Document EqeRdgd3R6vZ555MYM3Ymrm1R

i PROCEEDINGS OF THE BIOCHEMICAL SOCJETY formation of 3,5-dihydroxyphouylrvcotio ncid, tho fonnrr was ittoubntod tinder similar conditions with mtratmnl micro-organisms, tvlion tho formation of S.fl.dihydroxyphmylncctio ncid woa shown. Ad* mmiatmtion of 3,4,0-tfihydroxyphcnyJacrtio ncid incubated for 48h in a medium containing n.AinxMCPA. Examination of tiwmo axtrnou showed tho prosonceofall threeproviounly otmorvcU metabolites. Repeated t.I.o. of uthor-solublo fractions lod to the iaolation of products MX j and MX3 oh crystalline phenyl-* ami. pvniMU'iMw i concern; it m U<-ire<J pr to rot* roAtiUod in tho excretion of 3,0-dihydroxy- compounds. Thnirki* phmylncctio acid. Both subatances aro non-phonolic acids with u.v. nu|i]M,rting Ik To determine whether myricotin breakdown is spectra identical will) that of MCI'A. Compound Dm fire, J. I, * , dependent on the activity of tho intestinal micro MX, lias an i .r. spectrum characteristic ofa hydroxy, l flora in vim, the antibiotic neomycin <100mg) was latcd compound. It won shown to bo 4rchloro-S- fed daily to rats before and during tho period of liydroxymethylphcnoxyacotio acid, after syntliesis royricrlin Administration, when the formation of of this compound. Compound MX, hns on i.r. . Cinnamic / 3,8-dihydroxy phonylacotio acid was suppressed. spootrum characteristic of an umido acid. Acid j cursoro of W 1 Incubation of tho glycoside myricitrm (myricotin and alknlin hydrolyum yielded MCPA snd 'l Algu and 9 I 3-rhnmnosido) with intostinal micro-organisms gave rise to freo myricotin, 3,4,6-trihydroxyphonylscetio acid and 3,6-dihydroxyphonylacetio acid, indicating that rat intestinal micro-organisms are capable of efloeting tho cleavage of the glyoosidio a ninhydrin-positivo ether-insolublo component. Though still impure, tho chomical, chromatographic and spoctrosoopio properties of compound MX* are similar to thoeo of synthetic AT.(4-chloro-2-metl))iphenoxyaootyl)-L-aspartic acid. 1 ByD.U.Timl (Pej">rtmeH* liiochemietry, yetwy/h, U.K. 1 linkage as wall ns the hetorooyelio ring system of Tlie othor-insoluble metabolite is also acidic snd Previously myricitrin. unstablo to hydrolysis. Its purificstion ami of p-hydmxy chomical characterisation ore currently under This work was supported by s grsnt from tbs Agricul tural Research Council. investigation. higher plants algan Engitm D*. N. P. k Griffiths, L. A. (IMS). BioeMem.J. 110, 440. Griffiths, L. A. (1004). M, 173. Nakngawa, Y., 8hetlnr, M. It. k Wendsr, 8. H. (1MB). Oiockim. tiojAy> Acta, 47, 13*. D.J.C. is indebted to Ranks Hovlt McDougall fnr financial support. Tiiia work was in part supported bjr grants from the Agricultural Research Council aaii Imperial Chemical Industries Ltd. moulds Agars leeanu*, amt (Whiatanco, Thrclfnll ft\V 1070). UV r 'I ubiquinone fc Tho Metabolic Fate of 4-Chloro-2-methylphenoxyacetlc Acid In Paae The Microbial Metabolism of Biphenyl By D. Lunt and W. C. Evans. (Department <>j Biochemistry and Soil Science, Univereity College j and hnkrr's y acid, compoii benzoic new! i fungi (Youhid t By D. J. CoLUits and J. K. Gaunt. (Department oj North Wales, Bangor, Caerne., U.K.) Mnizc shod Biochetnielry attd Soil Science, University College oj North Wale*, Bangor, Caomo., U.K.) , Pure culturos of Gram-negative bactoria were isolated from tropical and temporate soils capable baker's-yrnst leenn inyeel lsh (mime) < Thia report deneribos preliminary results from ai of utilizing biphenyl os sole carbon sourco in nn 1>. blakeelee* investigation into tho metabolism of tho solootivi otherwise mineral salts medium. 2,3-Dihydmxv- tiona (U hiata herbicide, MCPA,* in plants. biphenyl was dotoctod and isolated from the culture; Tbrelfull * W t Radioaetivo MCPA, ''Cl- or oar6oxy-14C-laboilod was applied to tho loavos of 2J-day^>)d pea plants (Pisum sativum var. Progress no. 0). After 24h tho plants were extracted with boiling ethanol. Ex- this was cleaved by a partioulate fraction from biphenyl-grown cells to give a-hydroxy-^-plimjlraueonio semialdehyde ((m.p. 122*C, AMI, 350nm (acid) and 430nm (alkali)j. A solublo edi-fnv lU-,4C)ciniumi p-(U-*4C]ams pared by inet L-JTJ-"C)tyrea troots wore concentrated and fractionated by extract converted this initial ring-fission product ammonia-iyaai partition into ether. Fractions were examined by into phonylpyruvate through some, ss yet, un (Ogata, Uchiy t.I.o. for labelled metabolites. Two of the break identified intermediates. Tho subsequent metabolic At the end a * down products (MX| and MXj) Were found to be pathways of phonylpyruvate are known. Uu- were isolated t i ether-soluble, whereas a third was not. In each substituted biphenyl is therefore hydroxylated in ease both the chlorine atom and the carboxyl the 2,3-positions, to be followed by a `msta'-typv radioactivity ( 1068;ThreUbl) oarbon remained on the molecule. oloavage between C-3 and C-4 of the aromatic ring; In all ease*) This application teohniquo did not permit the this is to bo contrasted with the fate ofnaphthnlriH*. ubiquinone, t) uso of onough horbicido to isolate sufficient of any where a `mefa'-typo fission ocoura between tho olm-rvcd in the metabolite for its ohaimoal identification. Thui angular carbon atom and C-l of 1,2-dihydrnny- (percentage of incubation experiments with exoisod plant tissui naphthalene (Daviea ft Evans, 1004). Several plant varied from k* were set up. Light-grown pea shoot sections wan * Abbreviation! MCPA, 4-eUoro-t-msthylphenoxy Mtie arid. aromatio polymers oontain the biphenyl type bond (e.g. the biflavanoids). Multioomponent mixtures of ohlorinated bi yeast; that Ac from o.ll in bs specific radioaol MONS 084138 II PROCEEDINGS OF THE BIOCHEMICAL SOCIETY r,flr ling O.&mM* , liiowni the metabolite*. *s Uni in thn phenyl* are tinod in the plastics industry, And their prnHiMrnco in tho environment is giving enuso for concern; it may ho poMibio to select those with tho [ desired properties and still bo biodegradabJo. labelled from (U-,4C]cinnamic acid wore 1800,1228, 203 and 12 for maiy.o, gracilis, P, blakeslrwnus and baker's yeast respectively; those of tho samples labelled from p-(U-,4C)cmimaric acid woro 4460, S r,VNUillinn itlt* with u.v. CoiM|KKMIll . Thanks nro due to the Sricnce IW*cnroll Council for lupporting D.L, ] Davies, J. I. Jt Kvnns, W. C. (1964). tfiocAcm. J. 91, 251. 6310, 2030 and 800 respoctivdy. Ozonolytia degradations and comparison of thn apocific radio activities of tho ubiquinone HiwipleN with those of tho t-dcmcthylstorols established that groator than ifaliydroxy- ; 00% of tho radioaetivity in thn vibiquinono rnolo- 4-vhlnro-2> culcs was in tlw p-bcv/.>quinoTo ring. ;<*r synthesis ha* an l.r, Arid. Arid HCPA tl Cinnamic Acid and p-Coumarlc Add, Pre . : cursors of Ubiquinone in Higher Plante. Green Algae and Fungi Tho rrwilU ahow that in nil organism* examined tho p-Uydroxybcnr.oio acid utilixed for ubiquinone biosynthesis can bo formed from p-oouinanc acid and, witlt the possibie oxoeption of yeast, cinnamic rmnptMiiiii. matograidiw' ByD.R. Tiirblfaix,Ah Law end G. R.Whistawc*. acid. However, they do not rule out the possibility - (Department of Biochemistry and Agricultural that some of this p-hydroxybenzoic aoid is also mil JJX> w m-3-meth>l< BiocJtemietry, University CoUege 0/ Wales, Aber formed directly from chorismio aoid. ytfwyth, U.K.) This work wss supported in pert by tho Seienco Ressireh to neidle mid bntioa ami mtly wmlrr lrJ>ow*U supported l*y Council mmI . Previously wo hove reported on tho incorporation of p-hydroxybonzoks acid into ubiquinone by tha higher plants meiso and French boon, the green ilgno Euglena gracilis and OcAromonos danieo, the moulds Agaricus 00mpsetris and Phycomyees blokes /Means, and the yoest Saocharomyecs cerixnsia* (Whistanos, Throlfoll A Goodwin, 1067; Spillor, Threlfall A Whistanoe, 1068; Threlfall A Whistance, 1170). We now report on tho incorporation into ubiquinone by inaizo, E. gracilis, P. blaktsieeanue Council. A. L. wss in receipt offinancial support from tbs British Council. We thank Mias Marian B. Williams far vslusble technical assistance. Ogata, X., Ueliiyama, K., Ysmads, H. k Techikura, T. (1007). J. agric. biol. Chem, 31, 000. Spilter, G. H., Threlfall, D. R. k WhisUnco, G. R.<l68t). Arche Bioehtm, BiopAye. 125, 780. Threlfall, D. R. k Whlstanoo, G. R. (1970). Phytochsmietry (in*the Tress). Whistonee, G. R.,ThreffolJ, D. R. k Goodwin, T. W. (1967). Biothtm.J. 105, lifi. ind bakor's yoest of ainnamio aoid and p-oouraorto Yoshida. 8. (1980). A. Bee. PI. Phytiol. 29, 41. uiiyl cpurtuiCMl "f !ty College <! Mid, compounds known to give rise to p-hydroxyhmsoio acid in higher plants (Zenk, 1600) and some fungi (Yoshide, 1000). Maize shoots (200), E. gracilis oells (20g wet wt.), Zenk, M- H. (1988). In Bioeytttheei* 0/ Aromatic Compoutufe, p, 46. Ed. by Billok, G. Oxford: Pergsmoo Press Ltd. .ctorin w *t*' mmIm niphl* nim*e in bsker's-yrnst cells (lOOg wot wt.) and P. bloke tonus myoelia (35g wet wt.) wore inoubated for l*h (innlee) or Oh (B. gracilis, baker's yoast and P, bhkeslceantit) under our standard condi- Oxidation of Furan-2-Carboxylate to 2-Oxoglutarate by Pseudomonas putido F2: Studies ofEnzymblogyand Electron Transport Mhliyih"*)'* the eult lire; rnctiun fr*"" xy-/J*|*ln*nyl* liens (Whistance et al. 1007; Spillor et ol. 1060; Hirolfall A Whistonon, 1070) with either 4^Ci of i'U-l4CJcinnamio aoid (lOmCi/mmol) or 4pCi of MU-,4C]ooumeric ' aoid (12.9raCi/mmol) [pre By J. P. Kxtohxb and P. W. Tkuooill. {Depart ment of Biochemistry and Agricultural Bioehsmistry, University College of Wales, Aberystwyth, U.K.) Aim*. SWmm wired by inoubating i.-[U-,4C]phenylalanmo and Pseudomonas putido F2 grown with furan-2- ,|,lo eell-frw* t-fU-^CJtyrosino respectively with phenylalouino oarboxyiato oxidizos this compound to 2-oxoglut- siuii prmlmt umnonin-lynno from Ilhvtloiorvlo glulinis IKO660 amto through tho following socjik'iico of inter I AM yi'i. ..... lOgata, Uchiynmn. Yuuvula A Toohikura, 1007)). mediates: furan-2-carboxyinto - 2-furoyl-CoA - Ill M**1 *l**ll<* U tho nnd of tlto incubations tho ubiquinones 6-hydroxy-2-furoyl-CoA -* 5- oxo -A3-dihydro - 2 * ittmvii. Vn* we isolated from the organisms and assayed for furoyl-CoA O-oxoghitaryl-CoA -> 2-oxoglutarste. IrnKylni**! ,,k adioactivity (Whistanos et al. 1967; Spilfor et al. A key reaction of this sequence, the hydroxylation a 'tnrla'Ay|* UMi Threlfall AWhistanco, 1970). of 2-furoyi-CoA, draws its oxygon from wator with mmalie ringi nApIdlMilt-M'*. In all cases radioactivity was incorporated into the resultant obligatory requirement for an electron ibiquinono, the major radioactive compound Skooeptor, which may bo satisfied by Methyleno Blue lMtW4r ! ^diliydnu)'* ^served in the lipid fractions. The incorporation or the membrane fraction from furan-2-carboxy'percentage of doee) from [U-,4C)cinnamie aoid late-grown oells (Jones k Trudgill, 1907; Trudgill, govern! plant tried from 0.008 in maize to 0.0006 in baker's 1969). yl vyi* l***`l <tut; that from p-(U-I4C]eoumario acid varied Descriptions of other hydroxylations that draw tun 0.11 in baker's yoast to 0.03 in maise. The thoir oxygon from wator are oonfined to nitrogen- .orinnutl b* VesiAo radioaotivitios (a.p.m./fimol) of the samples containing hydrophilio ring structures (Degloy A MQNS 084139