Glycine 0.175 0.0325 2.37 0.350 0.0650 10.92 0.075 0.014 2.12 0.030 0.0050 0.73 0.030 0.005 0.61 0.025 0.0045 0.62 O.O45 O.OO85 1.17 Isoleucine 0.141 0.0150 1.09 0.025 0.0025 0.42 trace trace trace trace trace trace trace trace trace trace trace trace trace trace trace Lysine 0.547 0.105 7.66 0.200 O.O385 6.47 0.340 O.O65 9.85 0.561 0.1050 15.44 0.687 0.0130 15.95 0.596 0.1150 15.86 0.641 0.1250 17.24 Methionine 0.559 0.052 3.83 0.147 0.0120 2.02 trace trace trace 0.250 0.0235 3.45 trace trace trace 0.216 0.0205 2.83 trace trace trace Ornithine 0.310 0.0650 4.74 0.465 0.1030 17.31 0.280 0.059 8.94 0.240 0.0505 7.43 0.285 0.060 7.36 0.435 0.090 12.a O.47O 0.0995 13.72 Phenylalanine 0.990 0.084 6.13 0.0155 0.0015 0.25 0.007 0.0005 0.07 trace trace trace 0.010 0.001 0.12 trace trace trace trace trace trace Proline Not detected in Mycelial extracts Serine Not present 0.137 0.0185 3.11 0.015 0.002 0.30 0.014 0.002 0.29 0.046 0.005 6.13 0.060 0.008 1.10 0.104 0.014 1.93 Threonine 0.222 0.026 1.90 0.093 0.011 1.85 0.148 0.0175 2.65 0.023 0.0025 0.37 0.026 0.003 0.36 0.066 0.0075 1.00 0.131 0.0155 2.13 Tryptophan trace trace trace trace trace trace trace trace trace trace trace trace trace trace trace trace trace trace trace trace trace Tyrosine 4.210 0.3250 23.72 0.807 0.0625 10.50 0.495 0.0380 5.76 0.810 0.0625 9.19 0.526 O.O4O5 4.97 0.459 0.0355 4.90 1.130 0.0875 12.07 Urea 0.133 0.0620 4.52 Not detected 0.004 0.002 0.30 0.008 O.OO45 0.73 0.011 0.005 0.61 0.013 0.005 0.69 0.059 0.025 3.44 Amide Nitrogen 0.040 0.0350 2.55 0.045 0.0400 6.70 0.065 0.055 8.33 0.065 0.055 8.09 0.130 0.1150 14.11 0.090 0.080 11.03 0.040 0.035 4.83 TOTAL 8.201 1.0517 76.74 2.805 0.4455 74.50 2.28 0.4345 65.81 3.252 0.664 97.7 2.98 0.594 72.85 3.07 0.685 94.51 3.60 0.690 95.15 Kjeldahl Nitrogen 1.370 0.595 0.660 0.680 0.815 0.725 0.725 of the Mycelial extract in grams of nitrogen/100 g. of anhydrous Mycelium. a.a. = Grams of amino acid/100 grams mycelium extracted. a.a.N = Grams of amino acid nitrogen/100 grams mycelium extracted. %N a Amino acid nitrogen expressed as a percentage of the total nitrogen of the mycelial extract. - 161

QUANTITATIVE STUDIES OF THE AMINO ACIQ COMPOSITION OF

P. TUMULOSUS FRACTIONS

Results and Discussion

For these investigations P. tumulosus was cultured on WD5> a medium uncomplicated by the initial presence of an amino acid. However, the studies with WD5-Glycine and-

Alanine provided evidence for the amino acid nature of fractions, together with an indication of the tfunknown amino acid" in mycelial extracts, and were directly responsible for the decision to grow the organism on WD5. Crowden*s investigation of phamlic acid metabolism in P. tumulosus was a parallel study and this worker measured the accumulation of phenolic acids in P, tumulosus on WD5, concurrently with the authorTs amino acid analyses which are classified in Tables

No. 25, 26 and 27.

While it is realised that mycelium contains a complex of proteins, it was of interest to conduct a detailed amino acid analysis of this tissue in order to contrast its composition with the amino acid make-up of the cell wall protein.

The main characteristic of the mycelial amino acids was the high concentration of alanine (6fo) which accounted for

26fo of its total nitrogen. The possibility of the occurrence of amino acids in atypical concentration in mycelia of basidiomycete fungi may be of use in studies on their taxonomy, since the systematics of the higher fungi are TABLE No. 26.

The Amino Acid Analysis of the Mycelium of Polyporus tumulosus

The following analysis was performed on acid hydrolysed 30 day (mature mycelium.

a. a • a.a.N.

Alanine 6.10 0.961 25.76 Arginine 1.07 0.346 9.28 Aspartic Acid 2.05 0.214 5.74 Cystine trace Glutamic Acid 1.07 0.102 2.73 Glucosamine 4.50 0.352 9.43 Glycine 1.80 0.337 9.03 Isoleucine 2.73 0.292 7.83 Lysine 0.49 0.092 2.47 Methionine 1.80 0.170 4.56 Phenylalanine 1.17 0.098 2.63 Proline 1.07 0.131 3.51 Serine 0.78 0.102 2.73 Threonine 1.51 0.175 4.69 Tryptophan 0.63 0.088 2.35 Tyrosine 2.19 0.170 4.55 Amide Nitrogen 0.02 0.109 0.50 TOTAL 28.98 3.649 97.79 a.a. = Grams of Amino Acid/100 grains of anhydrous mycelium. a.a.N. s Grams of Amino Acid Nitrogen/lOO grams of anhydrous mycelium.

%N = Amino Acid Nitrogen expressed as a percentage of the total Kjeldahl Nitrogen of the anhydrous mycelium. The mycelium contains 3.73 grams of Nitrogen/100 grams. The Acid hydrolysed mycelium

contains 3*33 grams of Nitrogen/lOO grams. TABLE No. 27.

Amino Acid Analysis of the Cell 'Jail Protein of Polyporus tumulosus

a.a. a.a.N. 2$N Alanine 2.79 0.440 9.09 Arginine 1.39 •) .446 9.21 Aspartic Acid 1.83 0.193 3.99 Cystine trace trace Glutamic Acid 1.95 0.185 3.82 Glucosamine 1.60 0.125 2.58 Glycine 1.50 0.280 5.78 Isoleucine 3.94 0.420 $. 66 Lysine 1.83 0.350 7.23 Methionine 5.22 0.490 10.12 Phenylalanine 2.06 0.175 3.61 Proline 1.32 0.160 3.30 Serine 1.15 0.153 3.16 Threonine 1.04 0.122 2.52 Tryptophan 1.83 0.251 5.18 Tyrosine 7.43 0.575 11.88 Amide Nitrogen 0.03 0.028 0.57 TOTAL 36.91 4.352 90.72 a„a. = Grams of Amino Acid/lOO grams of anhydrous cell wall fraction. a.a.N. = Grams of Amino Acid Nitrogen/lOO grams of anhydrous ceil wall fraction. y£N = Amino Acid Nitrogen expressed as a percentage of total Kjeldahl Nitrogen of the cell wall

fraction. - 164 -

incomplete and uncertain. The estimated amino acid nitrogen of the mycelium

accounted for 9&fo of the total nitrogen of this fraction. This was not in conflict, within the limits of experimental

error of the method of analysis, with the estimated nucleic

acid nitrogen of fungal mycelium reported by Smithies (515)

as 4$. There are a number of possibilities arising from a

consideration of the data in Table Mo. 25. Primarily the

incidence of a particular amino acid suggests the presence of known enzymes and pathways associated with the metabolism of

the amino acid in question. Particularly is this so when a metabolic path has been established in fungi. Within limits

this approach will be made, but the inclination to propose

from the general to the particular will be cautioned by the knowledge that these quantitative data represent the dynamic overall picture at time intervals, and not the sequential progress of hypothetical individual reactions. It is a peculiarity with fungi, in contradiction to other microorganisms that particular metabolite accumulation occurs. This is an axiomatic concept in fungal biochemistry and results from an interruption or imbalance in cycles, causing the pile-up of compounds representing one or more

stages on the metabolic path. In an appropriate nutritional environment, where the disturbance in balance of assimilatory and dissimilatory - 165 -

mechanisms results in the accumulation of one or more metabolites, the concept suggests that the accumulation stems directly from an imbalance in a substrate concentration in growth media. A depression in the rate of cell synthesis results. It is a distinct possibility that the data in

Table No. 25 is evidence of such substrate (nitrogen) imbalance.

Reference to Table No. 25 shows that, quantitatively, arginine is the major metabolic product. Its presence was complemented by other amino acids of the ornithine cycle and, urea, which suggests the mediation of this nitrogen path in

P. tumulosus. Argininosuccinic acid was not detected in any fractions of this organism in spite of a deliberate search with the sensitive and extremely selective chromatographic technique (491). This does not suggest that this acid does not occur, but rather that its absence merely pictures its low concnetration which would be governed by the equilibrium relation with its precursors and products.

The low concentration of citrulline in contrast to that of its metabolically related amino acids is very probably a manifestation of the same requirement of trace quantities of metabolites to promulgate certain reactions.

The presence of the tTunknown amino acid” at times, which corresponded to the highest arginine concentration, indicated a possible relation between these two amino acids. It was established that the "unknown amino acid" possessed a - 166 -

monosubstituted guanidino group. The accumulation of

arginine to a critical level could thus introduce a shunt,

resulting in the transfer of the guanidino group of arginine

to another amino acid via a transamidinase-catalyzed reaction.

For this hypothesis to be tenable, ornithine as one of the

products of the reaction should accumulate and such is seen

to be the case.

The accumulation of such basic compounds as arginine

and the "unknown amino acid" could also be a mechanism on

the part of the organism to protect itself from the extremely low pH of the growth medium. There is a time-dependent

relationship between drop in pH and the increase in arginine concentration.

The quantitative relation of ornithine and arginine is also consistent with the participation of arginine desimidase.

It has been previously stated that arginine can serve as a source of energy if carbohydrate is limited or exhausted, and it is possible that part of the purpose of arginine accumulation is the provision of an energy storage compound.

The presence of urea in the mycelial extract of a

13 day culture reflected the possibility of arginase activity; the concentration of this metabolite diminished in older cultures and increased at autolysis. The ureides, allantoin and allantoic acid, were detected in autolysing cell extracts and the presence of urea with these materials at this time suggested that the ureides and urea were products of nucleic - 167 -

acid breakdown. It seems possible that arginase may be

present but not active due to the stress of carbon/nitrogen

ratio imbalance.

The sequential accumulation of aromatic amino acids as

evidenced with cultures of P. tumulosus on WD5-Glycine and-

Alanine was faithfully followed by the organism grown on

WD5. The quantitative data were consistent with the explanation previously given that aromatic amino acid decline

was coupled to phenolic accumulation. Fig. 18 shows CrowdenTs

measurements of phenolic acids plotted against the concen­ tration of tyrosine in ageing WD5 cultures. Tyrosine at

7.43io accounted for approximately 12jo of the nitrogen of

the cell wall protein (Table 27) which suggests that during synthesis of the wall considerable ,rmetabolic pressure” was exerted on the biosynthetic mechanism of this metabolite.

This, coupled with the increasing depletion of nitrogen

from the growth medium, provided circumstances conducive to synthesis of phenolic acids.

Little constructive comment is attempted on the

accumulation of lysine in mycelial extracts. This amino acid

is remarkably inert in biological systems, having little

relation with the molecular traffic of the established

pathways. Associated compounds of the intermediary metabolism of this amino acid, viz. aminoadipic acid, pipecolic acid, were not found in extracts of P. tumulosus.

The presence of glucosamine in the extracts intimates FIGURE No. IS.

Graph Showing Sequential Changes in the

Concentration of Tyrosine and Phenolic Acid;

in Polyporus tumulosus 5-0 T QIDV

i na U-

OHON3Hd

NO. OF DAYS - 169 -

that the formation of this amino sugar is probably active in the cytoplasm of hyphal cells and does not occur as the result of direct synthesis and incorporation into the nexosamine polymer at the site of cell wall formation.

Proline, on the other hand, would seem to exemplify amino acid synthesis directly at its site of incorporation into insoluble mycelial and cell wall proteins since this most obvious amino acid was not detected on chromatograms of mycelial extracts in spite of a deliberate search and the awareness of its possible presence at low concentration.

^-Alanine is a known component of pantothenic acid (516), coenzyme A (517), anserine (518), carnosine (519), spermine

(520), and spermidine, and it is a known intermediate in the metabolism of dihydrouracil, ft -ureidopropionic acid (521) and aspartic acid (522). The involvement of this amino acid in the metabolism on none, some, or all of these compounds in P. tumulosus is of course a theoretical possibility.

Methionine was the only sulphur amino acid consistently detected in quantity in fractions of P. tumulosus. The trace appearance of cystine- was an accurate description of the evidence found for its presence, but the well known difficulty in resolving, and identifying, these acids, engenders lack of confidence in suggesting that sulphur metabolism in this organism confines itself to the accum­ ulation of these two compounds. - 170 -

The mycelial extracts of 27, 34, and 41 day old cultures contained a ninhydrin positive spot which was resolvable by the N/Q solvents (491). Acid hydrolysis of the extracts, before application to the chromatograms removed the presence of this spot. On this evidence it is assumed that the spot was caused by a peptide. The amino acid nature of the extract

(with the exception of tryptophan) did not alter with acid hydrolysis.

Tyrarnine was found in mycelial extracts of 13 day old cultures of P, tumulosus. No other amines have been detected in extracts of this organism. - 171 -

THE AMINO ACID COMPOSITION OF THE CULTURE MEDIUM Results and Discussion

The amino acids present in the culture medium, (Table No. 2£), were identified by paper chromatography and the

magnitude of the ninhydrin-amino acid colour yield was

visually estimated as a quantitative index of the amount of amino acid.

Some amino acids at low concentration, among them,

glutamic acid, arginine, lysine and glycine have high colour

yield on chromatograms. For this reason some discretion was necessary in evaluating the tabulated information.

The data in the table were collected from chromatograms which had been loaded with 100 til of culture fluid. This unusually high loading, coupled with the qualifying remarks

above, confirms the belief that there is little accumulation of amino acid in the culture liquor of P. tumulosus.

This could result from failure of the organism to back-

flow amino acid into the media of young actively growing cultures, or there may be fast reassimilation of material which would otherwise be expected to accumulate.

The low concentration of amino acid, resulting in the need to spot a high loading on chromatograms was responsible

for the failure to analyse the amino acid composition of the culture fluids. - 172 -

THE CELL 7/ALL PROTEIN

Results and Discussion

The same amino acids were found in both cell wall and mycelial proteins (Table No. 27). The high concentration of five amino acids together with the anomalous finding of diminished glucosamine concentration in cell wall, contrasts the composition of these fractions. The alkaline treatment of mycelium during isolation of !twallTt is a suggested reason for the considerable difference in the amino sugar analysis.

The high concentration of alanine in mycelium has received previous comment. The uniquely high concentration of tyrosine in "wall" distinguishes this fraction, and it is probable that the ultimate fate of the large amount of tyrosine in mycelial extracts is incorporation into cell wall protein. - 173 -

TABLE No. 28. W5

The Amino Acid Composition of the Culture Fluid of

Poly porous turnulosus

No. of days of incubation: 13 20 27 34 41 49 56

Alanine - T T T 4- -

Arginine - + + 4-4- 4-4- 4-4- 4-4-

Glutamic Acid - + + 4-4- 4- 4-4- T

Glycine - 4 4-4- 4-4- 4-4- 4-4- 4-4-

Lysine + 4- 4-4- 4- 4-4- 4-4- 4-4-

Ornithine - - 4- 4- 4- 4- 4-4-

Serine - 4- - T - T -

Threonine - - T T T T

= absent 4- = present

T = trace 4-4- = very prominent

FIGURE No. 19.

Photograph of a Chromatogram of Ureides

Isolated from Extracts of Autolysing

Cells of P. tumulosus

FIGURE No. 20.

Two Dimensional Chromatogram of Ureides

Isolated from Extracts of Autolysing

Cells of P. tumulosus

- 175

UREA AND UREIDES

Results and Discussion The mycelial extracts of autolysing cultures of P. tumulosus were found to contain urea and the ureides:

allantoin, allantoic acid, ureidosuccinic acid, and

A citrulline. An unidentified ureide was also found. Paper

chromatography was used to resolve and identify these

compounds (see Figs. No. 19 and 20). The sequential incidence

of the ureides identified in mycelial extracts of P. tumulosus

cultured on WD5 is shown in Table No. 29.

TABLE No. 29. Days of Culture 13 20 27 2k 41 49 56

Allantoin - - F F + +

Allantoic Acid - - F F + +

Unidentified Ureide - - FF + + *Urea + F F FF F +

Ureidosuccinic Acid , F FF F F

+ = present

F = faint trace

- = not detected A The quantitative data for citrulline and urea are presented

in Table No. 25.

Ureidosuccinic acid is a known intermediate in the metabolism of orotic acid and it is possible that its

incidence in ageing cultures of P. tumulosus is related to the biosynthesis and catabolism of pyrimidines. - 176 -

A time sequence chromatogram of the reaction mixture

(sampled at intervals) of a carefully controlled acid hydrolysis of allantoin indicated that four p-dime thy}.amino - benzaldehyde-visualised compounds were among the products of hydrolysis. These are allantoin, allantoic acid, and an unidentified ureide which may be allantoxanic acid and/or uroxanic acid, both intermediates in the hydrolysis of allantoin. Urea, the final reaction product, increases in concentration as hydrolysis proceeds. The mycelial extracts of P, tumulosus showed the presence of compounds which behave similarly on chromatograms to the ureides isolated as hydrolysis products of allantoin.

It is quite probable that the acid medium of autolysing cultures of this organism may chemically hydrolyse allantoin, a compound on the path of purine catabolism, to yield the ureides described, or it is equally possible that the enzymes associated with the autolysis of the nucleoprotein of fungal cells may produce the same compounds. FIGURE No, 21.

Photographs showing Separation of

"Unknown Amino Acid" with

the Solvents N/Q of Hardy et al. (491)

A Visualised with Kinhydrin Reagent.

B Visualised with Sabaguchi Reagent. A

B - 178 -

"THE UNKNOWN AMINO ACID"

Results and Discussion

There are two 1-guanidino monosubstituted amino acids in mycelial extracts of P. tumulosus. Arginine is one of these and its presence and possible significance has been discussed. The other is that compound referred to in this thesis as the "unknown amino acid".

Resolution of this unknown acid has been achieved chromatographically with the six amino acid solvents, and its presence on chromatograms detected with ninhydrin and the three guanidino visualising agents (see Fig. 21). It was possible to separate the unknown amino acid from arginine on a Dowex 50 column.

The positive test with Dakaguchi reagent and its response to the Diacetyl, and Pentacyanoaquoferriate reagents is presumptive evidence for the amidine group.

The intense purple colour imparted with the chromogenic- ninhydrin reagents (containing dicyclohexylamine or collidine, Pages 112, 115) is considered good evidence for an cC-amino group on the unknown acid, since these agents, besides characteristically colouring some (tf-amino acids, invariably visualise amino acids substituted in other than the *\-amino position with quite striking colours, e.g.,

^-alanine shows light green, flt-and £ -amino butyric acids, pale blue.

The unknown acid was able to survive prolonged acid - 179 -

hydrolysis, showing it was not a peptide, and when developed

chromatographically in phenol solvents it was found on

visualisation to streak as arginine does.

Separation of the unknown acid from arginine and other

amino acids was achieved by resolution on a Dowex 50-X8

column (50 cm. high X 0.6 cm. diam.). Arginine was eluted

from the column over the range 212-289 mis. with 0.4 M

citrate buffer pH 5.27 (523). Further elution with this

buffer to a volume of 1,528 mis. failed to remove the other

guanidino compound.

The unknown amino acid was isolated in the effluent

fraction 169-179 mis. with a pH 3.25 buffer (523). The

lower pH required to displace this acid from the column

suggests that there is greater dissociation of its basic

groups. Evidence suggesting that the unknown acid has a

shorter carbon chain than arginine is discussed below. If

it is conceded that the unknown acid possesses a shorter

carbon chain, then an additional consequence is the observed

increased basicity of the unknown acid in comparison with

arginine.

In an effort to establish the identity of the unknown

amino acid a number of the guanidino compounds reported in

biological systems were screened chromatographically, these

are listed below.

Homoarginine Glycocyamine Guanidinobutyramide

Argininosuccinic Acid Canavanine Agmatine

Creatine Creatinine Lombricine - ISO

Although none of these proved to be the unknown acid, these compounds were useful in establishing chromatographic maps of guanidino compounds. From the position of the unknown acid in relation to other Sakaguchi positive compoundj on the map, it would seem that the unknown is either an

Oi-amlno-A or d - (1-guanidino ) propionic or butyric acid.

The substituted butyric acid would seem the more likely compound, for if the transamidination theory (Page 166) is tenable, then the acceptor of the amidine group from arginine would be (X,X -diaminobutyric acid, a reported metabolite in microorganisms (524); OtrS -diaminobutyric acid however was not found in P. tumulosus.

Visual inspection of equally loaded chromatograms of the mycelial extract of P. tumulosus, cultured on WD5,

T./D5-Glycine, and WD5-Alanine showed that the unknown acid was produced in significantly greater yields in the WD5-Alanine and AD5-Glycine media than in WD5. This would seem to be another manifestation of the stress of carbon/nitrogen ratio imbalance. - 131 -

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