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.