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P-6 Microorganisms in and Biofactor Research

E .E. SNELL

Departments of Microbiology and Chemistry, The University of Texas, Austin, Texas 78712 (U. S. A.)

I. INTRODUCTION Because I retired fully almost two years ago, and spoke in Osaka one year ago about our most recent findings on histidine decarboxylase, I was undecided about accepting an invitation to speak here. However, since few of us are aware of older developmental findings outside our own field, I thought it might be useful to recall some of the many roles microorganisms have played in vitamin and biofactor research, then consider briefly their use in study of biosynthesis and degradation of , using and vitamin B6 as examples.

II. MICROORGANISMSAND THE DISCOVERY AND/OR ISOLATION OF VITAMINS Table 1 illustrates the almost dominant role played by microorganisms as test organisms in the discovery and/or isolation of various vitamins and other biofactors. Of the currently known B- vitamins, only , and were discovered and isolated exclusively through studies of animal nutrition, while essentially all of the alternate forms of the vitamins (e. g. pyridoxal, , pantetheine, reduced and conjugated forms of , etc.) were discovered through studies of microbial nutrition.

TABLE 1 Microorganisms and progress in recognition of vitamins and related compounds

For references, see [1, 2].

Because microbial growth, unlike animal growth, is rapid and easily controlled, such studies enormously expedited completion of this chapter in nutrition. They also contributed to discovery and isolation of additional biofactors not shown in Table 1, e.g. lipoic acid, mevalonic acid and many metabolic intermediates. E. E. SNELL 35

III. USE OF MICROORGANISMSFOR THE DETERMINATIONOF VITAMINS The studies referred to in Section II provided crude assay procedures for vitamins which, upon further refinement, could be used for the quantitative determination of each of the B-vitamins. The first such method to gain wide acceptance was that using Lactobacillus casei for the assay of riboflavin [3]. Many subsequent methods were patterned after it. Much of our current knowledge of vitamin distribution was obtained by such methods. Quantitative discrepancies observed when different assay organisms were used sometimes led to the discovery of important new forms of a given nutrient (e. g. pyridoxal, pyridoxamine, folic acid conjugates, biocytin, etc.).

IV. MICROORGANISMS AS PROVIDERS OF, OR COMPETITORS FOR, VARIOUS VITAMINS IN ANIMAL NUTRITION The intestinal flora of animals is a complex mixture that includes coliform bacteria, lactic acid bacteria, yeasts, and other organisms. Some of these are nutritionally non-demanding, and are able to synthesize whatever vitamins they need for growth. Others (e. g. the lactic acid bacteria) require most of the B-vitamins for growth. Still others may accumulate or even destroy some of the vitamins. Thus one might expect to find circumstances where the flora supplies part of the vitamin requirement of animals, and other circumstances where competition for limited amounts of a vitamin occurs. Both phenomena have been observed. In early studies of rat nutrition, requirements for , folic acid, and were most easily observed when antibiotics were included in the diet to suppress their synthesis by the intestinal flora. We observed the opposite relationship among the vitamers of B6 (see Table 2): feeding antibiotics increased the activity of pyridoxal and pyridoxamine relative to that of pyridoxine for rats.

TABLE 2 Effect of aureomycin on comparative activities of pyridoxine, pyridoxal and pyridoxamine for rats [4].

On this diet, the three vitamers showed equal activity only in the presence of the antibiotic. We interpreted this to mean that the antibiotic suppressed organisms that decreased the availability of pyridoxal and pyridoxamine either by partial destruction, or by accumulation within their own cells. Many lactic acid bacteria require pyridoxal or pyridoxamine for growth, but cannot utilize pyridoxine for this purpose, a situation that might account for part of the observed differential effect.

V. MICROORGANISMS AND ELUCIDATION OF VITAMIN FUNCTION Early clues to the functions of some of the B-vitamins were provided by nutritional differences observed in the presence and absence of the vitamin. For example, a role for folic acid in synthesis of thymine and purine bases was first suspected when it was observed that Plenary Lecture 6 36

this vitamin was not required for growth of certain lactic acid bacteria when the latter compounds were included in the growth medium (pantothenic acid and methionine, in whose synthesis folic acid also plays a role, were both present in the medium used). Similarly, a role for biotin in fatty acid synthesis was presaged by the observation that oleate replaced biotin for growth of L. casei in complex medium (such media contain aspartate, for whose synthesis later findings showed that biotin also was required in this organism). Finally, an essential role for vitamin B6 in synthesis of each of the amino acids (including D- alanine, necessary for cell wall synthesis) could be shown by the fact that amino acids that were not essential in the presence of the vitamin became so in its absence (see Table 3), and that these amino acids did not permit the organism to synthesize the vitamin. Similar observations cannot be made with animals, where vitamin B6 is needed for degradation as well as for biosynthesis of amino acids.

TABLE 3 Comparative vitamin B6 requirements for growth of S. faecalis with amino acids or with the corresponding keto acids [5].

*Growth corresponding to about 1 mg of cells/ml was observed in all cases except those indicated by "no growth".

VI. MICROORGANISMS AS "FACTORIES" FOR PRODUCTION OF BIOFACTORS Microorganism have been used for the commercial synthesis of a wide variety of amino acids, some vitamins (e. g. riboflavin and vitamin B12), and many enzymes. Through use of the more recent technique of cloning, they have become major sources of enzymes of other organisms, including those of the human species. Indeed, it is through use of microorganisms in this way that extensive study of many types of human proteins has become possible.

VII. MICROORGANISMS AS TOOLS FOR STUDY OF BIOSYNTHESIS AND DEGRADATION OF VITAMINS AND OTHER METABOLITES Microorganisms play a dominant role in the degradation of organic materials in soil. Their degradative and synthetic abilities, the ready control of their growth environment, and the ease with which genetic modifications can be effected, have made them preferred experimental organisms for studying the mechanism of both synthesis and degradation of amino acids and vitamins. The literature in this area is extensive; I shall provide only two examples drawn in part from our own work with vitamins. 1. Synthesis of Pantothenic Acid. The general scheme for biosynthesis of this vitamin became clear from studies of mutants of E. coli E. E. SNELL 37

auxotrophic for pantothenic acid but blocked at various steps in its synthesis. Subsequent studies at the enzymic level then established the details of each of these steps. ƒÀ-Alanine and pantoic acid moieties are synthesized separately, then coupled by reactions shown in Fig. 1.

Fig. 1. Steps in biosynthesis (narrow arrows) and degradation (broad arrows) of pantothenic acid [6,7].

2. Degradation of Pantothenic acid. Cells of Pseudomonas P-2 grown with pantothenate as carbon-nitrogen source hydrolyse this vitamin to ƒÀ- alanine and pantoic acid. Further degradative steps were established by use of partially purified enzymes from cell extracts, and are shown in Fig. 1. Obviously synthesis and degradation of micronutrients, like that of macronutrients, occur by related but distinct routes. 3. Biosynthesis of Vitamin B6. The carbon skeleton of pyridoxine arises from glyceraldehyde by the route shown in Fig. 2. In some strains of E. coli, carbon atoms 5 and 5' also can arise from glycolaldehyde. Mutational studies show that at least six steps are involved; their nature is not known. The thiazole portion of thiamine

I

II

Fig. 2. Relationship between synthesis of the thiazole portion of thiamine (I) and pyridoxine (II) in E. coli [8, 9]. Plenary Lecture 6 38

also arises in part from glyceraldehyde, and Fig. 2 emphasizes the probability that these two molecules share one or more steps in their biosynthesis. This fact may explain some curious older observations (Fig. 3) that some strains of Saccharomyces carlsbergensis widely used for assay of vitamin B6 did not require this vitamin unless thiamine was present in the medium. By interfering with uptake or use of thiamine, neopyrithiamine promoted growth in the presence of thiamine just as did pyridoxine. These relationships would be readily explained if added thiamine suppressed synthesis of cellular thiamine by inhibiting steps also required for synthesis of pyridoxine.

Fig. 3. Inhibition of growth of S. carlsbergensis by thiamine (A) and its reversal by pyridoxine or neopyrithiamine (B) [10].

Fig. 4. Degradation of vitamin B6 by Pseudomonas MA-1 (Pathway I, numbered steps) and by Arthrobacter Cr-7 (Pathway II, lettered steps). Single purified enzymes catalyze each step shown [11]. E. E. SNELL 39

4. Degradation of Vitamin B6. Breakdown of pyridoxine via pathway I

(Fig. 4) is initiated by oxidation at the 4-hydroxymethyl group and was found in a pseudomad isolated by enrichment culture with pyridoxamine as the carbon-nitrogen source. Since pyridoxal is an intermediate, this

path is presumably followed for its degradation as well. Pathway II, initiated by oxidation at the 5-hydroxymethyl group, was found in both a pseudomonad and an Arthrobacter that were isolated with pyridoxine as the carbon-nitrogen source. Almost all of these enzymes have been

purified to homogeneity; their further study might extend current knowledge concerning the induction of long inducible degradative

pathways, now based almost entirely on studies of the, ƒÀ-ketoadipate pathway for degradation of homocyclic aromatic compounds [12].

VIII. CONCLUDING REMARKS

This skeletal summary emphasizes the dominant role studies of microbial biochemistry have played in developing our knowledge of many areas of vitamin and biofactor research. Their similar role in development of molecular genetics and molecular biology is sufficiently recent to be common knowledge, and has finally brought us the ability to

study biochemical events in man at the molecular level. Such studies are certain to expand knowledge of nutritional interrelationships far beyond their current level.

REFERENCES

[1] Peterson, W. H. and Peterson, M. S. (1945) Bact. Revs. 9, 49-109.

[2] Snell, E. E. (1956): Currents in Biochemical Research, ed. by Green, D. E., Interscience Publishers, N. Y., pp.87-114.

[3] Snell, E. E. and Strong, F. M. (1939) Ind. Eng. Chem., Anal, Ed. 11,346-350.

[4] Linkswiler, H. M., Baumann, C. A., and Snell, E. E. (1951) J. Nutr. 43, 565-573.

[5] Holden, J. T., Wildman, R. B., and Snell, E. E. (1951) J. Biol. Chem. 191,559-576.

[6] Brown, G. M. (1960) Physiol. Revs. 40,331-368.

[7] Magee, P. T. and Snell, E. E. (1966) Biochemistry 8,409-416. [8] Hill, R. E., Sayer, B. G., and Spenser, I. D. (1989) J. Amer. Chem. Soc. 111,1916-1917.

[9] Julliard, J.-H. and Douce, R. (1991) Proc. Nat. Acad. Sci. (U. S. A.) 88,2041-2045.

[10] Rabinowitz, J. C. and Snell, E. E. (1951) Arch. Biochem. Biophys. 33, 472-481.

[11] Nelson, M. J. K. and Snell, E. E. (1986) J. Biol. Chem. 261, 15115-15120.

[12] Cook, K. A. and Cain, R. B. (1974) J. Gen. Microbiol, 85,37- 46.