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,,*<lr>l/L,In(/n)"100, O:t20', O:tL2", wherelh,r is the Intensityof an indrvrdualmeasurement, and (/6) rs the meanintensrty of that reflection. f - Percent rsomorphous difference ls calculatedusing the expresstonI Fo" lFpJ,l Fe x 100, whereFp and Fpsrefer to the nativeand the denvativestructure factor. I Thephasing power of a derivattveis definedas the ratioof the amplitudeof the r,m.s.heavy-atom scattenng factor to the r.m.s,lack of closure. $ Ethylmercury phosphate. 1,4-Dracetoxymercuri-2,3-d imethoxybuta n6. 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