insights into cephamycin biosynthesis: the crystal structure of cmci from streptomyces clavuligerus

doi:10.1016/j.jmb.2006.02.004 J. Mol. Biol. (2006) 358, 546–558

Insights into Cephamycin Biosynthesis: the CrystalStructure of CmcI from Streptomyces clavuligerus

Linda M. Oster1, Diane R. Lester2, Anke Terwisscha van Scheltinga1

Martin Svenda2, Michiel van Lun1, Catherine Genereux3 andInger Andersson1*

1Department of MolecularBiology, Swedish University ofAgricultural Sciences, Box 590S-751 24 Uppsala, Sweden

2Department of Cell andMolecular Biology, UppsalaUniversity, Box 596, S-751 24Uppsala, Sweden

3Centre for protein EngineeringInstitute of Chemistry B6aUniversity of Liege, B6-4000Liege, Belgium

0022-2836/$ - see front matter q 2006 E

Present addresses: L. M. Oster, StrLester, School of Plant Science, UnivScheltinga, Department of Structuraam Main, Germany; C. Genereux, LInfectious Parasitic Diseases, FacultLiege, Belgium.

Abbreviations used: DAC, deacetcysteine; NADH, nicotinamide adenroot-mean-square; COMT, cathecho

E-mail address of the [email protected]

Cephamycin C-producing microorganisms use two enzymes to convertcephalosporins to their 7a-methoxy derivatives. Here we report theX-ray structure of one of these enzymes, CmcI, from Streptomycesclavuligerus. The polypeptide chain of the enzyme folds into aC-terminal Rossmann domain and a smaller N-terminal domain, andthe molecule packs as a hexamer in the crystal. The Rossmann domainbinds S-adenosyl-L-methionine (SAM) and the demethylated product,S-adenosyl-L-homocysteine, in a fashion similar to the common bindingmode of this cofactor in SAM-dependent methyltransferases. There is amagnesium-binding site in the vicinity of the SAM site with a boundmagnesium ion ligated by residues Asp160, Glu186 and Asp187. Theexpected cephalosporin binding site near the magnesium ion isoccupied by polyethyleneglycol (PEG) from the crystallisation medium.The geometry of the SAM and the magnesium binding sites is similarto that found in cathechol O-methyltransferase. The results suggestCmcI is a methyltransferase, and its most likely function is to catalysethe transfer of a methyl group from SAM to the 7a-hydroxycephalosporin in the second catalytic reaction of cephamycin formation.Based on the docking of the putative substrate, 7a-hydroxy-O-carbamoyldeacetylcephalosporin C, to the structure of the ternaryCmcI–Mg2C–SAM complex, we propose a model for substrate bindingand catalysis. In this model, the 7-hydroxy group of the b-lactam ringligates the Mg2C with its a-side facing the methyl group of SAM at adistance that would allow methylation of the hydroxyl-group.

q 2006 Elsevier Ltd. All rights reserved.

Keywords: 7a-methoxycephalosporin; cephamycin C; methyltransferase;S-adenosylmethionine; b-lactam
*Corresponding author
lsevier Ltd. All rights reserved.

uctural Chemistry Laboratory, AstraZeneca R&D, S-431 83 Molndal, Sweden; D. R.ersity of Tasmania, GPO Box 252-55, TAS 7005, Australia; A. C. Terwisscha van

l Biology, Max Planck Institute of Biophysics, Marie Curie Str. 15, D-60439 Frankfurtaboratory of Parasitology and Pathology of Parasitic Disease, Department ofy of Veterinary Medicine, University of Liege, Boulevard de Colonster, 20. B-4000

ylcephalosporin C; SAM, S-adenosyl-L-methionine; SAH, S-adenosyl-L-homo-ine dinucleotide, reduced form; NCS, non-crystallographic symmetry; r.m.s.,

l O-methyltransferase.ing author:

Crystal Structure of CmcI from S. clavuligerus 547

Introduction

The increasing occurrence of bacterial resistanceto antibiotics is a serious threat to human health. Inthe case of b-lactam antibiotics, the most commonmechanism of resistance is the synthesis by bacteriaof b-lactamases that hydrolyse the b-lactam ring.1

Cephamycins are b-lactam antibiotics substitutedwith a methoxyl group at the 7a-position of thecephalosporin nucleus, a modification that con-siderably reduces inactivation by b-lactam hydro-lysing enzymes.2

In cephamycin-producing organisms, synthesisof 7a-methoxylated cephems is via the route leadingto the b-lactam antibiotics, penicillins and cepha-losporins (Figure 1; reviewed by Baldwin &Schofield3), but the details of the late reactionsleading to 7a-methoxylation are not well under-stood. For example, biosynthesis of cephamycin Cinvolves methoxylation at C-7 and carbamoylationat C-3 0 of deacetylcephalosporin C (DAC). Thesequential order of the methoxylation and carba-moylation has not yet been elucidated, but carba-moylation has been implied to take place beforemethoxylation.3 Methoxylation at the 7a position ofthe cephem nucleus occurs in two steps, hydroxy-lation at C-7, followed by methylation of thehydroxylated intermediate.4,5 The hydroxyl groupat C-7 derives from molecular oxygen6 andmethionine is the source of the new methylgroup.7 Conversion of cephalosporin C and O-car-bamoyl DAC to their 7a-methoxy derivatives wasdemonstrated in cell-free extracts of Streptomycesclavuligerus on addition of Fe(II), 2-oxoglutarate,S-adenosyl-L-methionine (SAM), and a reducingagent.4 In the absence of SAM only the hydroxyl-ated intermediate was formed.5

Few details are known about the enzymesinvolved in cephamycin production. Xiao et al.8

purified an enzyme from S. clavuligerus with theproperties of a 2-oxoglutarate-dependent dioxygen-ase and an estimated molecular mass of 32 kDa.Judging by its cofactor requirement and substrate/product specificity in activity assays, it wasconcluded that this enzyme catalysed the hydroxy-lation step in cephamycin biosynthesis. Two genes(cmcI and cmcJ) in Amycolatopsis lactamdurans(previously Nocardia lactamdurans) were sub-sequently identified to encode two enzymesresponsible for the methoxylation reaction at theC-7 position.9,10 The cmcI and cmcJ genes are alsopresent in S. clavuligerus.11

CmcI encodes a 28 kDa protein with fragments ofsequence characteristic of methyltransferases andmonooxygenases (Figure 2). The 32 kDa product ofcmcJ shows weak similarity to a gibberellindesaturase with hydroxylase activity12 and to anumber of hypothetical proteins in the sequencedatabase. Neither cmcJ, nor the gibberellin desatur-ase show high similarity with the 2-oxoglutaratedioxygenases that catalyse the earlier steps ofcephalosporin biosynthesis. Coque et al.9 trans-formed a DNA fragment containing cmcI and cmcJ

into S. lividans and showed resulting transformantscould convert cephalosporin C to the C-7 methoxy-lated product. Both genes were necessary forefficient methoxylation. The cmcI gene alone con-ferred weak hydroxylase activity with either of theelectron donors 2-oxoglutarate or NADH present,whereas transformants carrying cmcJ only showedno activity. Based on these experiments, Coque et al.proposed that 7a-cephem-methoxylation is per-formed by a two-component system composed ofboth proteins CmcI and CmcJ.9 Studies withrecombinant and native CmcI and CmcJ proteinssupported this theory and it was suggested that theactive site of the methoxylation complex is on CmcIwith CmcJ working as a “helper” protein.10 CmcI ismost often annotated as the hydroxylase and CmcJas the methyltransferase.11,13–15 Results presentedhere give reason to question this assignment.

As a step towards the elucidation of thebiosynthetic sequence and the individual roles ofthe enzymes involved, we report here the crystalstructures of CmcI in its apo-form, in complex withmagnesium, SAM and S-adenosyl-L-homocysteine(SAH). The mode of cofactor binding and the foldstrongly point to a role for CmcI as a methyltrans-ferase. Based on docking experiments, a model forthe binding of the putative substrate 7a-hydroxy-O-carbamoyl DAC is presented.

Results

The sequence of cmcI in the pET32 expressionplasmid described by Lester et al.16 differed fromthe sequence reported by Alexander & Jensen11 atresidues 10, 160 and 200. It was not immediatelyobvious whether these differences arose from PCRerrors, sequencing errors, the presence of CmcIisoenzymes or different strain origins because atleast one of the differences was encountered whencmcI was independently recloned from S. clavuli-gerus genomic DNA. We amplified cmcI and cmcJ intandem from genomic DNA multiple times andconcluded that the alterations at positions 10 and160 were mutations caused by PCR errors andpromoted by high GC content in the region. Wefound no evidence that a conservative substitutionat position 200 was a PCR error and thereforeattributed it to differences in wild-type S. clavuli-gerus strains similar to those recorded by Alexander& Jensen.11

Thus, a new pET32 expression plasmid wasgenerated by site-directed mutagenesis for theproduction of wild-type CmcI protein. Thissequence differs from the GenBank accessionAF073896 by a phenylalanine instead of a leucineat residue 200. The plasmid was used to produceprotein for the majority of structural studiesdescribed here. We will refer to the product asCmcI or wild-type. The product of the originalconstruct16 became L10Q/D160N and is sometimesreferred to in the text as the mutant. The mutant isof interest because residue 160 is situated in the

Figure 1. The cephamycin biosynthetic pathway in S. clavuligerus. Biosynthesis of naturally occurring penicillins andcephalosporins begins with a condensation of three amino acids (L-cysteine, L-valine, and the unusual L-a-aminoadipicacid) to form the tripeptide d-(L-a-aminoadipyl)-L-cysteinyl-D-valine (ACV). This non-ribosomal peptide biosynthesis iscatalysed by a large multifunctional enzyme, ACV synthetase. The ring closure of the linear ACV to form the bicyclicisopenicillin N (IPN) is catalysed by IPN synthase27 in an unusual oxidation reaction. Cephalosporin biosynthesisbegins with the isomerisation of the L-a-aminoadipyl side-chain of IPN by IPN epimerase to form penicillin N.Expansion of the five-membered thiazolidine ring of penicillin N catalysed by deacetoxycephalosporin C synthase(DAOCS) in prokaryotes49,50 forms the six-membered dihydrothiazine ring common to all cephalosporins. Theresulting compound, deacetoxycephalosporin C, is hydroxylated at the C-3 0 position by DAC synthase to form DAC.In the final step of cephalosporin C biosynthesis an acetyl moiety is transferred to the –OH group ofdeacetylcephalosporin C by acetyl-CoA:DAC acetyltransferase. In the cephamycin specific biosynthetic pathway ofactinomycetes the C-3 0 hydroxyl group of DAC is carbamoylated by DAC-O-carbamoyltransferase51 and the C-7position is methoxylated in two steps, hydroxylation, followed by methylation of the hydroxylated intermediate toform Cephamycin C.4,5

548 Crystal Structure of CmcI from S. clavuligerus

Figure 2. Alignment of selectedCmcI homologs identified during aPSI-BLAST search. Numberswithin parenthesis are the acces-sion codes from the Swiss ProteinDatabank. From above, CmcI fromStreptomyces clavuligerus (O85726),CmcI from Amycolatopsis lactam-durans (Q51079) 78% identity,Cephalosporin hydroxylase fromRhizobium loti (Q98ID5) 28% iden-tity, hypothetical protein slr1619from Synechocystis sp. (P72897)24% identity and hypothetical pro-tein ML0127 from Mycobacteriumleprae (Q9CD89) 25% identity. Iden-tical amino acid residues aredepicted in red, similar aminoacid residues in orange and resi-dues of S. clavuligerus CmcIinvolved in binding of SAM aremarked by asterisks. The positionsof the mutated amino acid residuesof the original CmcI are bold andmarked by their number. a-Helicesof CmcI are depicted as greencylinders and b-strands as bluearrows.


active site and is conserved between species asaspartate.

Structure solution and quality of the model

Crystals of CmcI display non-isomorphism,resulting in widely differing unit cell constantsand switches between different space groups. Non-isomorphism was observed between crystals ofdifferent ligand complexes, different recombinantclones, but also within the same complexes andclones. By employing single-wavelength anoma-lous diffraction and a powerful program to locatethe numerous selenium sites,17 the structure couldbe solved. Non-isomorphism is likely to be causedby the slightly different packing of the hexamerswith respect to each other, whereas the hexameritself appears as a rigid unit.

The monomers comprise residues 2–234, butthere are slight variations in each monomer in thequality of the density at the N and C termini (non-crystallographic symmetry (NCS) restraints weregradually relaxed during refinement).A superposition of all monomers in the asymmetricunit using the algorithms encoded in O18 gave r.m.s.deviations in the range between 0.25–0.65 A for allCa atoms. Quality checks using PROCHECK19

show good geometry and good agreement betweenmodels and data. All residues except Tyr91 andAsp187 are within the most favoured or addition-ally allowed regions of the Ramachandran plot inall data sets. Tyr91 and Asp187 are in the disallowedregion in all monomers and have very well defineddensity. These residues bind SAM and Mg2C inthe cofactor complexes (see below). Tyr91 is found

in a loop-region between b-strand 4 and a-helix 4, atone side of the SAM binding site. Asp187 ispositioned on the opposite side in the loop-regionbetween b-strand 8 and a-helix 8. A cis-peptidebond connects residues His153 and Pro154. Pro154is found at the start of b-strand 7, away from theactive site cleft.

Overall structure and topology

The refined structure of CmcI is shown inFigures 3 and 4. The protein is a hexamer, arrangedas a trimer of dimers with 32 point symmetry(Figure 3 (a) and (b)). The final model has an Rcryst

of 18.9 and an Rfree of 24.6. Biochemical analysis bynative sodium dodecyl sulfate/polyacrylamide gelelectrophoresis, gel filtration and native massspectrometry indicate CmcI forms a hexamer insolution.16

The monomer is built up of nine a-helices (a1–a9)and ten b-strands (b1–b10) (Figure 4). It consists ofan N-terminal domain involved in oligomerisationand a C-terminal domain, which we proposeharbours the active site. The C-terminal domain,comprising amino acid residues 67–236, has amodified a/b fold (Rossmann-fold) common tonucleotide binding proteins.20 The Rossmann-likefold is built up of a central parallel six-strandedb-sheet (b4–b9) in which one antiparallel strand(b10) is inserted. A total of seven helices packagainst the central sheet. The N-terminal domain,comprising residues 1–67, consists of two shorta-helices (a1 and a2), two small anti-parallel bstrands (b2-b3), and one b strand (b1) that forms anextension of the central b-sheet of the C-terminal

Figure 3. Quaternary structure of CmcI. (a) The hexamer. (b) The hexamer in (a) rotated 908 clockwise around thevertical axis. (c) Type one interactions between subunits. The two monomers in the dimer are in red and blue, and a thirdmonomer (yellow) is in the background. (d) Type two interactions between subunits (in blue and yellow) with a thirdmonomer (red) in the background. Structure elements involved in interactions are labelled in (c) and (d).


domain of an adjacent molecule. The presence of theN-terminal excursion makes it unlikely that themonomer exists as a soluble entity on its own.

Subunit interactions

The N-terminal domain is responsible for theoligomerisation by forming two types of inter-actions with neighbouring molecules (Figure 3(c)and (d)). Type one interactions involve residues24–59 from one molecule that pack against the a3,a8 and a9-helices and the loop comprising residues215 to 226 of an adjacent molecule (Figure 3(c)). Anextension of the central b-sheet is formed by theinteraction of the b1-strand of one molecule withthe b9-strand of the adjacent molecule and viceversa, to form a dimer (Figure 3(c)). Further tightinteractions between the two monomers involvehelices a3 and a4 from both monomers.

In the second type of interactions, residues 1–20interact with a third molecule by packing againsthelices a7, a8 and a9 and vice versa (Figure 3(d)).

The type two interactions are weaker than the typeone interactions, because the interface contains alarge cavity that harbours the active site (see below).

These oligomerisation interactions result in ahexameric arrangement of the molecules, whereeach molecule interacts with two neighbouringmolecules (Figure 3(a) and (b)). The hexamer canbe described as a trimer of dimers and has the shapeof a puckered doughnut, with a diameter of 100 Ain the widest dimension and 50 A in the narrowest.A solvent channel with a diameter of approximately30 A runs through the middle of the hexamer.Residues 24–58 from the N-terminal domain facethe inside of the channel.

SAM/SAH binding

Crystals of complexes of wild-type and mutantenzymes with SAM were obtained by co-crystal-lisation and were approximately isomorphous withthe crystals of the Se-methionine derivative(Table 1). The electron density for the cofactor is

Figure 4. The fold of the monomer.(a) Ribbon diagram colour rampedfrom blue at the N terminus to redat the C terminus and with second-ary structure elements labelled.(b) Topology diagram with a-helicesand b-strands coloured green andblue, respectively. The beginningand the end of the secondary struc-ture elements are labelled.


well defined in the structure and allowed unam-biguous modelling of SAM into density in aposition that corresponds well with the commonbinding site for the cofactor in SAM-dependentmethyltransferases.21 SAM is bound through anextensive network of hydrogen bonds and hydro-phobic interactions (Figure 5). The adenine ringinteracts with the side-chains of Arg117, Asp138,Cys139 and Ala163. The ribose moiety formshydrogen bond interactions via the two hydroxylgroups to the side-chain of Asp116 and alsointeracts with Arg121 and Ala161. The sulphuratom and the S-methyl group make hydrophobicinteractions with Leu18, Leu64 and Tyr91. Thecarbonyl and amide groups of the methioninemoiety form hydrogen bonds to Lys65, Glu87,Gly89, Ser95 and Asp160.

The structure of a complex with SAH, thedemethylated product of SAM, was also deter-mined. SAH binds in the same position as SAM andall interactions of SAH with the protein are identicalto those of the SAM-complex (not shown).

Magnesium binding and the importanceof Asp160

Crystals of wild-type CmcI could only beobtained by including MgCl2 (instead of sodiumacetate) in the crystallisation solution. This resultedin a different packing (Table 1) and the resultingelectron density maps clearly showed density for abound magnesium ion close to the SAM bindingsite. The magnesium ion is ligated by Asp160,

Glu186 and Asp187 (Figures 6 and 7). No mag-nesium could be observed in the structure of themutant containing the D160N substitution. In themaps of the wild-type enzyme with magnesiumbound, density shaped as two elongated cylinderswas observed (Figure 6). This density extends intothe cavity between the subunits (Figure 3(d)).Considering the shape and length of the density, itis most likely polyethyleneglycol (PEG), which wasused as precipitating agent. Two PEG unitsconsisting of 4-C (C4H8O3) and 12-C (C12H24O7)were modelled into the density (Figure 6). In thebinary E–Mg2C complex the density for the Mg2C

was relatively weak and no direct binding of PEG tothe metal ion could be observed. However, in theternary E–Mg2C–SAM complex, Asp160 ligatesboth Mg2C and SAM and the density for PEGextends all the way to the Mg2C. No density foreither Mg2C or PEG were observed in the structuresobtained from crystals of the L10Q/D160N mutantobtained by soaking or co-crystallisation withMg2C.

Structural homologies

The Protein Data Bank (PDB) database wasanalysed for structures similar to CmcI usingDALI,22 with the monomer as the search model.Searches using the dimer or a trimer gave similarresults, whereas the use of a hexamer as a searchitem did not produce any hits. The result showshigh structural homology to SAM-dependent meth-yltransferases, with the top 39 hits belonging to this

Table 1. Statistics for data collection, phasing and refinement

Type

L10Q/D160N

apo

L10Q/D160N

Se peak

Wild-type

Mg2CWild-type

Mg2C–SAM

L10Q/D160N

SAM

L10Q/D160N

SAH

Data collection

Space group P21 P212121 P212121 P212121 P212121 P212121

Cell a, b, c (A) 93.6, 182.6, 103.2 92.8, 103.0 182.0 77.9, 145.5, 162.1 91.5, 102.4, 181.9 92.8, 103.0, 182.0 90.9, 102.5, 181.4

b (deg.) 91.05 90.0 90.0 90.0 90.0 90.0

Resolution (A) 95.3–2.5 40–3.6 54.2–2.8 51.3–2.6 91.3–2.9 91.3–2.8Outer shell (A) 2.59–2.50 3.73–3.60 2.90–2.80 2.69–2.60 3.0–2.90 2.90–2.80

Wavelength (A) 0.9393 0.9793 0.933 0.933 0.931 0.931

No. measurements 2,198,107 1,222,209 893,572 876,420 1,152,624 404,706

No. unique reflections 119,750 38,449 46,430 53,933 37,586 40,910Redundancy 8 9 7 7 6 5

I/s(I)a 25.7 (3.4) 20.0 (10.0) 16.0 (2.5) 13.4 (1.9) 14 (2.2) 13.6 (2.4)

R-merge (%)b 7.6 (33) 12.0 (23) 8.9 (48) 9.9 (49) 9.2 (57) 6.3 (43)ESRF Beam line ID14-EH4 ID29 ID14-EH2 ID14-EH2 ID14-EH3 ID14-EH3

Phasing statistics

Number of sites 55Phasing powerc 3.9

Figure of merit

(acentric/centric)

0.62/0.33

Refinement

Residues in model 2787 1387 1389 1391 1338

Number of solvent

molecules

760 191 315 230 263

B factor (A2) 49 68 52 80 70

Rcrystd 18.9 21.2 19.6 26.4 22.9

Rfreed 24.6 26.5 25.9 32.9 30.1

r.m.s. deviation from ideal geometry

Bond lengths (A) 0.011 0.009 0.008 0.011 0.011

Bond angles (deg.) 1.28 1.16 1.14 1.31 1.32

a Number in parentheses indicate the outer resolution shell.b R-mergeZ

Phkl

Pi jIiðhklÞKhIiðhklÞij=

Phkl

Pi IiðhklÞ, where Ii(hkl) is the intensity of the ith observation and hIi(hkl)i is the mean intensity of

reflection hkl, respectively.c Phasing powerZthe r.m.s heavy atom structure amplitude divided by the lack of closure error.d RZ

Phkl jjFojKjFcjj=

Phkl jFoj, where Fo and Fc are the observed and calculated structure factor amplitudes, respectively.


family of enzymes (Table 2). However, sequenceidentities are low (in the range of 8%–23%).Sequence alignment based on structural super-position of methyl transferases has shown that theonly highly conserved residues in the SAM-bindingregion of the core fold are part of a glycine-richsequence (named motif I) between b1 and aA (b4and a4 of CmcI), which interacts with the amino

acid portion of SAM, and an acidic loop between b2and aB (motif II) (b5 and a5 of CmcI), whichinteracts with the ribose hydroxyls.21 These motifsare highlighted in Figure 2.

The structure with the highest similarity to CmcIis cathechol O-methyltransferase (COMT).23 Super-position of a monomer of CmcI and COMT showsthat the nucleotide-binding motifs overlap (r.m.s.

Figure 5. SAM-binding to CmcI.Stereo view with SAM carbonatoms depicted in green. Electrondensity for the cofactor from a2FoKFc map is contoured at 1s.

Figure 6. The binding of PEG toCmcI. Close-up of the dimer inter-face between two subunits (in blueand yellow) in the same orientationas in Figure 3(d). Two PEG mole-cules (carbon atoms in white) arebound. The long PEG moleculeligates the Mg ions shown asgrey spheres. The bound cofactorSAM is shown in green. Electrondensity for the PEG molecules wascalculated from a 2FoKFc map andis contoured at 1s.


deviation 3.1 A for 167 Ca atoms of the nucleotide-binding domain). The SAM-binding site (Figure 5)is found in an equivalent position in all knownmethyltransferases although the residues that bindSAM are poorly conserved.21

The similarity of CmcI to COMT extends to themagnesium-binding site. In COMT, the Mg2C wasshown to be crucial for cofactor/substrate bindingand catalysis, although the Mg2C did not interactdirectly with SAM.23 The Mg2C in COMT iscoordinated by the carboxyl oxygen atoms of

Asp141, Asp169 and Asn170. Structural super-position of COMT and CmcI revealed that CmcIbinds Mg2C at the same position, coordinatedby Asp160, Glu186 and Asp187, where Asp160corresponds to Asp141 in COMT.

CmcI also shows a high structural similarity toNAD-dependent dehydrogenases. The NAD-dependent dehydrogenases display a low sequencesimilarity in the NAD-binding residues, and inthe glycine-rich motif at the same position as motif Iof the methyltransferases.21 At place 40 in our

Figure 7. Docking of 7a-hydroxy-O-carbamoyl DAC in the structureof CmcI. (a) Close-up of the activesite. 7a-Hydroxy-O-carbamoylDAC is in magenta, the cofactorSAM is in green and the Mg2C isdepicted as a grey sphere. Residuesfrom one monomer are in blueand from the second monomer inyellow. (b) Overview of a dimershowing the overlap of 7a-hydroxy-O-carbamoyl DAC withPEG. Colour coding as in (a) withPEG carbon atoms depicted inwhite. For clarity, binding of thedocked substrate to one of theMg2C sites only is shown.

Table 2. DALI search results for structural homologs to CmcI

Pos. PDB ID Z-score RMSD RA %SI Description

1 1vid 13.8 3.1 167 16 Cathechol O-methyltransferase (R. norvegicus)2 1af7 10.6 3.8 154 17 CheR chemotaxis receptor methyltransferase (S. typhimurium)3 1i9g 10.4 3.8 161 20 Rv2118c methyltransferase (M. tuberculosis)4 1xva 10.0 5.0 169 21 Glycine N-methyltransferase (R. norvegicus)5 1fbn 10.0 4.8 162 18 Fibrillarin homolog (M. jannaschii)6 1ej0 10.0 3.1 151 17 FtsJ RNA methyltransferase (E. coli)7 1qzz 9.9 3.2 156 23 Aclacinomycin-10-hydroxylase (S. purpurascens)8 1fpq 9.8 4.1 153 20 Isoliquiritigenin 2-O-methyltransferase (M. sativa)9 1nv8 9.6 6.6 168 22 N5-Glutamine methyltransferase (T. maritima)10 1kxz 9.5 3.0 138 19 Precorrin-6y methyltransferase (M. thermoautotrophicum)40 1kyq 6.7 3.0 101 12 met8P, dehydrogenase and ferrochelatase (S. cerevisiae)

PDB, Protein Data Bank; RMSD, root-mean-square deviation; RA, number of residues aligned; %SI, percent sequence identity over thealigned fragments.


homology search is Met8p,24 a bifunctional enzymethat performs dehydrogenation and ferrochelationin sirohaem biosynthesis. Met8p aligns with CmcIby overlapping its NAD-binding site on theC-terminal domain of CmcI.

Binding of NADH

To probe the binding of NADH, both wild-typeand L10Q/D160N mutant enzymes were co-crystal-lised with 20 mM NADH. NADH binding was onlyobserved in the mutant and its density was weak.The density corresponds to the central ribose-phosphate moiety, whereas the nicotinamide andadenine parts are only partly visible in the maps.NADH is positioned in the cavity between themolecules (Figure 3(d)), hence there are only threemolecules of NADH bound per hexamer. Thisbinding does not correspond to the expectedmode of binding of NAD/NADH to the nucleo-tide-binding motif20 and does not coincide with theSAM-binding site. The maps from wild-typeenzyme crystallised with NADH do not featuredensity for NADH, instead continuous density forPEG is observed at the corresponding position.

Computational docking

Attempts to co-crystallise CmcI with SAM, Mg2C

and cephalosporin C (Na-salt) or O-carbamoyl DACwere unsuccessful, probably because of thehydrolysis of the cephalosporin and/or the bindingof PEG in the expected cephalosporin-binding site.

We therefore attempted docking of three differentligands: 7a-hydroxy-O-carbamoyl DAC (the pro-posed substrate), O-carbamoyl DAC (its precursor)and cephamycin C (the proposed methylatedproduct) to the ternary CmcI–SAM–Mg2C complexby computational methods based on AutoDock3.25

The results show that all three ligands dock well.Docking energies for the hydroxylated intermedi-ate, 7a-hydroxy-O-carbamoyl DAC rank betweenK12 kcal/mol (top score) and K7.8 kcal/mol(lowest score). Docking energies of the other ligandsare in comparable range. The docked ligands with

their hydroxy group close (!3.5 A) to the Mg2C

have docking energies between K10.0 andK9.0 kcal/mol. Based on these results, a modelfor substrate binding can be deduced (Figure 7(a))where the 7a-hydroxyl is coordinated to the Mg2C

(2.1 A distance) and 3.2 A away from the methylgroup of SAM with the a-side facing the SAMmolecule. The binding site for the intermediateoverlaps partly with the binding site of the PEGmolecule observed in the crystal structure(Figure 7(b)). The adipyl side-chain of the cepha-losporin is held in place by Asp187 and Tyr191,whereas the carbamoyl side-chain interacts withArg16.

Discussion

In sequence databases, the product of cmcI isannotated as a cephalosporin hydroxylase,15

belonging to a family of 2-oxoglutarate-dependentdioxygenases. Data presented here indicate that thisannotation is incorrect. The structure of CmcIsuggests that the most likely function of thisenzyme is to work as a methyltransferase. TheC-terminal domain contains an a/b nucleotide-binding fold typical for SAM-dependent methyl-transferases, and binds SAM or SAH in a geometryexpected for these enzymes21 in both wild-type andmutant enzymes. The fold has features similar tosmall molecule methyltransferases, with the patternof additions/insertions to the core typical for thissubclass.21 The sequence identity of CmcI tomethyltransferases is weak but significant.

The structure also shows CmcI binds a Mg2C closeto the bound SAM, while no magnesium binding wasdetected in the L10Q/D160N mutant, indicating thatAsp160 is necessary for magnesium binding. Thismode of Mg2C binding was also found in COMT, amethyltransferase that gave the highest similarityscore in structural homology searches with the CmcIstructure (Table 2). In COMT, Mg2C binds thesubstrate and it is essential for activity. While Mg2C

is not documented as an explicit cofactor for7a-hydroxy-cephem methylation, the conditions for


the methylation reaction were established using assaysystems containing endogenous Mg2C.4,8,9 Thestructural data clearly suggest magnesium boundto CmcI.

We tried to co-crystallise CmcI with Fe(II) butdid not get diffracting crystals. Trials to soakL10Q/D160N mutant crystals with Fe(II) resultedin no density for iron in the electron density maps.Members of the family of 2-oxoglutarate-dependentdioxygenases share a common fold,26 and featurea common 2-His-1-carboxylate motif that bindsFe(II).27–29 While 2-oxoglutarate was a necessaryadditive to obtain crystals of the L10Q/D160Nmutant,16 no 2-oxoglutarate binding could beobserved in the crystal structure. The structure ofCmcI does not contain the jelly-roll fold common to2-oxoglutarate-dependent dioxygenases26,27 or the2-His-1-carboxylate Fe(II) binding motif. Weconclude that CmcI alone is not a 2-oxoglutarate-dependent enzyme, and it is unlikely that it is theenzyme purified by Xiao et al.8

The structure of the magnesium complexsuggests a way for cephalosporin binding nearthe active site. However, attempts to obtain acomplex with bound cephalosporin (in thepresence or absence of SAM) by co-crystallisationfailed. This is most likely due to the hydrolyticbreakdown of cephalosporin during crystallisation,and/or the presence of PEG (from the crystal-lisation medium) at the expected cephalosporin-binding site near the magnesium and SAM sites. Incrystals of the wild-type enzyme, PEG binds at thissite, and ligates the magnesium ion via a hydroxylgroup. In the mutant, Asp160 is replaced byAsn160, and this substitution hinders Mg2C

binding, which in turns abolishes PEG bindingnear this area.

The binding of the cephalosporin substrate to theternary CmcI–SAM–Mg2C complex was thereforesimulated. In these docking simulations, the boundPEG was removed from the structure. Results with7a-hydroxy-O-carbamoyl DAC indicate a plausiblebinding site for the putative cephalosporin sub-strate with its bicyclic core overlapping with one ofthe PEG molecules (Figure 7(b)). In this model, thecephalosporin is bound with its 7a-hydroxyl groupcoordinated to the Mg2C and its a-side facing theSAM molecule in an orientation that would allowmethylation of the hydroxyl-group. The reactionwould be facilitated by proximity to the Mg2C thatwould lower the pKa of the hydroxyl group,allowing the resulting ionised hydroxyl to make anucleophilic attack on the methyl group of SAM.The docked conformation of the cephalosporin–Mg2C complex shows cephalosporin bound in asimilar position as a bound inhibitor in the crystalstructure of COMT.23

The structural evidence strongly suggests thatCmcI acts as a methyltransferase in the biosynthesisof cephamycins. The substrate for this reaction is the7a-hydroxylated cephalosporin. Historically,the methyltransferase activity of CmcI/CmcJ ofA. lactamdurans was characterised using assays that

included both CmcI and CmcJ in a recombinantform.10 Unfortunately, we could not express solubleCmcJ from S. clavuligerus, and thus we were unable toperform similar experiments on CmcI/CmcJ fromS. clavuligerus. We also had problems with assayingCmcI directly for the relevant methyltransferasereaction because of a lack of the 7a-hydroxylatedsubstrate, which is not available commercially.

Other possible scenarios for CmcI catalysisinclude radical SAM reactions and/or the formationof a multi-enzyme complex between CmcI and CmcJ(see below). Known SAM radical enzymes possessan essential Fe4S4 cluster and display a characteristicCxxxCxxC sequence motif (C, Cys; x any aminoacid) that coordinates the Fe4S4 cluster.30 Theabsence of these features in CmcI argues against aclassical radical mechanism here. However, othertypes of reactions with one-electron donors maylead to radical formation on SAM. One of theproteins with a high score (7) in structural homologysearches against CmcI is aclacinomycin-10-hydroxylase from S. purpurascens (Table 2). Thisprotein shares the fold and mode of cofactor bindingof methyltransferases, but it is a hydroxylaserequiring SAM, molecular oxygen, and a reducingagent (GSH or DTT) for activity.31

If CmcI is a methyltransferase, then CmcJ is mostlikely the hydroxylase required for the first reactionin the sequence. The sequence of CmcJ contains anHXD motif common to the iron-binding site in2-oxoacid-dependent oxygenases. There are datain the literature, suggesting that CmcI and CmcJform a complex to perform the hydroxylation andmethylation reaction together.9,10 While there is nodirect evidence in the structure of CmcI for thissuggestion, we cannot exclude the possibility ofcomplex formation between CmcI and CmcJ.

Materials and Methods

Cloning, mutagenesis and purification

The cloning and purification of the cmcI gene producthas been described in detail earlier.16 In brief, the genewas amplified by PCR from Streptomyces clavuligerusgenomic DNA, using gene-specific primers with thecorrect overhangs for cloning into pET32 Xa/LIC(Novagen). The correction of the two positions disagree-ing with the previously reported sequence data was doneusing the QuickChange method (Stratagene) usingappropriate primers. The resulting expression plasmids(cmcI-N160pET32 and cmcI-D160pET32) were thentransformed into Escherichia coli BL21 (DE3) and thecells were grown, induced and harvested. The pET32 tagwas cleaved off with factor Xa (Amersham Biosciences)and the resulting native cmcI gene product purified aspreviously described.16

Crystallisation

Crystals were grown by the hanging-drop vapour-diffusion method at 20 8C. Crystals of unligandedL10Q/D160N mutant were obtained by mixing equal


volumes of the protein sample with a precipitatingsolution containing 8–12% (w/v) PEG 4000, 10 mM2-oxoglutarate, 0.2 M sodium-acetate and 0.1 M Tris(pH 7.5).16 Crystals of complexes with SAM and SAHwere obtained using similar conditions. The protein wasincubated with 10 mM SAM or SAH in room temperaturefor 1 h prior to crystallisation. Se-methionine substitutedmutant enzyme was crystallised using the sameconditions, except including 5 mM b-mercaptoethanolin the solutions.

Crystals of the wild-type enzyme were obtained using aprecipitating solution containing 0.2 M MgCl2, 0.1 M Tris(pH 7.5) and 1:1 mixtures of PEG with different molecularmass cut-off: 10–14% PEG 4000, 4–7% PEG 1000/8000, or5–7% PEG 550/20,000. Crystals of complexes with Mg2C

and SAM were obtained using similar conditions, butonly appeared from the 550/20,000 mixture. The proteinwas incubated with 10 mM SAM in room temperature for1 h prior to crystallisation. Crystals of wild-type andmutant enzymes with NADH were grown under similarconditions as the respective apo-enzymes. The sampleswere incubated with 20 mM NADH for 1 h prior tocrystallisation.

X-ray data collection

For data collection at cryogenic temperatures thecrystals were transferred to a solution of 25% ethyleneglycol in respective mother liquor and flash-frozen inliquid nitrogen. Data processing and scaling wereperformed with the programs Denzo and Scalepack.32

Data collection statistics are summarised in Table 1.Data to 2.5 A resolution of the apo-form of L10Q/

D160N were collected at 100 K on beam line ID14-EH4 ofthe European Synchrotron Radiation Facility (ESRF,Grenoble, France) (Table 1). The crystal belongs to spacegroup P21 with cell dimensions aZ93.6 A, bZ182.6 A,cZ103.2 A and bZ91.08. Assuming 12 molecules in theasymmetric unit gives a VM

33 of 2.7 A3 DaK1.A single-wavelength anomalous diffraction data set

to 3.6 A resolution was collected from a crystal ofSe-methionine derivatised mutant enzyme at beam lineID29 of ESRF using a wavelength of 0.9793 A (Table 1).The crystal belongs to space group P212121 with celldimension aZ92.7 A, bZ101.9 A and cZ182.0 A. Thiscrystal form contains six molecules in the asymmetricunit with a corresponding VM

33 of 2.7 A3 DaK1.Diffraction data from crystals of wild-type CmcI with

Mg2C and Mg2C–SAM were collected at ID14-EH2 at theESRF (Table 1). Crystals of CmcI–Mg2C belong to spacegroup P212121 with cell dimensions aZ77.9 A, bZ145.5 Aand cZ162.1 A. There are six molecules in the asymmetricunit. Crystals of CmcI–Mg2C–SAM belong to space groupP212121 with cell dimensions aZ91.5 A, bZ102.4 A andcZ181.9 A, and have six molecules in the asymmetricunit.

Diffraction data from crystals of the mutant enzymewith SAM and SAH were collected at beam line ID14-EH3at the ESRF. Both crystals belong to space group P212121

and have cell parameters similar to the correspondingcomplex with the wild-type enzyme (Table 1).

Structure determination from single-wavelengthanomalous diffraction data

The structure was solved from a Se-methioninederivative. The asymmetric unit contains six polypeptidechains, and each polypeptide contains ten methionine

residues. Using a resolution cut-off of 3.8 A, 52 seleniumsites (out of a total of 60 in the asymmetric unit) werelocated with a correlation coefficient of 41% using theprogram HySS.17 Initial phases were obtained withSHARP,34 extending the resolution to 3.6 A. Final phasesin SHARP were based on a total of 55 sites, and thesewere subsequently improved by density modificationprocedures in SOLOMON,35 implemented in SHARP, andgave an excellent and interpretable electron density map.This map was further improved by exploiting the 6-foldnon-crystallographic symmetry (NCS) of the asymmetricunit. Initial local symmetry operators were determined inO,18 refined with IMP,36 and used in local averagingperformed within O. An initial model was built into thismap in O, including residues 9–230 (out of 236 in total).Knowledge of the location of nine selenium sites allowedthe unequivocal assignment of the position and directionof the polypeptide chain.

Structure refinement and analysis

Five percent of the data were set aside for calculationsof a free R factor. The initial model of Se-methioninesubstituted CmcI was refined to 3.6 A resolution usingNCS Phased Refinement in CCP4i,37 which cyclesbetween Refmac538 and DM,39 using 6-fold averaging.This resulted in R-factors RcrystZ28.5% and RfreeZ36%.For phase extension to 2.5 A, the hexameric model wasplaced in the apo-CmcI cell using MOLREP,40 resulting ina correlation coefficient of 39% and R-factor of 51%. Thedata to higher resolution (2.5 A) come from the under-ivatised mutant enzyme, which has a P21 cell andcontains two hexamers in the asymmetric unit. Thisdata set was used for further refinement and rebuilding.The model was refined with NCS phased refinementusing 12-fold averaging and the structure was inspectedand rebuilt in O. Further refinement was done by themaximum likelihood method in Refmac5,38 using strongNCS restraints that were gradually released, and TLSparameter refinement with each monomer as a TLSgroup. Water molecules were added using the programarp/warp41 and were manually inspected in O. Thequality of the final model was assessed with PRO-CHECK.19 A summary of refinement and final modelstatistics is given in Table 1.

The structures of complexes of wild-type enzyme withMg2C and Mg2C–SAM, and mutant enzyme with SAMand SAH were determined by molecular replacementusing MOLREP, with a hexamer of the refined apo-CmcIas the search model. The structures were refined usingRefmac5 following the same protocol used for the apo-enzyme structure (Table 1).

Computational docking

Docking studies were performed using AutoDock3,25

with the CmcI–Mg2C–SAM structure and three differentligands: 7a-hydroxy-O-carbamoyl DAC, O-carbamoylDAC and cephamycin C. The ligand structures wereobtained from the Dundee PRODRG2 server.42 Ligandsand protein were prepared for docking by adding hydrogenatoms and charges using AutoDockTools.43 The search forthe best interaction energy was carried out using theLamarckian genetic algorithm. Each docking simulationconsisted of 100 independent runs, with a population size of200, 500 generations, and a maximum of 25 million energyevaluations. Initially, docking was done over the entireCmcI structure and each ligand, in order to determine


the proper docking site and ligand. Subsequently, thedocking area could be reduced to the cleft between the Mgions of two adjacent chains in a 33 A cubic box. Solutionswere ranked based on their docking energies and visuallyinspected in O.18

Sequence comparison and analysis of the structures

Multiple sequence alignments were performed usingCLUSTAL-W44 and secondary structure assignment wasmade with DSSP.45

Figures were prepared using the programs Molscript,46

Raster3D47 and PyMol.48

Protein Data Bank accession numbers

Atomic coordinates and structure factor amplitudeshave been deposited with the RCSB Protein Data Bank,accession codes 2bm8 (L10Q/D160N apo-enzyme), 2bm9(L10Q/D160N SAM-complex) 2br5 (L10Q/D160N SAH-complex), 2br3 (wild-type Mg2C-complex) and 2br4(wild-type SAM/Mg2C-complex).

Acknowledgements

We thank Christopher J. Schofield for discussions;T. Alwyn Jones for help with new options in O; RalfGrosse-Kunstleve for help with and access to HySSprior to publication; ESRF and the EMBL Outstationin Grenoble for providing beam time and datacollection facilities. This work was supported bygrants from the Swedish Natural Science ResearchCouncil (VR), and the European Union (no. QLK3-CT-2000-00513).

References

1. Matagne,A., Dubus,A.,Galleni,M.&Frere, J.-M. (1999).The beta-lactamase cycle: a tale of selective pressure andbacterial ingenuity. Nature Prod. Rep. 16, 1–19.

2. Stapley, E. O., Birnbaum, J., Miller, A., Wallick, H.,Hendlin, D. & Woodruff, H. (1979). Cefoxitin andcephamycins: microbiological studies. Ref. Infect. Dis.1, 73–87.

3. Baldwin, J. E. & Schofield, C. J. (1992). The biosyn-thesis of b-Lactams. In The Chemistry of b-Lactams(Page, M. I., ed.), pp. 1–78, Blackie, London.

4. O’Sullivan, J. & Abraham, E. P. (1980). The conversionof cephalosporins to 7a-methoxycephalosporins bycell-free extracts of Streptomyces clavuligerus. Biochem.J. 186, 613–616.

5. Hood, J. D., Elson, A., Gilpin, M. L. & Brown, A. G.(1983). Identification of 7a-hydroxycephalosporin Cas an intermediate in the methoxylation of cephalos-porin C by a cell free extract of Streptomycesclavuligerus. J. Chem. Soc. Chem. Commun., 1187–1188.

6. O’Sullivan, J., Aplin, R. T., Stevens, C. M. & Abraham,E. P. (1979). Biosynthesis of a 7-alpha-methoxycepha-losporin. Incorporation of molecular oxygen. Biochem.J. 179, 47–52.

7. Whitney, J. G., Brannon, D. R., Mabe, J. A. & Wicker,K. J. (1972). Incorporation of labeled precursors into

A16886B, a novel b-lactam antibiotic produced byStreptomyces clavuligerus. Antimicrob. Agents Che-mother. 1, 247–251.

8. Xiao, X., Wolfe, S. & Demain, A. L. (1991). Purificationand characterization of cephalosporin 7a-hydroxylasefrom Streptomyces clavuligerus. Biochem. J. 280, 471–474.

9. Coque, J. J. R., Enguita, F. J., Martin, J. F. & Liras, P.(1995). A two-protein component 7a-cephem-meth-oxylase encoded by two genes of the Cephamycin Ccluster converts Cephalosporin C to 7-methoxyce-phalosporin C. J. Bacteriol. 177, 2230–2235.

10. Enguita, F. J., Liras, P., Leitao, A. L. & Martin, J. F.(1996). Interaction of the two proteins of themethoxylation system involved in Cephamycin Cbiosynthesis. J. Biol. Chem. 271, 33225–33230.

11. Alexander, D. C. & Jensen, S. E. (1998). Investigationof the Streptomyces clavuligerus Cephamycin C genecluster and its regulation by the CcaR protein.J. Bacteriol. 180, 4068–4079.

12. Tudzynski, B., Mihlan, M., Rojas, M. C.,Linnemannstons, P., Gaskin, P. & Hedden, P. (2003).CLUSTAL W: improving the sensitivity of progressivemultiple sequence alignment through sequenceweighting, position-specific gap penalties and weightmatrix choice. J. Biol. Chem. 278, 28635–28643.

13. Brakhage, A. A. (1998). Molecular regulation of beta-lactam biosynthesis in filamentous fungi. Microbiol.Mol. Biol. Rev. 62, 547–585.

14. Elander, R. P. (2003). Industrial production of beta-lactam antibiotics. Appl. Microbiol. Biotechnol. 61,385–392.

15. Tatusova, T., Karsch-Mizrachi, I. & Ostell, J. (1999).Complete genomes in WWW Entrez: data represen-tation and analysis. Bioinformatics, 15, 536–543.

16. Lester, D. R., Oster, L. M., Svenda, M. & Andersson, I.(2004). Expression, purification, crystallizationand preliminary X-ray diffraction studies of thecmcI component of Streptomyces clavuligerus 7a-cephem-methoxylase. Acta Crystallog. sect. D, 60,1618–1621.

17. Grosse-Kunstleve, R. W. & Adams, P. D. (2003).Substructure search procedures for macromolecularstructures. Acta Crystallog. sect. D, 59, 1966–1973.

18. Jones, T. A., Bergdoll, M. & Kjeldgaard, M. (1990). O: amacromolecule modelling environment. In Crystallo-graphic and Modeling Methods (in Molecular Design)(Bugg, C. & Ealick, S., eds), pp. 189–190, Springer,New York.

19. Laskowski, R. A., MacArthur, M. W., Moss, D. S. &Thornton, J. M. (1993). PROCHECK: a program tocheck the stereochemical quality of protein structures.J. Appl. Crystallog. 26, 283–291.

20. Rossmann, M. G., Moras, D. & Olsen, K. W. (1974).Chemical and biological evolution of a nucleotide-binding protein. Nature, 25, 194–199.

21. Martin, J. L. & McMillan, F. (2002). SAM (dependent) Iam: the S-adenosylmethionine-dependent methyl-transferase fold. Curr. Opin. Struct. Biol. 12, 783–793.

22. Holm, L. & Sander, C. (1993). Protein structurecomparison by alignment of distance matrices.J. Mol. Biol. 233, 123–138.

23. Vidgren, J., Svensson, A. L. & Liljas, A. (1994). Crystalstructure of catechol O-methyltransferase. Nature,368, 354–358.

24. Schubert, H. L., Raux, E., Brindley, A. A., Leech, H. K.,Wilson, K. S., Hill, C. P. & Warren, M. J. (2002).The structure of Saccharomyces cerevisiae Met8p, abifunctional dehydrogenase and ferrochelatase.EMBO J. 21, 2068–2075.


25. Morris, G. M., Goodsell, D. S., Halliday, R. S., Huey, R.,Hart, W. E., Belew, R. K. & Olsson, A. J. (1998).Automated docking using a Lamarckian geneticalgorithm and an empirical binding free energyfunction. J. Comput. Chem. 19, 1639–1662.

26. Prescott, A. G. (1993). A dilemma of dioxygenases(or where biochemistry and molecular biology fail tomeet). J. Exp. Bot. 44, 849–861.

27. Roach, P. L., Clifton, I. J., Fulop, V., Harlos, K., Barton,G. J., Hajdu, J. et al. (1995). Crystal structure ofisopenicillin N synthase is the first from a newstructural family of enzymes. Nature, 375, 700–704.

28. Hegg, E. L. & Que, L. (1997). The 2-His-carboxylatefacial triad An emerging structural motif in mono-nuclear non-heme iron(II) enzymes. Eur. J. Biochem.250, 625–629.

29. Hausinger, R. P. (2004). FeII/alpha-ketoglutarate-dependent hydroxylases and related enzymes. Crit.Rev. Biochem. Mol. Biol. 39, 21–68.

30. Layer, G., Heinz, D. W., Jahn, D. & Schubert, W. D.(2004). Structure and function of radical SAMenzymes. Curr. Opin. Chem. Biol. 8, 468–476.

31. Jansson, A., Niemi, J., Lindqvist, Y., Mantsala, P. &Schneider, G. (2003). Crystal structure of aclacinomy-cin-10-hydroxylase, a S-adenosyl-L-methionine-dependent methyltransferase homolog involved inAnthracycline biosynthesis in Streptomyces purpuras-cens. J. Mol. Biol. 334, 269–280.

32. Otwinowski, Z. & Minor, W. (1997). Processing ofX-ray diffraction data collected in oscillation mode.Methods Enzymol. 276, 307–326.

33. Matthews, B. W. (1968). Solvent content of proteincrystals. J. Mol. Biol. 33, 491–497.

34. La Fortelle, E. & Bricogne, G. (1997). Maximum-likelihood heavy-atom parameter refinement for themultiple isomorphous replacement and multiwave-length anomalous diffraction methods. Methods Enzy-mol. 276, 472–492.

35. Abrahams, J. P. & Leslie, A. G. W. (1996). Methodsused in the structure determination of bovinemitochondrial F1 ATPase. Acta Crystallog. sect. D, 52,30–42.

36. Kleywegt, G. J. & Read, R. J. (1997). Not your averagedensity. Structure, 5, 1557–1569.

37. Collaborative Computational Project, Number 4(1994). The CCP4 suite of programs for proteincrystallography. Acta Crystallog. sect. D, 50, 760–763.

38. Murshudov, G. N., Vagin, A. A. & Dodson, E. J. (1997).Refinement of macromolecular structures by themaximum-likelihood method. Acta Crystallog. sect.D, 53, 240–255.

39. Cowtan, K. (1994). dm: an automated procedure forphase improvement by density modification. JointCCP4 and ESF-EACBM Newsletter on Protein Crystallo-graphy, 31, 34–38.

40. Vagin, A. & Teplyakov, A. (1997). MOLREP: anautomated program for molecular replacement.J. Appl. Crystallog. 30, 1022–1025.

41. Perrakis, A., Sixma, T. K., Wilson, K. S. & Lamzin, V. S.(1997). wARP: improvement and extension ofcrystallographic phases by weighted averaging ofmultiple-refined dummy atomic models. Acta Crystal-log. sect. D, 53, 448–455.

42. Schuettelkopf, A. W. & van Alten, D. M. F. (2004).PRODRG—a tool for high-throughput crystallo-graphy of protein–ligand complexes. Acta Crystallog.sect. D, 60, 1355–1363.

43. Sanner, M. F., Olsson, A. J. & Spehner, J. C. (1996).Reduced surface: an efficient way to computemolecular surfaces. Biopolymers, 38, 305–320.

44. Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994).CLUSTAL W: improving the sensitivity of progressivemultiple sequence alignment through sequenceweighting, position-specific gap penalties and weightmatrix choice. Nucl. Acids Res. 22, 4673–4680.

45. Kabsch, W. & Sander, C. (1983). Dictionary of proteinsecondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers, 22,2577–2637.

46. Kraulis, P. J. (1991). MOLSCRIPT: a program toproduce both detailed and schematic plots of protein.J. Appl. Crystallog. 24, 946–950.

47. Merrit, E. A. & Bacon, D. J. (1997). Raster3D:photorealistic molecular graphics. Methods Enzymol.277, 505–524.

48. DeLano, W. L. (2002). The Pymol Molecular GraphicsSystem, DeLano Scientific, San Carlos, CA, USA.

49. Valegard, K., Terwisscha van Scheltinga, A. C., Lloyd,M. D., Hara, T., Ramaswamy, S., Perrakis, A. et al.(1998). Structure of a cephalosporin synthase. Nature,394, 805–809.

50. Valegard, K., Terwisscha van Scheltinga, A. C.,Dubus, A., Ranghino, G., Oster, L. M., Hajdu, J. &Andersson, I. (2004). The structural basis of cepha-losporin formation in a mononuclear ferrous enzyme.Nature Struct. Mol. Biol. 11, 95–101.

51. Coque, J. J., Perez-Llarena, F. J., Enguita, F. J., Fuente,J. L., Martin, J. F. & Liras, P. (1995). Characterisationof the cmcH genes of Nocardia lactamdurans andStreptomyces clavuligerus encoding a functional 3 0-hydroxymethylcephem O-carbamoyltransferase forcephamycin biosynthesis. Gene, 162, 21–27.

Edited by R. Huber

(Received 22 December 2005; received in revised form 27 January 2006; accepted 2 February 2006)Available online 21 February 2006

insights into cephamycin biosynthesis: the crystal structure of cmci from streptomyces clavuligerus

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