Conversion of Coumarin to MaliloUc Acid b;r ~es
Isolated fram EBeudomonas Mac 291
by
Gerald Alan Lancaster, B. A. (Hm.)
A thesis submitted to the Facult,y of Graduate Studieô and Research in partial ful:filment 01' the requirements for the degree of Master 01' Science.
Department of Micrabiology, Macdonald College of McGill University, P.Q.. June, 1967
~ Gerald Alan Lancaster 1968 Table of Contents
Page
Introduction 1
Literature Review- Biological transformations of coumarin Bigher Plants 2 Animals 6 Fungi 8 Bacteria 9
MELterials and Methode Organism 13 Source of chemicals 13 Growth of cella 13 Chromatograph;y 14 Enzyme aas~ 15 Determina. tion of protein 15 Purification of the reductase 15
Resulta Detection of enzyme activit,y 16 Ass~ for the reductase 19 Ass~ for the hydrolase 19 Purification of the reductase 19 Properties of ooumarin oxidoreductase 29
Disoussion 40
Summary 42
Bibliograph;y 43
....-.., . @.... ,.. Aolalowledgements
The author expresses his sincere thanks to Dr. A. C. Blackwood for his help and his encouragement throughout the study.
Thanks are also due to the various other graduate students in the Department of Microbiology- notably Mr. Y. D. Ha.ng and Mr-. P.C. Chang for encouragement and rewarding discussions on research.
Financial assistance from the MCConnell Memorial Fellowship
Fun~ 18 gratefully acknowledged. lIlarobSolosr
ConnnioD of ~ 1io 118111otl0 .&oid li' BD. IaolaW tJoII JlaeudcllODU Rao 291
eDriohed l184ia. nclucea ooumann to d~l"OOO1JIIIlrlD ,. a Yeq epecUlo JaD&OÙ40ftdi1Otaae "Moh hae 1Hteft parified .8YeftteeDottcl~ ueing JlFAF-oellulo.. ohzoœatograpJv end caloium phosphate sel
at tte pH DtabiliV optlmlll of pB 6.0. !rh1a ~ :le iDh1bited 1V aulfh7d171 group :1Dh1bl tora 8Ild • cWo'dzoco1llU'1n. The 8D~ la stable at 4°0. for about a veetl it ftta1Da about 5~ of l'. ao'lY1V arter 25 lI11nutes at 5(JOO. D1b,rdl"oOOUlllllriD 101'1184 _ thie 8I12fID8 le
immediateq l'\Ydl'Oqzod by a ftq ao\1ve ..... dUJ.ydroco\lDBZ'1D
~drola8e. vhloh la J4"uent in oells g1'OWJ1 on ooumariD. Introduction
Coumarin (2-keto-1,2-benzopyran), the lactone of 2!!!.-g,,-h;rdroxy
cinnamic acid is synthesized by a number of plante includiDg commercialJ.y
important Meli1otus!UŒ.. The fact that it is one of the most potent naturally occurring germination inhibitors led to ear~ studies of its disappearance in the soil. Detailed etudies of its metabolism in animaIs were undertaken because it causes l1ver damage and dicumarol, a structurally related compound, is a potent anticoagulant. En~tic studies on its metabolism in a variety of biological systems ha8ecome\1 only recently.
This thesis demonstrates the initial attack on coumarin by Pseudomonas Mac 291 by the isolation and characterization of the enzymes involved. -2-
Literature Review
Biological transformations of coumarin
Higher plants 1;
Coumarin occurs naturally in a number of commercially important plants notably Meli1otus, Anthoxanthum and Lavendula, where its biosynhhesis and transformations have been studied int~ively as well as the genetic control of these processes.
The biosynthesis of coumarin in Melilotus and Hieroehloe odorata has been investigated b.Y Brown (1960, 1962a, 1963) and KOsuge (1959, 1961a, 1961b, 1964). In essence, eoumarin is derived from the shikimic acid pathwBiY via phen;ylalanine. An ammonia-lyase converts phenylalanine to ~-cinnamic acid which in turn is hydroxylated te ~-coumaric acid.
Free ~-coumaric acid i8 converted to o-coumaryl glycoside. A ~-~ isomerization occurs, a ~glucosidase removes the glucose moiety and the lactone forms. Coumarin content is controlled by a gene for the
2.-hydroxylation of ~-cinnamic aeid and a gene for the ~glucosidase of 2.-coumarin;yl glucoside. (Figure 1)
Brown (1962b, 1963) studied the formation of eoumarin and herniarin
(7-methoxycoumarin) in Lavendula officinalis. l3ased on C14-lebelling,
Brown proposed the route to herniarin to be analogous to the route to coumarin involving cinnamic acid, ~coumaric acid and .E,-methoxycinnamic acid. A ~-~ isomerization involving an 2.-glucoside oceurs just prior to lactone formation. -3-
Brown, Towers and Chen (1964) studied the biosynthesis of umbelliferone
(7-hydroxycoumarin) in Ilydra,pgea by C14-labelling and found the same pattern, i.e. cinnamic acid to ~coumaric acid to umbellic acid with a
~ to cis isomerization involving the ~glucoside of umbellic acid. Austin and Meyers (1965) studied umbelliferone biosynthesis in Ilydrangea and Lavendula and confirmed that ~glucosides were involved.
Kosuge and Conn (1962) have made extensive studies on the transformations of coumarin in Melilotus. They put forward a scheme whereby coumarin is reduced to dihydrocoumarin and then lvdrolyzed to melilotic acid (Figure II).
They isolated an enzyme, dihydrocoumarin hydrolase, from Melilotus ~
(white sweet clover) and Melilotus officinalis (yellow sweet clover).
This enzyme was present in small amounts in Hierochloe occidentalis
(sweet grass), Trifolium pratense (red clover), and Medicago sativa
(alfalfa). The en~e was purified 8O-fold by acetone precipitation, heat denaturation, ammonium sulfate fractionation and D~ceIIulose batch-wise adsorption and elution. A fluorometric ass~ was devised. One unit of enzymic activity was defined as the amount of enzyme which forma 1 pmole of meIiIotic acid in 1 min. under standard ass~ conditions.
An adaptation of the hydroxamate ass~ of Lip;)mann (1945) waa also used. Various substituted dihydrocoumarins and lactones were hydrolyzed by this enzyme including 7-hydroxydihydrocoumarin, homogentisic acid Iactone and
7-hydroxy-6-methoxydil\Ydrocoumarin. A pH optimum in the region of 7.8-8.2 was reported, but as nonenzymatic hydrolysia of dihydrocoumarin ia rapid abova pH 8.2, accurate determinations could not be made above that p~
No tests of activity below pH 7.0 were reported. reported for thia enzyme. A turnover number, if' the enzyme were asaigned a molecular weight of 100,000, was calculated to be 100,000 which would make it a very active en~e. -4-
Figure l Figure l
:Biosynthesia of coumarin in Meli10tus !:e.- (Kosuge, 1964)
.. IV~.. ~.;:.'.. :\ e
Figure
COOH OOH COOH H ----f 2 ---t ----1
phe nyl al anine trans· cinnamic o .. coumaric acid coumaryl acid glucoside 1
f ~ ~ '0 Vnu COOH f OH VnOglucose _, .. ~OOH CQumarm coumarinic acid coum arinyl glucosid e -6-
Kosuge and Conn (1962) also reported that the enzyme h;ydrogenatillg coumarin to d~drocoumarin ie stimulated by NADPH, but did IlOt purit'y the enzyme.
AnimaIs 1
Animal systems differ fundamentally from those of higher plante in that b,ydro~coumarine arise from coumarin itself. IVdroxylations followed by conjugatione with sulfate, glucuronic acid or glycine are common.
Zeitlen ll!l reported liver damage produced by coumarin in doge and rats in 1956. Detailed detoxification etudies were made shortly after.
Mead, Smith II ~ (1958a) etudied the detoxication of b,ydroxy coumarine in rabbits and found that }, 4, 5, 6 and 8-bydroxycoumarins are conjugated with glucuronic acid. The above compounds with the exception of 4-~droxycoumarin are also excreted as sulfate conjugates.
6-IVdroxycoumarin was also hydroxylated to aesculetin (6,7-dibydroxycoumarin).
Mead, Smith II ~ (1958b) further studied the metabolism of coumarin itself and Q,-coumaric acid in rabbits, ferrets, mice, rats and guinea pige. From a quantitative point of view, oruy 25% of the coumarin in~ested was recovered from the rabbit's urine, but no evidence of ring fission was obtained. The rabbit b,ydroxylated coumarin in the }, 7 and 8 positions and 5-hydroxycoumarin as weIl was isolated in the ferret, guinea pig and mouse. It was suggested that 1a2-dibydro-
1 .2-diols are precursors to hydroxycoumarins in vivo, which are dehydrated to give the hydroxycoumarin. Q,-Coumaric acid was conjugated directly to glucuronic acid at the phenolic hydroxyl group and to glycine at -7- the carbo:x:yl group. It wae also converted to 4-h;ydroxycoumarin and
7-h;ydroxycoumarin.
Booth .2i !!. (1958) studied the metabolism of coumarin in rats and rabbits. In rats fed coumarin oral~, splitting of the heterocyclic ring occurred to give a-coumaric acid, melilotic acid and a-h;ydroxyphezvlacetic acids in the urine. From rabbits, 3-h;ydro:xycoumarin and 7-h;ydroxycoumarin as well as a-h;ydroxyphenylacetic acid were isolated. When a-coumaric acid was fed to rats or rabbits, melilotic acid, 2.-coumaroylgl~cine, melilotoy19~cine, 4-h;ydroxycoumarin, o-h;ydroxyphenylh;ydracry lie acid and a-h;ydroxyphenylacetic acid were detected in the urine by paper chromatograph;y.
Kaighen and Williams (1961) conf'inned that 7 -h;ydroxycoumarin ia a significant metabolite of coumarin in the rabbit, b~t not the rat.
Creaven, Farke .2i al (1962) using a fluorometric ass~ presented evidence that a rabbit liver microsomal enzyme which converts coumarin to 7-h;ydro:x;ycoumarin is different from an enzyme which converts biphenyl to 4-h;ydroxybiphenyl. Age, sex and strain of rabbit were factors which inf'luenced 7-h;ydroxylation.
Kerekjarto (1966) UBing thin-lBiYer chromatograph;y on Kieselgel G studied the varioUB products formed by incubating varioUB coumarin derivatives with rat, dog and guinea pig liver microsomes to obtain an indication of the varioUB enzymes present. Evidence of mixed function oxygenas es , isomerases, d eh;ydras es , lactonases and oxidoreductases wae found. -8-
Fungi.
Coumarin ie degraded by several genera of fungi. Studies with fungi have been especial~ concerned with the formation of dicumaro1.
Be1lie (1958) reported vigoroUB growth of PenicilliUln ,ienseni and g. nigricans in a medium containing coumarin and sucrose; umbelliferone was detected as a product. Slow growth was obtained on 2,-coumaric acid;
4-~dro:xycoumarin was identified in the medium.
Shieh (1962) isolated severa1 strains of Fusarium solan! which could utilize coumarin and 2,-coumaric acid as sole carbon source.
On the basis of his data he proposed the sequence coumarin, dilvdrocoumarin, melilotic acid. As evidence Shieh showed that disappearance of coumarin by cell-free extracts was stimulated by NADH and NADPH. The pH optimum for the reduction of coumarin by cell free extracte was 6.5. Shieh purified diz\ydrocoumarin z\ydrolase two-fold by ammonium· sulfate fractionation. One unit of enzyme was defined as that amount of enzyme which forma 10 p.g of melilotic acid from d~dxocoumarin in 1.0 min.
The optimum pH for dih;ydrocoumarin h;ydrolase ws reported to be approximately 7.5. r- and ~ were reported to inhibit this en~e at 3 x 10-3M.
TheFusaria isolated by Shieh metabolized ~coumaric acid to
4-h;ydrox;y-coumarin. Dicumarol, the active agent in a hemorrhagic disease of cattle fed on spoiled clover silage, was formed nonenzymatica11y by exposing 4-~droxycoumarin to formaldeb;yde. This ie interesting in that it suggested that .Q.-coumaric acid and not coumarin i8 involved as a precursor to dicumarol.
Barran (1965) also using!. solan! was unable to obtain further en~atic degradation of meli10tic acid or .Q.-coumaric acid. -9-
:Bellis & !! (1967) reported the ability of various thermophilic fungi, Iiunicolor stellata, .!!- Lanuginosa and ~ pusillus, to grow on coumarin and 2.,-coumaric acid. Dioumarol, if produoed, alwa.ys was formed from 2.,-ooumaric aoid and not from ooumarin.
Baoteriaa
As early as 1917 Bobbins reported the disappearance of coumarin in the soil with a oorresponding increase in baeterial numbers. Audus and Quastel (1947) used cûu.marin ts property as a germination inhibitor to note its disappearance in the soil; seede grew after a five d~ lag. l'.o1'e recently Rivière and Chaussat (1966) studied the rhizosphere flora of Anthoxanthum odorata. According to their work, 16.2% of the organisme present there can destroy coumarin, whereas only 4.3% of the wheat rhizosphere organisms have this capacity. Organisms capable of destroying coumarin which they isolated included Arthrobacter pascens,
Xanthomonas, Eseudomona.s, No cardia, Streptomyces and the fungus, Penicillium. Intensive work has been done on coumarin metabolism by Levy using an Arthrobacter~. This etrain, isolated from the soil (1964a), utilizes coumarin as sole carbon source. Evidence of enzymic cleavage of the lactone rillg and conversion to 2.,-coumaric acid was obtained by incubat.ing coumarin-E? with extracts in the absence of NADE1 then chromatographieally separating the aromatics and counting the radioactivity of each product (1964b)
A NAD~oxidoreductase specifie for o-cournerie aeid was isolated (1964e) from cells grown on eoumarin and purified 20-fold by treatment with protemine sulfate, calcium phosphate gel adsorption, two ammonium sulfate fractionations followed by chromatograp~ on D~cellulose. This enzyme was sensitive to sulf~dr.Yl inhibitors e.g. ~chloromereuribGnzoate at -10-
,.3 x 10-4M gave 31% inhibition; it was not sensitive to KCN, NaN, or NaF at 3 x 10-3M. The oxidoreductase bad a ~ of 1.13 x 10-5M. The en~e was active over the pH range of 6.8 to 8.5.
Levy (1966) also isolated and purified 50-fold melilotate hydro~lase, which converts melilotic acid to 2,,-d1qydro~phenylpropionic acid and requiree
NADH, FAn and o~gen.
Coumarin metabolism has been studied in Pseudomonas~. Blackwood isolated 9 strains of Pseudomonas from spoiled sweet clover,which were capable of degrading coumarin in a complex medium. On the basis of chromatographie, manometric and radioactive-Iabelling evidenoe,
Halvorson (1961) postulated a scheme the same as Levy's except tbat ha gave an alternate pathway via salicylio acid to catechol. ~-Coumaric acid was postulated as an intermediate because,although it was not detected chromatographically, when it was used as substrate melilotic acid appeared in the fermentation Iiquor.
Chopra (1964) studied the initial attack on coumarin with cell free extracts. She obtained melilotic acid formation from coumarin in crude extracts and defined one unit of enzyme as the amount of enzyme required to produce 50 pg of melilotic acid from coumarin in one hour at 300 C.
She unsuccessfully attempted en~e purification by gel filtration, protamine sulfate precipitation and ammonium sulfate fractionation. -11-
Figure II Figure II
Conversion of coumarin to melilotic acid by biological systems. @ e
Figure Il
pl ant s, fun g i 1 Pseudomonus dihydrocoumarin ~ NAD/ H2 0
COOH '0 coumarin ~ H2 0 melilotic a cid
animais, /NADH Arthrobacter ~ COOH
1 -" fi.) 1 o cournerie acid -13-
Materiale and Methode
Organiem.:
FBeudomonas Mac 291 was ieolated by Blackwood fram spoiled sweet
clover bay.
Sources of Ohemicalsl
Coumarin wa.e obtained fram Fischer Scientific Company, M:mtreal.
Sigma Ohemical Co., St. louis, MJ. supplied NADH and NADPH, DEAE-cellulose,
Calcium Phosphate Gel, Protamine sulfate (Grade 1), Alumina 0 ~ and
~-~droxymercuribenzoate. K & K Laboratories, Jamaica, N.Y. were the
source of dihydrocoumarin and 4-hydroxycoumarin. ~-cinnamic acid and
2,-coumaric acid were fram Brickman and Company, Montreal. The various other
substrates tested i.e. 2,-methoxycinnamic acid, ~coumaric acid and 7-hydroxy-
coumarin were purchased fram J. T. :Baker Chemical Compal'.\Y, Phillipsburg, N. J.
8-lW'drcxycoumarin was a gift from llr. E.R. Blakely, Prairie Regional
Laboratories, Saskatoon, Sask. Melilotic acid was prepared by L. Barran
from dihydrocoumarin. Media were from Difco Laboratories, Detroit. Other
chemicals were obtained commercially.
Growth of cells 1
The organism was cultured on Balvorson's OIT (1961) medium,which was
composed as followsl 0.5% Yeast Extract, 0.5% Tryptone, 0.3% coumarin,
0.1% glucose and 10% (vol/vol) of a salt solution containing 1.3% N~HP04·12 ~6
0.9% K~P04' 1.0% KOI and 1.0% MgS04·7H20. The culture was maintained en a slant of OYT medium, without the glucose, and subcultured at monthly
intervals. For enzyme preparations the cells were grown in 10 liter -14- quantities on the liquid ClT medium in a New Brunswick Submerged
Fermenter, Model MF214, wi th aeration of one vol/vol/min and agitation or 400 r. p.m. Corning Antifoam B was automatioally fed in as required.
An inooulum of 0.25% was used. The oe11s were grown 17-18 hours and then harvested by oentrifugation at 10,000 g in a Serva11 refrigerated oentrifuge. In early studies cells were washed three times wi'1;h p1vsphate buffer at pH7.4 before freezing. Once fraotionation procedures were eEtablished, the oella were frozen in 10-15 gm (wet weight) quantities in sealed jars without prior washing and atored for usd at _200 C.
Chromatograph;y'.
Thin l~er chromatography was done on 200m2 glass plates ooated with Silica Gel G (E. Merok Co., Germany). The plates were activated 15 minutes at 1000 C. before use.
A solvent system containing 2-prc~anol, ammonia and water (811.1) was used to separate ooumarin from melilotic aoid and ~-coumario aoid.
For separating dïhydrocoumarin from melilotio acid and coumarin two- dimensional ohromatograph;y was used as followa 1 in direotion #1 the solvent system contained 2-propanol, water (8.1) and in direotion #2 the solvent system contained 2-propanol, water and ammonia (8.1.1).
Fluorescenoe under ultraviolet light under neutral ann a1kaline condi tions and the diazotized ~ni troaniline spr~ used by Halvorson
(1961) were sufficient to id ent ify the various oompounds tested. -15-
En~e ass~SI
(1) Coumarin NADH-oxidoreductase.
Once NADH was found to be the cofactor, an assE\}" was devised for this enzyme based on the loss of optical density of NADH at ,40 m"ji as i t is oxidized. For NADH, ~o = 6.20
(2) ~drocoumarin ~drolase.
This enzyme wa.s ass~ed by a modification of an assE\}" used by
~osuge and Conn (1962) for the plant enzyme. This is a colorimetric assE\}" based on the production of a ferric ion complex with the hydroxamic acid derivative of the substrate. This ass~ was a modification of work by Lipmann (1945), who described the reagents and procedure in detail.
Determination of Proteinl
Protein in the cell free extract was determined by the Biuret method using three times crystallized egg white lysosyme (Sigma) as standard. A new standard curve was run for each batch of reagent.
Other determinations were made by the spectrophotometric method described by DeMOss and Bard (1951).
Purification of coumarin NADB-oxidoreductase a
To de termine the reaction sequence a purification scheme for the oxidoreductase was worked out. The hydrolase was ass~ed along with the reductase at each step. DEAE-cellulose was prepared E}lld us~d as suggested by Peterson (1962). The various adsorbants (calcium phosphate gel and alumina 0 ~) were used as suggested by Colowick (1955). Gel filtration was performed according to the methods outlined by ?ha.rmacia Ltd. (1966).
AlI fractionation steps were carried out at 40 0. -16-
Results •.
AlI experiments were repeated at least twice.
Detection of Enzyme Activity and Eroduct Formation.
In the ear~ phases of this work en~e activity was detected by
chromatography before an assa;y method had been worked out. Coumarin,
enzyme and phosphate buffer, pH 1.4, were incubated together for 1 hour;
the mixture was extracted wi th ether and spotted on a chromatograph.
The enzyme was considered active if a spot for melilotic acid appeared.
To determine if ~-ooumario acid were present in the fermentation liquor, the following prooedure was used. a 10 liter batch of CYT was inoculated with FSeudomonas Mac 291. Two liter samples were taken at 0, 2.5 hours, 5 hours, 7.5 ho urs and 10.5 hours. Bach sample was saturated with NaCl and placed in a cold room. The last samples were centrifuged to remove bacteria. The samples were then acidified with HCI and extracted in a liquid-liquid extractor for a hours with diethyl ether. The extracts were taken to dr.yness, dissolved in alcoho1 and chroma tographed along wi th an ~-coumaric acid standard. No 2,-coumaric acid could be detected on the chromatographs.
To check that the en~e purified by the spectrophotometric assa;y actually did de grade coumarin, chromatography was employed. In these tests 1 ml of purified coumarin oxidoreductase (1.42 units), 1 ml of NADH
(2 prooles), 1 ml of coumarin (.1 umoles) and 1 ml of .05M phosphate buffer, pH 6.0, were incubated 10 minutes, then acidiried with 2 ml of 0.2 N Hel and extracted with ether; the ether extract was taken to dryness and dissolved in alcohol, then spotted on a plate along with standards of coumarin, dihydrocoumarin and melilotic acid. A two-dimensional -17-
Figure III Figure III
Two-dimensional chromatograph of coumarin incubated with purified en~.
Direction 1. 2-propanol/water, 8:1
Direction 2. 2-propanol!water/ammonia, 8.1.1
Spr~ 1 diazotized ~nitroaniline oversprB3'ed with 1N NaOH m.a. - melilotic acid; c - coumarinJ d- dihydrocoumarin r - reddieh; y - yellow; 0 - orange -Hl-
Figure III
1 1 1 1 6l2\• standards m.a. d c ,~ · ·1 1 ~. •••• _ ••• ___ ...... ----.... --- ....'t ---_. -... -_ . •• - ...... -_ •• --
Bi1 . 8 c,d
1• 1
m·o.8
,1 1 1
, ,1 !
direction 2 direction 1 sample standards -19-
chromatograph was run (Figure III).
Development of an Assay for Coumarin NADH-oxidoreducta.se a
One unit of enzyme is defined as the amount required to oxidize o one JlIIlole of NADH in 1 min at 27 0 at pH 6.0. The 0.5 cm ouvettes for the Zeiss R-f4QII spectrophotometer contained 0.1 ml of enzyme, 0.1 ml of coumarin solution (100 pg), 0.95 ml of 0.05M P04' pH 6.0, and 0.05 ml of NADH (0.25 pmoles). A referenee cuvette contained the above with
1.00 ml of P04 and no NADH. In the erude extract the reading had to be corrected for a NADBase present. Figure IV shows that loss of O.D. at
340 mp. i8 linear with time.
DiQydroeo~in hydrolase aetivit,ya:
C11e unit of enZ\Y1lle is defined as the hydrolysis of 1 p.unole of d~droeoumarin in 1 min at 27 0 0 at pH 5.4. This pH was used for con- venience sinee subsequent reaetions were earried out at the same pH.
A standard curve was plot'Ged with eaeh determination. Various dilutions of enzyme were incubated wi th 5 f.lID.oles of dihydrocoumarin and the substrate remaining wae determined. Figure V shows a typical standard curve for dihydrocoumarin.
Purification of coumarin NADH-oxidoreductaeea
Unless otherwise stated, the te:rm "purified en2'\Yllle" refers to material treated by steps (a) through (c), that is sixteen times purified. Figures VI and VII summarize the purification data on coumarin oxidoreductase and the behaviour of dihydrocoumarin hydrolase during this purification procedure. -20-
Figure IV Figure IV
Spectrophotometric ass~ for coumarin NAD~oxidoreductase showing the linear relationship when 108s of O.D. at 340 ~ is plotted against time. -21-
Fi 9 u re 1V
600
500
400
Q) 0' c: 0 300 .&; u
C 2 00 0
100
2 3 4 5 time (min') -22-
Fisure V e
Figure V
Colorimetrie Ass~ for dihydrocoumarin hydrolase
Standard curve 1 O. D. at 540 IIlJl plotted against pmoles of dihydrocoumarin. -23-
Figure V
.700
.600
.500
.400
o '0.300
.200
.100
dihydroGoumarin, IJ mole s ® -
Figure VI Purification of coumarin NADH-oxidoreductase
Step Unite/ml Total unite %Recover,y Protein Specifie Activity Purification (mg/ml) ( unite/mg protein)
Cell free 2.12 110.03 100 26.0 0.082' 1~0, extraat
DEAE-celluloee 0.58 40.88 19 0.83) 0.700 8.5
Calcium 1.52) 4.59 4 1.11 1.37 16.8 phoephate gel ® -
Figure VII
Activit,y of dihydrocoumarin ~drolaBe during purification of ooumarin NADH-oxidoreductase
Coumarin lUffiH-oxidoreductase ~droooumarin ~drolase Step unite/ml Speoific Activity Purifioation Unite/ml Speoifie Aetivity Purifioation
Cell free 2.12 0.082 1.0 10.5 0.4 1.0 extraot
DEAE-oellulose 0.58 0.700 8.5 0.96 1.2! ~.O.
Calcium 1.53 1.37 16.8 3.0 2.7 6.7 phosphate gel -26-
(a) Cell free extraots- 10-15 gma of frozen oells were ruptured as a thiok suspension by ultrasonioation (MD Ultrasonio Os 0 illator ) in 25 ml lots in 0.02M aoetate buffer, pH 5.4. The preparations were then oentrifuged for 10 minutes at 10,000 g. to remove the majorit,y of unbroken cells, and then at 55,000 g.for 30 minutes to remove wall debris.
(b) D~cellulose fractionation- ce Il free extract equivalent to 1350 mg of proteinwas diluted to 200 ml with 0.02M acetate,pH 5~4, and applied to a 25-45 oolumn (Pharmacia) of JlEA&.oellulosEl (0.84 meq/g), medium mesh, equilibrated with 0.02M acetate buffer, pH 5.4. After application of the sample the column was washed with acetate buffer and then eluted by a gradient system created by feeding 0.5M NaCl into a mixing ohamber con- taining 500 ml of the aoetate butfer. Fractions of 250 dropa were oollected with an LKB Fraction Collector, T.ype 1000. When necessar,y the active fractions were pooled and rechromatographed on a 15-30 oolumn (Pharmacia) after 1 hour dialysis against 0.02M acetate butfer, pH 5.4, using the elution gradient system. In this latter case 100 drop fractions were collected. Figure VIII shows the protein and enzyme elution pattern with the 25-45 column.
(0) Calcium phosphate gel adsorption- The gel sOlids/protein ratio required to adsorb ooumarin NAD~oxidoreductase is 2 at pH 5.5 as determined by titration. Adjustment of pH was made with 0.2M acetic aoid. E[ution is obtained using 0.02M P0 buffer, pH 1.0. The use of a small hand 4 homogenizer (A. Ho Thomas Company, Philadelphia, Penn.) wes found oonvenient for both eff:iderit: adsorption and elut:f.on. Figure VIII -27-
Figura VIII
Chromatograp~ of cell free extract on D~cellulose.
Upper curve- absorption at 280 ~ as a parameter of protein concentration.
Lower curve- activity of coumarin NAD~oxidoreductase. o w
'- ID ..0 - E > ::s c
CV '- c: ::s 0 0\ -() LL. c -'-
o C\I -29-
Misoellaneous purii'ioation methode investigated during this study 1
(a) Mo1ecular sieving- up to Sephadex G200 coumarin NADK-oxidoreductase comes out with the void volume. With G200, co1umn chromatograp~ is possible, but was not used routine1y due to massive 10ss of e~ activity.
(b) Protamine sulfate precipitation- this method ~ be used to remove nuc1eio acide, but due to considerable denaturation of en~e and variable results, it was IlOt used routinely. DEAE-cellu1ose performs the same funotion.
(c) Ammonium sulfate precipitation- this method was used occasionally, but a1w~s gave considerable 10ss of activity.
(d) Alumina C ~- the gel sOlids/protein ratio for adsorption of coumarin NADB-oxidoreductase was determined to be 4/5 at pH 5.5. E1ution ~ be carried out by 0.05M P04, pH 1.0. This method ~ be combined with the routine purii'ication scheme to get higher degrees of purii'ication than tbAt ttl;led routinely.
Properties of coumarin NADK-oxidoreductasel
1~ Cofactor specificity
Ea.rly in this study the enzyme was incubated wi th equimo1ar amounts of NADH and NADPH in the presence of substrate. NADPH was found to be 10% as act ive as NADH wi th the enzyme.
2. Substrate spec ificit Y
Purified enzyme was incubated with various ~droxy1ated coumarine and cinnamic acid derivatives. The cuvette contained 0.03 unite of -30-
en~e (0.1 ml), 1 pmole of substrate (0.2 ml), 0.25 pmoles of NADH (0.05 ml), and 42.5 pmoles of phosphate, pH 6.0 (0.85 ml). The reference ouvette contained the same constituents except the NADH was absent and an
additional 2.5 pmoles of phosphate (0.05 ml) were added. Figure IX sums up the results.
,. Sensitivit-,f ta inhibitors
Purified en~e was incubated with various inhibitors. The test
cuvette contained 0.06 units of en~e (O.'i ml), 0.7 f.lIlI.Oles of coumarin (0.1 ml), 0.25 pmoles of NADH (0.05 ml), 32.5 pmoles of phosphate at pH 6.0 (0.65 ml) and 0.1 ml of the inhibitor to be tested. The reference cuvette contained the same constituents except that an additional 2.5 pmoles of phosphate were substituted for the NADH. The par cent inhibition was calculated as followsa Units without inhibitor - units with inhibitor x 100 = %inhibition units without inhibitor
Figure X summarizes the sensitivity of coumarin NAD~oxidoreductase to various inhibitors.
4 • Inhibition by di~drocoumarin
Purified enzyme was incubated wi th dibydrocoumarin. The same general procedure was used as in the study of inhibi tors only this time
the cuvette contained 0.07 units of en~e (0.1 ml), 0.7 pmoles of
coumarin (0.1 ml), 0.25 pmoles of NADH (0.05 ml), 1.0 pmole of di~dro coumarin (0.2 ml) and 37.5 JllIloles of phosphate butfer, pH 6.0. The reference cuvette contained the same constituents except that 2.5 umoles of phosphate ,-lere substituted for the NADH. A control was included , -31-
Figure IX
Specificity of coumarin NAD~oxidoreductaae
Compound %Activity ooumarin 100
Q..-coumaric acid 0
:e,-coumaric acid 0 cinnamic acid 0
Q..-methoxycinnamic acid 0
B-hydroxycoumarin 0
7-~droxycoumarin 0
4-~droJÇY'coumarin 0 -32-
Figure X
Effect of inhibitors on Coumarin NAD~oxidoreductase
Inhibitor conc. (M) %Inhibition
Na~ 1 x 10-3 0 Na.F 1 x 10-3 0 eu"'" 1 x 10-3 100 eu# 1 x 10-4 63
PEMB* 1 x 10-4 100
PllMB* 5 x 10-5 89 Dilvdrocoumarin 8.3 x 10-5 47
* ~lvdroxymercuribenzoate with the same ingredients except that 0.2 ml of 9.5% ethanol were added instead of the dihydrocoumarin as the dihydrocoumarin contained 9.5%
ethanol to aid in its dissolution in water. Calculation of the
percentage inhibition was made calculating this control as equal to 100%
activity. The results are summarized at the bottom of Table X.
5. Sensitivit,y to heat
Tubes containing 4.1 units of e~e (5.0 ml) and 5.0 ml of 0.05M phosphate buffer, pH 6.0, were incubated in a water bath at 500 0. for
5, 10, 15, 20 and 25 minutes. Tubes containing 4.1 units of en~e (5.0 ml), and 5.0 ml of 0.05 M phosphate buffer, pH 6.0,containing 0.2% coumarin were incubated in a water bath at 500 0. for the same periods. Incubation
was terminated by placing the tubes in an icebath. The tubes were clarified
by centrifugation when necessary and then ass~ed in the usual manner. Figure XI shows the results of this experiment.
6. pH for optimum stability
OeIl free extract prepared in Tris-citrate butfer (0.05M citrate
neutralized with 0.1M Tris to pH 7.0) which had been treated with
protamine sulfate neutralized to pH 8.0 and had been centrifuged leaving
an extract with a 260 mu/280 mu absorbance ratio of approximately 1.0 was used in this experiment. To obtain the pH àesired 0.100 N HOI or NaOH was added to 5.0 ml of enzyme with rapid mixing using a pH meter. The amounts required of alkali or acid varied from 2.40 ml of 0.100 NaOH for pH 9.2 to no addition for pH 6.9. The extracts were then placed in centrifuge tubes in a water bath at 500 0. for 10 minutes, plunged into an icebath, neutralized by adding the complimentar,y amount of acid or base, -34-
Figure XI Figure XI
Beat sensitivity of coumarin NAD~oxidoreductase
Per cent activity remaining after treatment plotted against time.
triangles- no substrate added during treatment. circles- substrate added during treatment. -}5-
Figure XI
100
~ ':;- -(J 50 0
0~
5 10 15 20 25 ti m e (m in') -36-
Figure XII Figure XII
Effect of pH on coumarin NAD~oxidoreductase. circles- pH plotted against en~e activity. triangles- pH plotted against en~e stabilit,y. -37-
Fi 0 ure X Il
>.. .- ->
(,J -o
Q) >
3.0 5.0 7.0 9·0 pH -38.
made up to 10 ml, centrifuged and the supernatants ass~ed at pH 6.0.
Figure XII summarizes the data obtained by this method.
7. pH for optimum activity
Determination of a pH activi ty curve was attempted severaI times with unpurified en~e, but alw~s ran into difficulties due to turbidity in the lower pH ranges produced by protein precipitation.
With purified enzyme a curve with a distinct peak at pH 4.5 was obtained using the Tris-citrate buffer mentionned in #6. pH 4.5 was not used routinely for the reasons which follow. According to the Sigma Information
Bulletin, NADH is not stable at acid pR'S and can be chemically degraded with corresponding 10S6 of O.D. at 340 mp. The turbidity which developed in unpurified preparations made it unsuitable to use while working out a purification sbheme. Further, the enzyme was not stable at this pH. pH 6.0 was used routinely sinee at this pH the enzyme i6 most stable and becauae at thia pH aU of the coumarin is in the lactone forme
Figure XII ShOvlS the pH for optimum enzyme activity.
8. ReversaI of the reaction
Incubation of the purified enzyme, NAD and dih;ydrocoumarin in a number of trials failed to show any reversaI of the reaction.
9. Enzyme stability
Enzyme in frozen cells at _200 C retained over 75% of its activity during 1 month of storage. Unfractionated cell free extract deteriorated -39- quiokly (2 or 3 d~s) if stored unfrozen at refrigerator temperatures (4°0).
Purified enzyme after storage for 10 d~s at 4°0 retained 40° of its aotivity. -40- e Discussion
This work was undertaken to determine the mechanism of the
initial attack on coumarin by 1Seudomonas Mac 291. In other biological systems two meohanisms bad been shown, i.e. via 2,-ooumaric acid
(Arthrobaoter, a.n:imals) or via dihydroooumarin (fungi, plants). A complioation was the instability of the various compounds involved. For examp1e, coumarin in solutions above pH 6.8 exists as a mixture of the laotone and ooumarinic acid (Har 10, 1950); dihydroooumarin decompoaes within a few hours in solutions above pH 7.0. Since neither 2,-coumario acid nor dihydrocoumarin could be detected in the fermentation liquor, work was undertaken to purify the reductase.
Purified 2,-eoumaric NAD~oxidoreductase from Arthrobacter ~. would not
act on coumarin; whereas ooumarin NAD~oxidoreductase from Peeudomonas ~.
ia inactive on 2,-coumaric aoid. Purification of ooumarin NAD~oxidoreductase
was also pursued sinee charaeterization of this e~e bad not been aehieved from either the fungal or plant systems, where i t is c1aimed
to oceur. (KOsuge, 1962; Shieh, 1962). The evidence tbat 1Seudomonas Mac 291 degrades coumarin via dihydrocoumarin can be summarized as follows: Firstly, 2,-ooumaric acid does not appear as an intermediate in the fermentation liquor, nor
is it produced by cell free extracts. Secondly, a specifie en~e,
an oxidoreductase acting on coumarin, bas been isolated and oharaoterized. And finally a very active hydrolase acting on dihydroeoumarin and associated with the oxidoreductase during purifioation bas èeen -41-
demonstrated to be present in cells grown on coumarin.
In the plant system d~rocoumarin l:wdrolase is very active and
it ~as assumed by Kosuge that the reduction step was rate limiting.
In Pseudomonas!œ. the same situation is true--even when ass~ed at pH 5.4, which is far from the optimum for the plant or funga.l enzyme, the lvdrolase in the cell free extract of Pseudomonas !œ. was five times more active on a molar basis of substrate than the reductase.
Di~drocoumarin does not accumulate in the medium; melilotic acid is alwaya reoovered from chromatograms of the reaction carried out by cell free extracts or purified enz.yme. Attempts to remove the ~drolase, for example with Sephadex G200, resulted in drastically reduced activity.
The fact that d~drocoumarin inhibits the reductase is probably the reason for this. In properties, the reductase appears to be very negatively charged. This is seen from its elution pattern vith DEAE-cellulose. This probably explains its low pH activity optimum since much of the ionization would be suppressed at pH 4.5. Since coumarin is IlOt charged an alternative explanation might be SOir.::! sort of dissociation of the enzyme into fragments; the enzyme is not stable at its pH optimum. The reductase i8 not exceptionally heat stable nor i8 it stabilized by the presence of substrate. At refrigerated temperatures it maintains its activity for up to a week. The enzyme appears to be very sensitive to S~group inhibitors and dilvdrocoumarin. The enz.yme is very specifie for coumarin. -42-
Summary
This thesis establishes the mechanism of the initial attack on coumarin by Feeudomonas Mac 291, which is reduction of coumarin
to diqydrocoumarin followed by ~dro~sis to melilotic acid. An ass~ for coumarin NADR-oxidoreductase is outlined as well as a partial purification scheme for this en~e. Some of the properties of the coumarin NADR-oxidoreductase are discu6sed. The behaviour of the very active enzyme, dih;ydrocoumarin h;yd.:rolase, i6 noted during the purification of the reductase.
Résumé
ISeudoIDOUas Mac 291 détruit la coumarine dans les milieux enrichis. Dans cette thèse un enz,yme specifique, la coumarine
NADH-oxidoreductase, qui oatalyse la reduction de la coumarine en dilwdrocoumarine a été purifiée seize fois et oharacterisée. la dih;ydroooumarine est lwdrolysée par une laotonase très active. -43-
Bibliography
Audus, L.J. and. Qu.a.stel, J.B. 1947 Coumarin as a selective pqytocidal agent. Nature lli' ;20 Austin, D.J. and Meyers, M.B. 1965 Studiee on Glucoside Intermediates in umbelliferone Biosynthes ie • P.b;y'tochem. i .255 Barran, L.R. 1965 The Metaboliam of Coumarin and ~-Coumario acid b.Y Fusarium solam. 11. Sc. ~.!!!!!. eubmitted to the Dept. of Agricultural Bacteriology, McGill Unive.o:-aity
Bellie, D.M. 1958 Metabolism of coumarin and related compounds in cultures of Penicillium !U!.. ~Tature ~ .806
Bellis, D.M. 1967 The Biosyntheeis of Dicumarol Biochem. J.1.Ql1202
Booth, A. N., Masri, Me S., Ra bbine, D. J., Enerson, O. B., Jones, F. T. , and DeEds, F. 1958 Urinary Metabolites of Coumarin and o-Coumaric Acid. J.Biol.Chem,~'946 -
Brown, S.A., Towers, G. IL N., and Wright, D. 1960 Biosynthesie of the Coumarine 1 Tracer etudies on coumarin formation in Hierochloe odorata and Melilotus officinalis. Can.J.Biochem.P.qysiol.~.143
Brown, S.A. 1962a Biosynthesis of the coumarine III. The role of glycosides in the fonnation of coumarin by Hierochloe odorata. Can.J.Biochem.pqysiol.~1607
Brown, S.A. 1962b Biosynthesis of coumarin and herniarin in lavender. Science ill'977 Brown, S.A. 196;a Recent studies on the formation of natural coumarine. Llyodia 26.211 -44-
:Brown, S.A. 1963b Biosynthesie of the coumarine. The formation of coumarin and herniarin in lavender P.h;y'tochem. g,.137
:Brown, S.A., Towers, G. H. N. and Chen, D. 1964 Bioeyntheeis of the coumarine V. Pa.thw~s of umbelliferone formation in ~d~anse8 macrophylla. ~ochem.l'4 9
Cho pra, N. 1964 Pathway of ooumarin metabolism by enzyme extracts of FBeudomonas melilotica. M. Sc. Thesis submitted to the Dept. of Agricultural ~crobioloBY, MaGill Uhiversity
Colowick, S. P. 1955 Separation of Proteine by Use of Adsorbends. Methods in EnZ\Y'Illology 1'90, S.P. Colowick and N.D. Kaplan, eds.
Creaven, J. P., Parke, D. V. and Williams, Il. T.. 1962 Aromatic ~droxylation by liver microsomes Biochem. J ~'5p
DeMass, R.D., and Bard, R.C. 1957 Physiological and Biochemical Technics. Manual 2f. Microbiological Methods .169, Soc. of American Bacteriologists, ed. MCGraw-Hill Book Co., Toronto
Halvorson, H. 1962 The Metabolism of Coumarin by a Pseudomonas ~. ~. ~. Thesis submitted to the Dept. of Agricultural Microbiology, MoGill Uhiversity
Kaighen, M., and Williams, P..T. 1961 Metabolism of coumarin-3-C14. cl med. pharm. chem.l.25
Kerekjarto, B.V. 1966 Zur en~tischen ~droxylierung von Oumarin l 2'. ~siol. Chem. ~.264
Kosuge, T. and Conn, E.E. 1959 The metabolism of aromatic compounds in higher plants • .1 Biol. Cham. ~.2133
Kosuge, T., and Conn, E. E. 1961a The metabolism of aromatic compounds in higher plants iII. The ~glucosides of ~-coumaric, coumarinic and melilotic acids .I Biol .cheny' 236 11617 -45-
Kosuge, T. 1961b Studies on the Identity of Bound Coumarin in sweet clover. Arch.~ioohem.~iop~s.~1211
KosUe,""3 , T. and Conn, E.E. 1962 The Metabo!ism of Aromatic Compounds in Higher Plants V. Purification and properties of dihydrocoumarin ~drolase of Melilotus alba. J.~iol.Chem.237.165;
Kosuge, T. 1964 &bssible Routes of Biosynthesis of Phenolics in Diseased Tissue of Higher Plants. ~.~.~ ~ Phenolics Group 2f. North America V.C.Runeckles,ec
Levy, C.C. and Weinstein, GeD. 1964a Metabolism of Coumarin by a Microorganism Nature 292'596
Levy, C.C. 1964b Metabolism of Coumarin by a Microorganism. 2,-coumar1c acid as an intermediate between coumarin and me11lot1c acid. Nature 204.1059
Levy, C. c. and Weinstein, G. D. 1964c The Metabo !ism of Coumarin by a Microorganism II. The reduction of 2,-coumaric acid to melilotic acid. Biochem. 2,.1944
Levy, C.C. and Frost, P. 1966 The Metabolism of coumarin by a microorganism v.; Melilotate hydro:xylase. J.~iol.Chem.~1997
Lipmann, F. and Tuttle, L. C. 1945 A Specifie Mieromethod for the Determination of Acyl Phosphates. J.~iol.Chem·122,21
Mead, J.A.R., Smith, J.N. and Williams, R.T. 1958a Studies on Detoxication. The metabolism of bydroxycoumarins. ~iochem. J.68.61
Mead, J.A.R., Williams, R.T. 1958b Studies in Detoxication. The metabolism of coumarin and o-coumar1e acid. Biochem.J.68.67 Paterson, E.A. and Sober, !I.A. 1962 Column Chromatography of Proteine. substituted celluloses. ~thods in Enzymology- l';, S.P. Colowick and N.O. Kaplan, eds. -46-
Phaxmacia Ltd. 1966 Sephadex- ~ filtration ~ theory !!!! practice 110 Place Cremazie, Suite 412, Montreal 11, Canada
Riviere, J. and Chaussat, R. 1966 Destruction de la coumarine dans la Rhizosphere. Ann.lnat.Fasteur, Suppl.la155
Rabbins, W.J. 1917 Disappearance of oumarin, vanillin, pyridine and quinoline in the soil. Alabama Agr.exp.Stat.Bull.19bi65
Shieh,H. 1962 The Metabolism of Coumarin and ,2,-Coumaric acid by Soil Fungi • .Eh. ]. Thesis submitted to the Dept. of Agricultural Bacteriology, McGi1l University
Sigma Chemical Compaqf 1966 Beta-Diphosphop,yridine Nucleotide, reduced form ~rmation sheet, 3500 DeKalb St. St.Louis MO. 63118
';+1""... Ze ... v.a.-...~, B. R., Thiessen, R. and Murer, H.K. 1956 Toxicity of coumarin J.P.harmacol.EOCp.Therap.118a348