a TETTERSTO NATURE
Grystalstructure of lsopenlclllln fl synthasels the first from a new structural famlly of enzymeg
Potor L. Roachx, tan J. Gtlftonf, Vllmor Fiiliipt, Karl Harlort, Gooflrey J. Bartonf, Janot Halduf, lng.r Andcmontt, Ghrl$opher J. Schofleld* & Jack E. Baldwln*$
* DysonPerrins Laboratory and the OxfordCentre for MolecularSciences, University of Oxford,South parks Road, OxfordOX1 3QY, UK T Laboratoryof MolecularBiophysics and the OxfordCentre for MolecularSciences, University of Oxford,South parks Road, OxfordOX1 3QU, UK
PrNlclrrnq antibioticsare all producedfrom fermentationderived penicillins becausetheir chemical synthesisis not commercially viable.The key step in penicillin biosynthesis,in which both the plactam and thiazolidinerings of the nucleusare created,is medi- ated by isopenicillin N synthase(IPNS), which binds ferrous iron end usesdioxygen as a cosubstrate.In a uniqueenzymatic step, with no chemicalprecedent, IPNS catalysesthe transfer of four hydrogenatoms from its hipeptidesubstrate to dioxygenforming, in a singlereaction, the comptete bicyclic nucleus ofthe penicilinsr. We now report the structureof IPNS complexedwith manganese, whichreveals the activesite is unusuallyburied within a ,ielly-roll' motif and lined by hydrophobicresidues, and suggesthow this structure permits the process of penicillin formafion. Sequence analysesindicate IPNS, 1-aminocyclopropane-l-carboxylicacid oxidaseand many of the 2-oxo-acid-dependentoxygenases contain a conservedjelly-roll motif, forming a new structural family of enzymes. The story of penicillinis one repletewith surprises,from its serendipitousdiscovery2, its unusual chemical structure and efficacyas an antibiotic3'4,and more recentlythat its biosynthesis is dependentupon iron and dioxygent.The key stepin plnicillin biosynthesisis the transformationof the linear tripeptideZ-d- (a-aminoadipoyl)-I-cysteinyl-o-valine(ACV) into isopenicillin N (IPN) by the loss of four hydrogen atoms in a desaturative ring closure with concomitantreduction of dioxygento water.
s@ "'"-)l-Yt"*g^ rpNs.Fe(r)N3N\,^v^y"!fv coz --Z\* o o-H-^A^pY,,, io. "on*{6 coa tr),o L+(o-Ammoadrpoyl),4-cystenyl-D-vaLne (ACV) Isp€nrcilln N (IPN)
Thus, the full four-electronoxidizing power ofdioxygen is used in this IPNS-catalyseddesaturative step, unlike other non-haem ferrous iron-dependentoxygenases and oxidaseswhich require electrondonors or oxidizablecosubstrates6.
$ To whom correspondenceshould be addresd. fPermanentaddr6s SwedishUnjveEityofAgriculturalscrences,UppsataBiomedicatCentte, Departmentof N4olecularBrolog/, PO Box590, S-75124, Uppsata,Sweden. 700 NATURE. VOL 375 . 22 JUNE1995 LETTERSTO NATURE
TABLE1 Datacollection and phasingstattsttcs Resolution Numberof Unique completeness ar lsomorphous Numberof Phasrngpower{ Compound (A) observations reflections ek) fk) 6;6s1gnsst(o/o) sites (acentnc/centric) Native z.c L36,252 36,720 98.1 10.9 EMPS 3.0 79,890 27,416 97.9 !4.2 20.6 4 7.43,I.73 Baker'sdi-mercunal 3.5 44,943 13,961 94.4 72.5 30.2 4 7.56,'7.22
RecombinantIPNS from AspergiltusnidulansB was crystallizedin the presenceot 2.5mM MnCl,(ref.9). The crystals(0.5-1.0mm overall dimensions)belongto space group P2r2r21with unit cell dimensions of a:59.2 A,b=L27.O A, c:139.6 A.The asymmetric untt contains a ormer, and the solventcontent of the crystalsis 60%. Thestructure of the enzymewas solvedbythe methodof multipleisomorphous replacement (MlR) from two heavy-atomderivatives. The derivatives were prepared by soaking nattve crystals at 20-21 "C ln 0.5 mM solutionsof the respecttveheavy- atom reagentsfor 76-20 h. High-resolutionnatrve data were collectedustng 0.893A radiationat beamline4 of the ESRF(3Ocm MARresearcn lmageplate detector). All otherdata werecollected rn-house on an 18 cm MARResearch tmage plate detector ustng CuKa radratron from a Rigaku rotatinganode generator operating at 60 kV, 70 mA.The data were processedwtth the DENZOprogramle and the MIR phaseswere improvedby non-crystallographtcsymmetry averaging, solvent flattenrng and histogrammatching using the CCP4Surte of Programs(Program DM)2o. The two derlvativeshad two sitesIn common.Electron density maps were interpreted with the programO21. Skeletonized maps22 were calculated for chain traclng.Intermediate models of the enzymewere refinedby srmulatedannealing ustng the programX-PLOR23 wtth non-crystallographicsymmerry restraintsimposed. During 7 cyclesof refinementand rebuilding,328 resrdues(Gly2 and Ser3 werenot visiblein eachsubunit) could be fitted to the densityIn bothmolecules of the asymmetricunrt. In the finalcycle, the posittonsof all the atomsin the asymmetncunit includtng 194 water molecules,two active-site-boundmanganese ions, and two addrtronalmanagese tons (bound to Glu81 and His82 rn eachsubunrt) were refined withoutnon-crystallographic symmetry restratnts. At present,the crystallographicR factor (defined as R"o.1=f lF"5"l lF"u,"l,,IlF"o.l.100) in the resolutionrange of 8-2.5 A is 22.Oo/ofor all observationsand 21-.5o/oustns 33,229 reflectionswith Fobs>2o(F"0.).The free Rlalue2ois 26.5% basedon 1,449 randomlyselected reflections (4% of the total) and 26.40/ofor1,425 reflectionswith Fobs>2o(Fob").The r.m.s.deviation of the bondlengths,bondanglgsandtorsionanglesfromstandardvaluesis0.0lA,2and24',respectrvely.ThemeancoordtnateerrorisO.45Abased on the SIGIVIAAmethodz5. The r.m.s.difference between subuntts is 0.32 A for C, atoms andO32A for all atoms.Gln 330, which ts coordinated totheactive-sitemetal,rstheonlyRamachandranoutlrerrneachcharn(chainA,y=100';chainB, y=90). *R...e.=LLnln,,*
Initially IPNS from Cephalosporiumacremonium was crystal- from the external environment. Thus. the reaction can be lized,but the crystalso_btained were of insufficientquality for channelled along a single path. avoiding the many side structuredetermination'. Crystals of a recombinantAspergillus reactions potentially open to the highly reactive speciesresult- nidulansIPNSE complexed with manganeseewere then obtimea ing from the reduction ol dioxygen at the metal. The role of andwere used to determinethe structureof IPNS at a resolution enzymes.in such processeshas been characterized as negative of 2.5A (Table l, Fig. 1). Thesecrystals contain manganese catalysis''. insteadof iron at the activesite and are relativelystable under Previous sequencecomparisons betweenIPNS, 1-aminocyclo- aerobicconditions. The secondarystructure of the enzymecon- propane-l-carboxyhc acrd oxidase and some 2-oxo-acid-depen- sistsof 10 helicesand 16 B-strands(Fig. 1). Eight of the dent oxygenaseshave identified homologous regions, including B-r0. strands(85, 98-14,Figs I and2) fold to givea jelly-roll motif two containing the active site histidinesof IPNS (His 214 and The jelly-roll motif is common qngng viral capsidproteinslo His 270)'4. F'or some other 2-oxoglutarate-dependent oxy- andhas been identified in enzymes"'''.Unlike other known jelly- genases convincing sequence alignments have '', not been roll proteinsr0 in IPNS thejelly roll is not a completelyciosed achievedra'r5.In the light of the structure of IPNS, sequence )' structuralunit and the activesite is buriedwithin the B-barrel, comparisonru of IPNS with 1-aminocyclopropane-1-carbox- The sideof thejelly roll consistingof B-strands12,9, 14 and 5 ylic acid oxidase and related 2-oxo-acid-dependentoxygenases is extendedat both endsby strands1,2, and4, 5, respectively, (Figs I and 2) leads us to propose that many of them use the to form a largersheet (Fig. l). The continuationof the B-sheet same basic structural platform and have thus evolved through from B5 allows the C-terminal tail (324-331) extendingfrom a divergent process.In particular thejelly-roll core and the long- the flnal a-helix (alO) to enter betweenthe two facesof the est a-helix (a6) in IPNS are highly conserved.The presenceof jelly-roll,allowing Gln 330to ligateto themetal. This glutamine the a6 helix may be concernedwith stabilization of the distorted is presentin all independentlydetermined IPNS sequences.The jelly-roll motif (Fig. 1). These comparisonsalso indicate that otherside of thejelly roll consistsof strands8, 13,l0 and 11. the active site iron is likely to be coordinated by the sidechains of Strands6 andT form a hairpinloop on the surface.The longest the conservedaspartyl (Asp 216) and histidyl (His 214,His 270) a-helix(a6) straddlesB-strands 4,5,9,12 and 14on theoutside residuesfor the other members of the family. The conservation of the jelly roll and is linked by a sharp turn containingthe of structural and active-sitemotifs throughout a number of these conservedGly I 65to anotherstrand of chain( I 66-I 78) contain- enzymes suggests that they operate through closely related ing a7 (Fig. lb). mechanisms. Gln 330 which is also ligated to the manganese The active-sitestructure (Fig. 1d) revealsthe manganeseion, bound to the active site in the IPNS structure and is presumably substitutingfor the ferrousion at the metal binding site. It is involved in catalysisis conservedthroughout all reported IPNS attachedby four proteinligands (His214, Asp216, His270. sequences,but is not conserved through other members of the Gln 330) and bearstwo water molecules,the latter occupying family. It may be that this differencein coordination chemistry coordinationsites directed into a hydrophobiccavity within the between IPNS and the other members reflectsdifferences in their protein.It is likelythat ACV anddioxygen bind to thecoordina- mechanism and substrate specificity, in particular the fact that tion sitesoccupied by the watermolecules and Gln 330(Fig. 3). IPNS does not use a 2-oxo-acid cosubstrate. Sucha structuralcharacteristic (an iron-bindingsite within an IPNS, l-aminocyclopropane-1-carboxylic acid oxidase and unreactivehydrophobic substrate binding cavity) is probablya the related 2-oxo-acid-dependentoxygenases constitute a new requirementfor this classof enzyme,as it resultsin the isolation structural family which catalyses a wide range of oxidative chem- of the reactivecomplex and subsequentintermediates (Fig. 3) istry, including hydroxylation, desaturation and desaturative
NATURE.VOL 375 . 22 JUNE1995 70t NATIIRF VOt ?76 )) XrNtr 1qO6 TETTERSTO NATURE
< FlG.1 a-d, The structureof IPNS.a, The three-dimensionalstructure of isopenicillinN synthaseshowing the conservedjelly-roll motif in green.The structure is viewedfrom the entranceto the activesite. The active-sitebound metal and its ligandsare buriedwithin the jelly roll. A secondand weaker metal-bindingsite is locatedon the surfaceof the moleculein a non-conseryedarea with Glu 81 and His82 as orotein ligands.The secondarystructure elements of the moleculeare shown in greyexcept for the conservedB-strands of the jelly-rollmotif (green). Residue331 is the C-terminalthreonine, and residue4 is the first N- FId{ HAFVSI SAVPFIPTAiEAETNVRASFVREgOERPK terminalresidue that could be localized HgH ATFVSNWSTKSVSESFIAPLOKRA in the structure.b, Distribution FOL$ MKTAQGVSATLTMEVARVQAIA6LSKCMDTIPSEYIN$ of conservedresidues in IPNSand relatedenzymes. The moleculeis &.10 MALNSX!ATESAPARLNEEMKQgYRPPFSOSOESKVPgDFIVI' oAocs viewedfrom the back of the active site. Resultsfrom the sequence oAocnAos comparisonsof Fig.2 are mappedon the three-dimensionalimage of tpils the molecule.The colouringscheme follows the conventionof Fig.2: sequenceidentities are shown in redand residueswith similar physical- chemicalproperties in yellow.c, Structurecoloured accordingto crystal- ME NIFIPI JJ]NEI EKLN GOERANr ME dI'lK D lographictemperature factors. Blue shows the smallestvalues and red VPHDRFSDEIVIPVIVISltl Eor DG PRRRAEI RGRIVhA EKDVPVGNDUIPIIIIDllt ooH HHLL VOOilrK the highest.Comparison with c shows that the distributionof low B ENEOPAATTILIHONIVILI OVPVIDLR DPDTI'IKMVKLIIIAD factorresidues SEKFEASELILFVN-FIVIPTIOLEKFMSqoKsYves AT RLIVbE in the structurelargely follows the distributionof conser- DT TIVF TIFISiL! AEL QCgL HOOEIFIRB ved residuesin the family.d, Stereo-viewof the activesite regionof T 8 KIVIPVIFIR ILI DOL KSGKVLTEILhE MGSVSKANIYJPKIIJDtyl SpLFGDD OAAKMRvAoa&pA IPNS.The figure is takenfrom the sameorientation as b and c. Electron 10 rl| C* & ffiffiI densitywas contouredaI 1.4o, where o representsthe r.m.s.electron FI dl az densityfor the unit cell. The structureshows a distortedoctahedral l geometry around the manganese,with the ligandsformed from two E|L*VmH vMDrll/lEKrdflK MEo{HF K E LVASK water moleculesand the side chains dv v Dsl AAL VADMARLhlfi 3i DIKfi. R F D its EG KK of His 214, His270, Asp216 and qvt FE NFI L ML EIIIMEVFIK:t A! EiI(IEK F KPKSEAAKFELPLEO Gln 330 (Fig.2). The latterwas unanticipated.An analysisof the protein OIL I HFI A I A DS,HIKVBIIK poEIEIKE L I AKTP OSr{Dl FIV V NIH M MG RIVIHD cndN LOVKNR AKRKvG rt geometryaround the metal site indicatesstrain in the neighbouring YILT OP ELKSIAIKD DI SEAIEIK R A vT3PV PTMfr YILT EIS DHTSIAIFE pl SEEEKRAVTLAO BNAR polypeptidestructure. The carboxylateof Thr 331 forms a H bond with YIA V ilIH ilvoRnFo EI T9EEKWDL AI RAYNKEHQDOVN Arg87. The $0 IHfr 80 metal ligandGln330 is the only Ramachandranoutlier. :) I * Thecoordination chemistry of IPNSis to ourknowledge unique. probably Q' ai d4 \ the most relatedcoordination chemistry is found in the iron and man- ganesesuperoxide dismutases, where the metal ligandsare provided Eker voAEvTDLDmESTFFLRHL prsNr sqvFlDLD GIGFIVS6 HLOGEAVODMIREIVTYFSVPVKARDYSRI/VPIOKP by the side chains of an aspartyland three histidyl residues,with a KIAIKLYVe cE oL s NEE F L Yh'{KDT L AH6cHPL DoDL VNSWPIEK P glutaminyl EF{YGTSL OKEVEEKKGFiIVDIILFHKIWPPSAVNYRYTWIXNP nearby residuewhich is not direcflyligated to the metal.a, YFIYTN S F F GN F A S {tIL P WKE TFISL RCV AAON S S A A HOYV L OTL GP $ F b and c wereproduced using MOLSCRIPT26, RISIFTGLESESTAOITNTGSYSDIYISI'CYSMGTADN LFPI6GD e using021. RbIFSALEWESTAVVTETOKYSDMISTCYSMGIO6N LFFINRG AE|JYY L S I P O K K A V E $ F C Y L N P NEIT P D H P R I Q A K T P T H E V N V WIID E T K H rPt 1r0 120 1s -g __ F3S4(5
EKGYIKNAFYG SKOP D'EALARACVO M KLGYFONFLS O EOHFMIEAAG GDgI SflEYFKNF F€OI'I EPD GGV gAFLDC PLA GED IDDFVEC FlG.2 Sequencecomparisons between isopenicillin N synthase,1-ami- EENFFARHFKPDOTLAS r?oc t* nocyclopropane-1-carboxylicacid oxidase and related 2-oxo-acid- - dependentoxygenases. The figure was prepared using ALSCR|pTlT. a7 Numberingfollows the sequenceof IPNSfor whichthe secondarystruc- turesare shownas cylinders(helices) and arrows(B-strands). ldentities NF GltlK[glsffYFlPF-FlKP D L IKELRAE KV$G D OKIVIVIVSIFY PRICPIOPO L veq are shownwith red backgrounds,positions at whichamino acids with I OMT'NLITKIYY PIPh PIDP S $ +EEEGiiE LPS similarphysical-chemical properties are conserved yellow V Y LILIKIIINIY Y PIPIOPIR P D L v00 areshown in D S I ILIBILINIYYPTTPDIK P E V vsG as in Fig.1b. p-Strands5 and 8-14 which comprisethe jelly-roilmotif EPLIIIRFIRIYFPIOIVPIEHBSAEEO rEtn m A rd CANE DPLB-IRff.IFIYFPIEN'PEORVAEEE CANO are shown in green.The nletal ligandsare indicatedby red triangles. v v LUnMlpry L olpllP.l€A A t KT A A Dcr Aqor ery vail Otherresidues with side chainswithin 8 A of the metalare indicated ..61s {Dry} *g* H} 'l06 q8 06 F? 08 by black triangles.ACCO, 1-Aminocyclopropane-1-carboxylate oxidase I i/l esr from tomato (accessionnumber SwissProt P05116); F3OH, flavanone 3-p-hydroxylasefrcm Zea mays (GenbankUO4434l: H6H, hyoscyamine M +[ B3" - Mi ?flHF UEsFITXIfrFHBffi IITHEHFTffi b 6-p-hydroxylasefrom henbane(SwissProt P243971: FOLS, flavanot syn- ^ thasefrom Petuniahybrida (Genbank 567953); GC20,gibberellin C-20 " oxidaseftom Cucurbitamaxima (Genbank X73314); DAOCS, deacetoxy- cephalosporinC synthasefrom Streptomycesclavuligerus (SwissProt ',,ffieInl iIln i P18548); DAOC/DACS,deacetoxycephalosporin C synthase/deacetyt- F:o ffiHFlfll1 P12Affihi*frllff c9 cephalosporinC synthase,a bifunctionalenzyme from C. acremonium 1 (SwissProtP11935); IPNSfrom A. nidulans (SwissProtP05326). For someof the 2-oxo-acid-dependentoxygenases (such as clavaminic acid A OTO€T N sFhvlilYFlA KT L vE KE AEE sTevY P K t5; v t{gEcs N PTIATIVIYFILAVRDGEAPI LOHPT TFA synthaseto preliminarysequence alignments T DPTBD YFPTIIEFIAK E L L NADNPPLYKPYSYS did not indicate V NKDKT F SEF{ENrePIIPKLLSEAHP PKFKT sufficientsequence similarity with IPNSto predictthe presenceof the V I'ISUNA HDFVTFhIP E E L VE KSPP RKYPD APRBDQIAGSA ADIFTFISIVIP L A RE CGF DVS L DGET jelly-rollmotif with confidence(see also ref. 74). The conservationof SPGRDARVGSS R Pl)F SFNNiAqSREWGFNVFI PS E R WVHAE N YDFVIIOEF DPREPNGK S DREP structurethroughout the 2-oxo-acid-dependentand relatedoxygenases msn3l0 will require further structural r) analyses.Pairwise comparison of the Fr4 Fls sequencesfollowed by singlelinkage cluster analysis shows that the l proteinsall clusterwith standarddeviation scores >6.0. Scoresover r vFloovE& NATURE.VOL 375 . 22 JUNE1995 703 TETTERSTO NATURE +ACV,02 -H2O,-L4, -H- FlG.3 Proposedscheme for the overallreaction catalysed by IPNS. 114, Protein ligands (His214, Asp216, Hrs270, Gln330); R, L-d-(a-aminoadrpoyl).The schemeshows (a), the two substratesbound to the rron,b, the proposedB-lactam ferryl Intermedrateand c, the enzyme-productcomplex with IPNand two watermolecules. Mechanistic studies on the con- versionof ACVto lPN, using kinetic isotopeeffects and sub- strateanalogues, have led to the proposalof an enzyme-bound p-lactamintermediate directly attached to the iron centre,at the oxidationsate of rv (b). This complexmay then react to completecarbon-sulphur bond formatton by a free-radicaltype mechanisms18.Soectroscopic Investigations on the natureof the metalligands at the activesite of IPNShave indicated that the side chainsof 2 or 3 histidyland an aspartylresidue act as metal ligandsS2T-3o.EPR measurements have been inter- pretedas showingthe presenceof a water moleculecoordt- nated to the ferrous iron in an IPNS/ACV/NOcomplex analogousto a3o.Support for the proposediron-sulphur bond (in a) has come from UV-V|S27and EXAFSspectroscopy'". Indeed,the actrvesrte surrounding the metal-btndingsite of IPNSis a predominantlyhydrophobic cavity, consistent with the generatronof a highlyreactive intermediate, ltgated to the metal by residuesthat are highlyconserved throughout the family,His 2!4, His270 andAsp 216 (Fig.2). The intermedracy of a ferrylspecies such as b impliesa protein-boundltgand HrO,-H* | (La),presumably Gln 330, is releasedfrom the iron beforeits io formatton.Intttal modelling studies indicate substrate binding RHN^ ll .L' may inducesubstantral reorganization at the activesite. The presentlyproposed mechanism for the 2-oxo-acid-dependent c.f. cyclization reactions. Some members have been shown to have and improvethe biosyntheticroute to antibioticsof t8. BJactams a remarkably lax substrate specificityts The determination of medicinalimportance. Studies on the mechanismmay alsolead the structure of IPNS will help investigationsinto their catalytic to the designof new non-proteincatalysts capable of catalysing mechanisms and will allow systematic engineering to attempt in a stereoselectivemanner reactions that arecurrently synthet- the enzymatic synthesis of new types of otherwise inaccessible icallyimpossible. T Recerved24 January;accepted 26 Apnl1995 17 Barton,G. ) Prot.Engngat 37-40 (1993) 18 Baldwrn,J E & Bradley,M Chem Rev tO, 107+1088 (1990) 1. Pang,C.-P. et at. Btochem.J.222,749-795 t7984) 19 otwlnowskr,Z. n Data Collectionand Processing(eds Sawyer,L, lsaacs,N W & Barley. (7929J 2. Flemlng,A. Brit. J. exp.Path.LOt226-236 S ) 55-62 (DL SClrR34.Daresbury Laboratory. Warnngton, U K, 1993) 3, Florey.H W et a/. Antibtot,csVol. 2 ioxford Unrv Press,London, New York,1949) 20. coMan, K ActacrystalloEr D5O, 760-763 (1994) 4. Crowfoot,D., Bunn,C. W , Rogers-Low,B W & Turner.Jones,A lhe Chemistryof Penicillin 21. Jon6, T. A., Zou,J. Y., Cowan,S. W. & Kjeldgaard,MActa crystallo€lt Aa7,110-119 (edsClark, H. T, Johnson,J. R.& Robinson,R.) 310-321 (PnncetonUnrv Press, Pflnceton, (1991) NewJe6ey, 1949) 22 Jon6.T A. &Thrrup,S. EMBOJ.5, 819-€22 (1986) 5 Baldwln,J. E. & Schofield,C. J In rhe Chemrstryof P-tactams(ed. Page, M. l.) Chapter1 23 Brrlnger,A. T.,Kuriyan, J. & Karplus.l\4. Sc,ence 235,458-460 (1987). (Blackre, London,1992) 24 Btinget, A T Nature355, 472474 11992) 6. Ferg,A L. & Lrppard,S J. Chem.Rev ta, 75HO5 (1994). 25 Read,R J. ActacrystalloEr Aa2, 14G-149 (1986) 7. Fujlshlma,Y. et al. J molec Biol.212, 772-774 (7994) 26 Kraul,s.P ). J. appt Crystallogr24,946-950 (1991) 8. Baldwln,JE, Blackburn,JM,Sutherland,J D.&Wright,M C fetrahedron47,5991- 27 ONnle,A. lM et a/ Btochemtstry3'., 46O2-46L2 (7992). 6002 (1991). 28 Randall,C R eta/ BiochemistryS2t6664-6673 (1993). 9. Roach,P L.,Schofreld, C .J.,Baldwrn, J E, Clfton,l J. & Hajdu,J. Prot. Scr. a,1007-1009 29 Scott,R. A., Wang,S.,Erdsness, M K, Knaucrunas,A & Chen,V J Bjochemstry3t, (1995) 4596-4601 (1992J 10. Stuart, D I Curr Opin. sttuct. Biol 3, 167-174 (1993) 30 Chen,V. J. et at. ). biot. Chem 2|,,! 27677-27681 (7989) 11. Norns,G E, Sullrvan,T J, Anderson,B. F & Baker,E N Structure2,1049-1059 {1994) 12 Srlnlvasan,N. et a/. Structure2,70'1,7-7027 \7994). 13 Retey,). Angew.Chem.2t, 355-361 (1990) ACKNoWLEDGEIVIENTSWe thank Z H Zhangfor helpfuldrscussrons, J. W Keeprng,J Prtt, 14. Prescot,A. G. J. exp. Bot. aa,849-€61 (1993). M Samr,A. Hassanand W. J. Sobeyfor experttechnrcal assrstance, B Rasmussenand A 15. Marsh, E N , Chang,M.D I & Townsend,C A. Biochemistry'.'-,72644-72657 (1992) Abergfor the outstandrngfaclrtres at beam lrne4 of the ESRF(Grenoble. France) and the 16. Barton,G J Meth Enzym tt3,403-428 (1990) MRC,BBSRC, EPSRC and Zenecavta a DTILrnk Scheme for ftnanctalsupport of thls work 704 NATURE.VOL 375 . 22 JUNE1995