Structural Basis for Activation of the ThiaminDiphosphate-dependent Enzyme Oxalyl-CoADecarboxylase by Adenosine Diphosphate*□S

Received for publication, September 8, 2005, and in revised form, October 7, 2005 Published, JBC Papers in Press, October 10, 2005, DOI 10.1074/jbc.M509921200

Catrine L. Berthold‡, Patricia Moussatche§, Nigel G. J. Richards§, and Ylva Lindqvist‡1

From the ‡Molecular Structural Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-17177Stockholm, Sweden and the §Department of Chemistry, University of Florida, Gainesville, Florida 32611-7200

Oxalyl-coenzyme A decarboxylase is a thiamin diphosphate-de-pendent enzyme that plays an important role in the catabolism ofthe highly toxic compound oxalate.We have determined the crystalstructure of the enzyme fromOxalobacter formigenes from a hemi-hedrally twinned crystal to 1.73 A resolution and characterized thesteady-state kinetic behavior of the decarboxylase. Themonomer ofthe tetrameric enzyme consists of three �/�-type domains, com-monly seen in this class of enzymes, and the thiamin diphosphate-binding site is located at the expected subunit-subunit interfacebetween two of the domains with the cofactor bound in the con-served V-conformation. Although oxalyl-CoA decarboxylase isstructurally homologous to acetohydroxyacid synthase, a moleculeof ADP is bound in a region that is cognate to the FAD-binding siteobserved in acetohydroxyacid synthase and presumably fulfils asimilar role in stabilizing the protein structure. This differencebetween the two enzymes may have physiological importance sinceoxalyl-CoA decarboxylation is an essential step in ATP generationin O. formigenes, and the decarboxylase activity is stimulated byexogenousADP.Despite the significant degree of structural conser-vation between the two homologous enzymes and the similarity incatalytic mechanism to other thiamin diphosphate-dependentenzymes, the active site residues of oxalyl-CoA decarboxylase areunique. A suggestion for the reaction mechanism of the enzyme ispresented.

Oxalic acid is one of nature’s most highly oxidized organic com-pounds, and its dianion is a strong chelator of metal cations, especiallyCa2�, causing oxalate to be highly toxic to many organisms (1). Inhumans, elevated levels of oxalate are associated with several diseases,including the formation of calcium oxalate stones in the kidney (uroli-thiasis), renal failure, cardiomyopathy, and cardiac conductance disor-ders (1–3). Relatively large amounts of oxalate are introduced into thebody through the diet, although this diacidmay also arise as a byproductof normal cellular metabolism (4). Because humans, in common withothermammals, are not able to degrade oxalate, this compoundmust be

eliminated by excretion in the urine or via the intestine (5). The recentobservation that a symbiotic, gut-dwelling bacterium, Oxalobacter for-migenes, may regulate oxalate homeostasis in humans, therefore, hasimportant implications for efforts to develop new strategies for treatingoxalate-related diseases (6). O. formigenes is an obligate anaerobe bac-terium found in the gastrointestinal tracts of vertebrates, includinghumans, and is unusual in that it employs oxalate as the sole energysource for its survival (7, 8). As a result, this bacteriumnot only degradesfree oxalate entering the intestine lumen but also creates a transepithe-lial gradient favoring oxalate secretion and preventing absorption ofoxalic acid in the lower tract of the intestine (9). A direct correlationbetween the number of recurrent kidney stone episodes and a lack ofO.formigenes in the intestinal flora has recently been demonstrated (10,11). Unfortunately, efforts to re-colonize the gastrointestinal tract withO. formigenes in individuals who lack the organism have proven ineffec-tive to date, a finding stimulating interest in the development of oxalate-degrading enzyme-based therapies (11).Oxalate is anaerobically decarboxylated in O. formigenes to give CO2

and formate via a two-step pathway that is mediated by the coupledaction of two enzymes, formyl-CoA transferase (12–14) and oxalyl-CoAdecarboxylase (OXC)2 (15) (Fig. 1). The metabolic importance of thesetwo enzymes to the organism is evident from the observation that theymake up 20% of the total protein content in the cell (16). It has beenproposed that this oxalate degradation pathway, when coupled withoxalate/formate transport by an oxalate/formate transmembrane anti-porter (17, 18), is the origin of the transmembrane potential required forATP production in O. formigenes (19). The first step in oxalate catabo-lism involves formyl-CoA transferase-catalyzed transfer of CoA fromformyl-CoA to oxalate, which activates the oxalyl moiety for thiamin-dependent decarboxylation in the subsequent reaction mediated byOXC (15). The second step regenerates formyl-CoA and liberates CO2with consumption of one proton.Each subunit of nativeOXC consist of 568 amino acids, with amolec-

ular mass of 60,691 Da (20). The enzyme requires the cofactor thiamindiphosphate (ThDP) (15), the biologically active form of vitamin B1,which is common in proteins catalyzing the cleavage of carbon-carbonbonds adjacent to a carbonyl group. In this paper we report the 1.73 Åresolution crystal structure and kinetic characterization of recombinantOXC fromO. formigenes. This crystallographic study reveals a novel setof residues in the active site fromwhichwe propose amechanism for thedecarboxylation, together with an ADP-binding site. The ADP site

* This study was supported by grants from the Swedish Research Council-ScientificCouncil for Natural and Engineering Sciences (to Y. L.) and by National Institutes ofHealth Grant DK61666 (to N. G. J. R.). The costs of publication of this article weredefrayed in part by the payment of page charges. This article must therefore behereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.

□S The on-line version of this article (available at http://www.jbc.org) contains a purifica-tion table for recombinant, wild type OXC (Supplemental Table S1) and a structurebased sequence alignment of OXC and AHAS (Supplemental Fig. S1).

The atomic coordinates and structure factors (code 2C31) have been deposited in the ProteinData Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, NewBrunswick, NJ (http://www.rcsb.org/).

1 To whom correspondence should be addressed. Fax: 46-8-327626; E-mail:ylva.lindqvist@ki.se.

2 The abbreviations used are: OXC, oxalyl-CoA decarboxylase; ThDP, thiamin diphos-phate; POX, pyruvate oxidase; AHAS, acetohydroxyacid synthase; BFD, benzoylfor-mate decarboxylase; zPDC, pyruvate decarboxylase from Z. mobilis; yPDC, pyruvatedecarboxylase from yeast; MES, 4-morpholineethanesulfonic acid; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; HPLC, high performanceliquid chromatography.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 280, NO. 50, pp. 41645–41654, December 16, 2005© 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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http://www.jbc.org/cgi/content/full/M509921200/DC1Supplemental Material can be found at:

appears to have evolved from an FAD-binding site, which is present inthe related enzymes pyruvate oxidase (POX) (21) and acetohydroxyacidsynthase (AHAS) (22). Kinetic studies stimulated by this structuralobservation support the hypothesis that ADP is a high affinity activatorof the enzyme, a finding that may be of physiological relevance inO. formigenes.

MATERIALS AND METHODS

Expression and Purification—A BL21(DE3) Escherichia coli expres-sion strain transformed with pET-9a carrying the wild type oxalyl-CoAdecarboxylase (oxc) gene from O. formigenes (20) was generously sup-plied by Dr. Harmeet Sidhu (Ixion Biotechnology, Inc., Alachua, FL).Cells were grown in LB broth to an A600 of 0.3–0.6, and protein expres-sion was induced by the addition of isopropyl 1-thio-�-D-galactopy-ranoside to a final concentration of 0.4 mM. Cells were harvested bycentrifugation after 3 h at 37 °C (A600 1.8–2.0). Cell pellets from1 liter ofculture were suspended in 50 ml of lysis buffer (100 mM KH2PO4, pH7.2, 1 mM dithiothreitol, 10 mM MgCl2) and sonicated. The lysate wasclarified by centrifugation, and supernatant was loaded onto a 20-mlBlue-Sepharose fast flow affinity column equilibrated with Buffer A (25mM NaH2PO4, pH 7.2, 0.1 M NaCl) at a flow rate of 4 ml/min. Thecolumn was washed with Buffer A, and OXC was then eluted withBuffer B (25 mM NaH2PO4, pH 7.2, 2 M NaCl). Fractions containingOXC were combined, and buffer was exchanged using a Sephadex G25HiPrep 26/10 desalting column equilibrated with Buffer C (25 mM

NaH2PO4, pH 6.5) at a flow rate of 10 ml/min. OXC was eluted withBuffer C, and fractions containing the enzyme were loaded on aQ-Sepharose high performance anion exchange column equilibratedwith Buffer C at a flow rate 5 ml/min. Purified OXC was eluted using a10–40% gradient of Buffer D (25 mM NaH2PO4, pH 6.5, 0.5 M NaCl),and fractions were pooled. Purified OXC was concentrated in an Ami-con Stirred Cell and stored at �80 °C as previously described (23).

Crystallization—OXC was dialyzed against 50 mM MES buffer, pH6.5, and 10 mM ThDP and 10 mM MgCl2, and 1 mM coenzyme A wasadded. The solution was concentrated to a final protein concentrationof 5 mg/ml before crystallization by the hanging drop vapor-diffusiontechnique. Full details about crystallization screening and optimizationhave been published elsewhere (23). Useful diffraction-quality crystalswere obtained after �4 days, with a precipitating solution containing

27% polyethylene glycol 550monomethyl ether, 100mMBis-Tris buffer,pH 6.5, and 50 mM CaCl2.

Data Collection and Processing—Data were collected to 1.73 Å in anitrogen stream at 110 K at beamline ID23–1, European SynchrotronRadiation Facility, Grenoble, France, as described (23). No additionalcryoprotectant was needed, and the crystals were directly flash-frozenin the nitrogen stream. All images were processed usingMOSFLM (24),and the unit-cell parameters were determined using the autoindexingoption. The data set was scaled using the program SCALA (25) (TABLEONE).

Structure Solution and Crystallographic Refinement—Full detailsabout the structure solution procedure, including how twinning wascharacterized qualitatively and quantitatively, have been published else-where (23). Briefly, the data showed 622 symmetry, but hemihedraltwinning was detected using the intensity statistics and distributionsobtained from the CCP4 programs TRUNCATE (25, 26) and DETWIN(25, 27). The Yeates and FamMerohedral Crystal Twinning Server wasused as an additional indicator and for calculations of the twin fraction

TABLE ONE

Data collection statisticsValues in parentheses are for the highest resolution interval.

Resolution (Å) 29.24-1.73 (1.82-1.73)No. of observations 1,004,713 (145,215)No. of unique reflections 140,751 (20,434)Rsym (%)a 9.8 (49.2)Completeness (%) 99.8 (100.0)Mean (I/�(I)) 17.9 (3.8)Multiplicity (%) 7.1 (7.1)

aRsym � �hkl�i �Ii � �I��/�hkl�i� I�, where Ii is the intensity measurement for areflection, and �I� is the mean value for this reflection.

TABLE TWO

Structure refinement and final model statistics

Resolution (Å) 29.2-1.73Twinned Rwork (%) 15.1Twinned Rfree (%) 17.5Number of amino acids 2 � 546Number of atoms 8999Protein 8231Ligands 108Water 658Ion 2B-factors (Å2)Wilson plot 21.1Protein 24.6Main chain 23.4Side chain 25.9Ligands 23.5Water 32.9Ion 17.8Root mean square deviation from ideal geometryBond length (Å) 0.007Bond angles (°) 1.29Ramachandran plot (%)Residues in most favored regions 89.6Residues in additional allowed 10.0Residues in generously allowed 0.2Residues in disallowed 0.2

FIGURE 1. Catabolism of oxalate by O. formigenes.

Crystal Structure of Oxalyl-CoA Decarboxylase

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(�) (28). An incomplete low resolution model of OXC has previouslybeen determined fromcrystals in space group P42212 diffracting to 4.1Åresolution (23). This model, covering 468 of 568 residues, was obtainedby molecular replacement using a polyalanine model of AHAS (22),omitting loops and theC-terminal domain. The low resolutionmodel ofOXC was subsequently used for molecular replacement in the highresolution data with the program MOLREP (25, 29) searching for adimer in all possible true space groups. The molecular replacementsearch was initially performed in twinned data without any success andwas, therefore, continued in detwinned data generated by DETWIN.The true space group P3121 was deduced based on packing consid-

erations and statistical comparisons. The structure of OXC in the P3121crystal form was refined to 1.73 Å with a fixed twin fraction of � � 0.43using the protocol for twinned data in the CNS software (30). A test setof 4.6% of the reflections was excluded to monitor the Rfree value. Carewas taken to keep the twin-related reflections together in either the testor work set in order not to bias the refinement. Refinement with simu-lated-annealing, individual B-factor, and energy minimization proce-dures was interspersed with rounds of manual model building in�A-weighted 2Fo � Fc electron density maps with the graphics programO (31) (TABLE TWO). Initially, non-crystallographic symmetryrestraints were applied but were later abandoned. All ligand library fileswere created with the Dundee PRODRG2 server (32). Water moleculeswere added automatically in CNS at Fo � Fc difference density peaks3� followed by manual inspection and additional assignment in themolecular graphics programCoot (33). After convergence of the refine-ment in CNS, refinement of both the coordinates and the twin fractionwas performed in SHELXL (34). The test set was kept intact during allrefinement steps. The refined twin faction � � 0.440 was used in thefinal refinement steps inCNSwhen double conformations for some sidechains were added.

Structural Analysis—The final model was validated with the PRO-CHECK (35, 36) and CNS (30) programs. Structural comparison withhomologous structures were carried out using the SSM superposition inCoot (33) and the least square facility inOusing default parameters (31).All figures portraying protein models were prepared using PyMOL(www.pymol.org).

Size Exclusion Chromatography Measurements—Size exclusionchromatography was carried out following the procedure described inJonsson et al. (14) using Buffer E (100mMKH2PO4, pH7.0, 100mMKCl)at a flow rate 1ml/min.Molecular weight standards usedwere lysozyme(14.4 kDa), carbonic anhydrase (29 kDa), peroxidase (44 kDa), bovineserum albumin (66 kDa), alcohol dehydrogenase (150 kDa), apoferritin(443 kDa), and thyroglobulin (669 kDa). Void volume (vo) was deter-mined with blue dextran (2000 kDa).

Enzymatic Assay—Recombinant OXC (0.775 �g/ml) was assayed in60mMKH2PO4, pH 6.7, 60�MThDP, 6mMMgCl2, with a final reactionvolume of 100 �l. The reaction was started by adding 10 �l of oxalyl-CoA at appropriate concentrations to reactionmixture preincubated at30 °C for 2.5 min. Oxalyl-CoA was synthesized and purified followingliterature protocols (14, 37) and diluted in 50mMNaH2PO4, pH 4.5. Thereaction was quenched by adding 11.1 �l of 20% acetic acid to assaymixture, and the initial rate of formyl-CoA formation was analyzed byreverse-phaseHPLCusing amodification of a previously published pro-cedure (14). Briefly, 75-�l aliquots of quenched reaction mixture wereinjected onto a C18 analytical column (Dynamax Microsorb 60–8 C18,250 � 4.6 mm) equilibrated with 98% Buffer F (25 mM sodium acetate,pH 4.5) and 2% Buffer G (20% Buffer F; 80% CH3CN) at a flow rate of 1ml/min.After injection, the proportion of BufferGwas raised to 6%overa 12-min period followed by a step to 95% Buffer G that was continuedfor 2min before returning to 2%BufferG.Under these conditionsThDPeluted after 3.7 min, oxalyl-CoA eluted after 6.1 min, free CoA eluted

FIGURE 2. Kinetic characterization of OXC.Assays were performed as described under “Mate-rials and Methods.” A, recombinant OXC analysisby size exclusion chromatography. Ratiosbetween elution volume (ve) and void volume (vo)were determined for OXC (open circle) and com-pared with the ratios for the molecular mass stand-ards (filled circles). These values were used to cal-culate the estimated molecular mass of OXC insolution, as described under “Materials and Meth-ods.” B, formyl-CoA production over time. Enzymewas assayed in 500 �M oxalyl-CoA. Reactions werecarried out in duplicate and quenched after 0.5, 1,2, 5, and 10 min at 30 °C. Linear extrapolation isshown by a dashed line. C, Michaelis-Menten Plotfor OXC. Enzyme was assayed in 10, 15, 30, 50, 100,and 500 �M oxalyl-CoA. Reactions were performedin triplicate and quenched after 30 s at 30 °C. Curvefitting to v � Vmax [oxalyl-CoA]/(km � [oxalyl-CoA])gave a Vmax of 87.4 3.9 �mol/min/mg and a km

of 23.3 3.4 �M, with an R value of 0.96. Errorsrepresent errors of fit. D, OXC inhibition by CoA.Enzyme was assayed in the presence of 30 (Œ), 100(● ), and 300 (f) �M CoA at 15, 30, 50, 100, and 300�M oxalyl-CoA. Reactions were performed in dupli-cate and quenched after 30 s at 30 °C. Curves werefitted to v � Vmax

app [oxalyl-CoA]/(kmapp �

[oxalyl-CoA]).

Crystal Structure of Oxalyl-CoA Decarboxylase

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after 9.9 min, and formyl-CoA eluted after 11.3 min. The concentra-tion of CoA derivatives in the aliquots was determined as describedpreviously (14).

Activation and Inhibition Studies—Recombinant OXC was assayedas described above in the presence of varying concentrations of ADP,ATP, and FAD suspended in 100 mM KH2PO4, pH 6.7, and free CoAdiluted in 50 mM NaH2PO4, pH 4.5.

RESULTS AND DISCUSSION

Expression, Purification, and Characterization of RecombinantOXC—The recombinant, wild type enzyme was expressed inBL21(DE3) E. coli cells and purified using a two-step purificationprocedure that was significantly different from that published previ-ously (15). Thus, after an initial dye-affinity chromatography step,wild type OXC could be purified to near homogeneity by anionexchange chromatography in yields of typically 5–10 mg/liter of cul-ture (Supplemental Table S1). Gel filtration experiments were per-formed to determine the oligomeric state of the recombinant OXCusing a size exclusion chromatography column calibrated with avariety of molecular mass standards. OXC eluted in a single peakwith a retention time corresponding to 243 kDa (Fig. 2A). Becausethe OXC monomer has a molecular mass of 60,691 Da (20), thisfinding suggests that the purified enzyme is a tetramer in solutionrather than the homodimer suggested in earlier studies of recombi-nant OXC (20).The activity of the recombinant, wild type OXC was measured using

anHPLC-based assay rather than the coupled assay used to characterizethe native enzyme (15) so as to measure formyl-CoA productiondirectly. All reactions were performed at pH 6.7 to minimize the rate ofuncatalyzed thioester hydrolysis. Under these conditions, product for-mation was linear over a period of 3 min (Fig. 2B), permitting the deri-vation ofMichaelis-Menten steady-state kinetic parameters by fitting tostandard equations (Fig. 2C) (39). Under our assay conditions, theapparentKm of oxalyl-CoAwas 23 3.5�M,which is 10-fold lower thanreported for native OXC (15), and the turnover number (kcat) was 88s�1. This difference in the apparent Km values likely reflects technicaldifficulties in the coupled assay used in the earlier study (15) becauseformate dehydrogenase activity appears to be affected by the presence ofCoA thioesters.Sequence alignments (Supplemental Fig. S1) are consistent with the

hypothesis that OXC is evolutionarily related to AHAS (22, 40), theThDP-dependent enzyme that plays an important role in the biosynthe-sis of branched chain amino acids (41) and which is the target for sulfo-nylurea-based herbicides (42). This observation was intriguing in thatAHAS contains a “vestigial” FAD-binding site even though the redoxco-factor plays no role in the catalytic mechanism and presumably ispresentmerely to stabilize the three-dimensional fold of the protein (43,44). Because electronic spectroscopy provided no evidence for the pres-ence of bound flavin in OXC,3 we hypothesized that the FAD-bindingsite in AHAS had been “co-opted” as a binding site for the CoA portionof the substrate and examined the ability of free CoA and FAD to inhibitOXC activity. These experiments showed that although FAD does notinhibit OXC (data not shown), free CoA is a mixed inhibitor of OXCwith respect to oxalyl-CoA, exhibiting Ki and Ki� values of 400 and 270�M, respectively (Fig. 2D).

Significant functional insights have been provided by the availabilityof crystal structures of many different ThDP-dependent enzymes, ofwhich some of the most extensively studied are transketolase (45), POX

(21), AHAS (22), the E1-component of pyruvate dehydrogenase (46),branched-chain �-keto acid dehydrogenase (47), benzoylformatedecarboxylase (BFD) (48), and the pyruvate decarboxylases fromZymomonas mobilis (zPDC) (49) and yeast (yPDC) (50). To provide ahigh resolution three-dimensional structure of OXC from O. formi-genes, we have developed crystallization conditions for the enzyme andwere able to phase the collected x-ray data, as reported elsewhere (23).

Quality of the ElectronDensityMap and theModel—Electron densitymaps calculated from the crudemodels at different stages of refinementallowed tracing of the gaps in the polypeptide chain and assignment ofthe sequence where not previously clear. The asymmetric unit containsa homodimer of two OXCmonomers related by a non-crystallographic2-fold symmetry parallel to the c axis. The C� atoms of the two mono-mers superimpose with a root mean square deviation of 0.22 Å. Themodel contains 2 � 546 amino acids (comprising residues 7–552), two3 P. Moussatche, unpublished results.

FIGURE 3. The crystal structure of OXC. A, schematic diagram of the monomer. Thehelices of the three domains are colored pink, orange, and green, respectively. ThDP andADP are depicted as ball-and-stick models. B, the dimer of OXC dimers. The coloringscheme is as in panel A.

Crystal Structure of Oxalyl-CoA Decarboxylase

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thiamin-2-thiazolone diphosphate molecules, twoMg(II) ions, and twoADPmolecules. A total of 655 ordered water molecules and 10 residueswith alternative conformations were built. No density could be seen forthe 6 first and 16 last residues, which are likely disordered. The structureofOXCwas refined to a twinnedR-factor4 of 15.1% and anRfree of 17.5%with good stereochemistry (TABLE TWO). 89.6% of the amino acidresidues in the final model were located in themost favorable regions ofthe Ramachandran plot. We note that one residue in the active site,Tyr-483, is located in the disallowed region in both subunits but is welldefined in the electron density.

Overall Structure—The monomer of OXC shows the general ThDPbinding fold containing three �/�-type domains, designated PYR (resi-due 1–192), R (residue 193–368), and PP (residue 369–568) (51). Eachdomain is composed of a central six-stranded parallel �-sheet sur-rounded by �-helices on both sides (Fig. 3A). The functional dimerarrangement is common to all ThDP-dependent enzymes (51) and isconserved also in OXC. In the dimer the C-terminal ends of the�-strands of the PYR domain in one subunit faces the C-terminal endsof the �-strands of the PP-domain in the second subunit, thus formingtwo equivalent active sites (Fig. 3B). The overall structure is highly sim-ilar to that observed for othermembers of theThDP-dependent enzymefamily. OXC shows the highest sequence identity, 23%, to AHAS (seeSupplemental Fig. 1), and despite this moderate overall sequencehomology, superimposition of theC� trace of a subunit ofOXCwith theholo-structure of AHAS from Saccharomyces cerevisiae (PDB code 1jsc)matches 465 residues with an rootmean square deviation of 1.74 Å (Fig.4A) and 898 C� atoms with an root mean square deviation of 1.89 Å forthe dimer. Comparison of OXC with BFD (1bfd), zPDC (1zpd), andPOX (1pow) shows slightly less similarity (1.88 Å for 432 C� atoms, 1.96Å for 414 C� atoms, and 1.99 Å for 443 C� atoms, respectively). Because

of the marked structural similarity between OXC and AHAS, we inves-tigated ifOXCwas also a target for herbicide inhibition.Under our assayconditions, no changes in enzyme activitywere observed in the presenceof chlorimuron ethyl or sulfometuron methyl (data not shown). Theseresults were consistent with the absence in OXC of the amino acidsrequired for herbicide inhibition of AHAS, Arg-380, Lys-251, Trp-586,Pro-192, and Val-583 (52, 53). Modeling of the herbicide chlorimuronethyl into the OXC crystal structure also resulted in stereochemicalclashes.The structure of a mobile loop, comprised of residues 479–504, is

unique to OXC in that it is opened up and lacks any discernible second-ary structure. In other homologous ThDP-dependent decarboxylasestructures, this region folds down over the active site, forming a helix-loop structure. Close to where the helix-loop is found in the homolo-gous structures, the last ordered C-terminal residue of OXC is located(Fig. 4B). The remaining 16 residues are disordered. In AHAS, the helix-loop as well as the C terminus is disordered in the holo structure (22),but in complex with several different herbicides these regions becomeordered and cover the active site (54). This finding on AHAS was fol-lowed up in a recent study where the “mobile” loop and C-terminal “lid”were deleted and shown to be important for stabilization of the activedimer and ThDP binding (55). It is probable that, during the catalysis ofoxalyl-CoA decarboxylation, the mobile loop in OXC changes confor-mation, and the 16C-terminal disordered residues fold up and forma lidover the opening to the active site, forming the required hydrophobicenvironment.

Quaternary Structure—There are differences in the overall quater-nary structure between ThDP-dependent enzymes. For example,althoughAHAS (22) and transketolase (46) are dimers, POX (21), zPDC(49), and BFD (48) are all tetramers. As described above, gel filtrationexperiments suggest that the oligomeric state of OXC is a tetramer insolution. This proposal is further substantiated by the crystal structure4 Rtwin � ��[F2

calc(h1) � F2calc(h2)]1/2 � Fobs(h1)�/��Fobs(h1)�.

FIGURE 4. Comparison of the structure of OXCwith AHAS. A, stereoview of the C� trace of OXC(green) superimposed on the holo-structure ofAHAS from S. cerevisiae (PDB code 1jsc) (blue). B,stereoview of the mobile loop. OXC is green, andAHAS (PDB code 1t9b) is blue.

Crystal Structure of Oxalyl-CoA Decarboxylase

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in which the crystallographic 2-fold axis generates the biological tet-ramer from the dimer in the asymmetric unit (Fig. 3B). The arrange-ment of the tetramers differs among POX, BFD, and zPDC (21, 48, 49).Superimposition of the OXC tetramer with each of these three struc-tures shows that OXC forms the same compact dimer of dimers as POXand BFD.

ThDP Binding—The ThDP cofactor is bound in a cleft between thePYR and PP domain from two different subunits (Fig. 3B). A strongelectron density feature close to the C2 position on the thiazolium ringwas interpreted as the thiazolone derivative of ThDP (Fig. 5A) (56),which is likely a result of exposure to the intense x-ray radiation and is,therefore, an artifact of the experiment. Despite considerable differ-ences inquaternary structureand lackof sequencehomology, theThDP-dependent enzymes bind ThDP and the divalent Mg2� ion cofactorwithin a similar framework (57), which is crucial for proper orientationof the coenzyme in the V-shaped conformation. All of the identifiedcommon family features are shared by OXC. The pyrophosphate isbound to the PP domain at the cleft formed when the loops from two�-strands cross over to different sides of the sheet and the N termini ofthe following two helices provide hydrogen bonds. Further stabilizationis obtained through hydrogen bonds to the polar residues Asn-402 andTyr-377 and via the Mg2� ion, which is bound to the PP domain by theconserved ThDP fingerprint (58, 57). The thiazolium ring is locatedbetween the subunits, and Met-428 contributes with a conservedhydrophobic interaction that stabilizes the V conformation. The pyrim-idine ring is predominantly bound by the PYR domain. The bindingincludes only two conserved interactions, a hydrogen bond from a glu-tamic acid (Glu-56 in OXC) to the N1� of the ring and a hydrogen bondfrom N4� to the carbonyl of Gly-426 in the PP domain.

Active Site Residues—Although the mode of cofactor binding andfold is highly conserved among ThDP-dependent enzymes throughoutevolution, there are significant differences in the active sites, and noconserved residues other than the few involved in cofactor binding canbe found. The putative active site of OXC (see below) contains threeresidues that could possibly contribute to catalysis; Tyr-120, Glu-121,and Tyr-483 are all located in the proximity of the catalytic site (Fig. 6).All these residues are conserved among oxalyl-CoA decarboxylasesfrom different bacterial species, with the exception of Glu-121, which isexchanged for a glutamine in Mycobacterium tuberculosis. The corre-sponding residues that have been implicated to assist in the reaction inPDC are His-113, His-114, and Glu-473 (49, 50), but in BFD, anotherdecarboxylating ThDP-dependent enzyme of known structure, thereare no corresponding polar residues.

Catalytic Mechanism—The first steps in OXC-catalyzed decarboxy-lation, as in all ThDP-dependent enzymes, involves activation of thecofactor by the enzyme (59–62) (Fig. 7). Thus, the conserved Glu-56-N1� hydrogen bond interaction stabilizes the tautomeric form of thecofactor in which the N4� amino group is converted to an imino group,and in the process one proton is displaced from the N4� atom to thesolvent, perhaps assisted by Glu-121. The V conformation of the ThDP,constrained by Met-428, positions the 4�-imino group sterically closefor direct deprotonation of the C2 atom of the thiazolium ring. A con-served hydrogen bond between the formed imino group and the car-bonyl group of Gly-426may further assist by positioning the lone pair ofthe imino group favorably for proton abstraction at C2 and therebyform the highly nucleophilic ylid structure.After the C2 carbanion is formed, the cofactor attacks the carbonyl

carbon of the thioester in oxalyl-CoA, and the covalently attached inter-mediate can be modeled into the active site (Fig. 6). The carboxyl groupof the intermediate forms hydrogen bonds to Tyr-483 and to the main

chain amino group of Ile-34. Upon formation of the covalent bond, thedeveloping negative charge of the carbonyl oxygen on the � carbonatom of the substrate has to be electrostatically stabilized. In the modelthere are two candidate functional groups thatmight act to perform thistask, the Tyr-120 side chain and the protonated 4�-imino group ofThDP. The 4�-NH2was proposed earlier to stabilize the negative chargeof this oxygen in transketolase and other enzymes (61, 63).Cleavage of the substrate with the formation of CO2 gives a covalent

intermediate, the �-carbanion/enamine. The resonance contribution ofthe reactive high energy �-carbanion form and the low energy planarenamine to this enzyme intermediate has long been debated in ThDPchemistry (60) andmight indeed be different for ThdP enzymes catalyzingdifferent reactions. Recent studies ofBFDsuggest that the enzymepreventsthe intermediate from taking the planar enamine form by avoiding orbitaloverlap through hydrogen bonding to the hydroxyl group on the � carbonatom (64). In OXC this stabilization can be performed by Tyr-120 and the4�-NH2. From this, it would follow that the intermediate stays partly tetra-hedral, and protonation of the �-carbon in the next step has to take placefrom the same direction as the CO2 previously left. None of the three resi-dues, Tyr-120, Glu-121, and Tyr-483, is in proper distance from the�-car-bon to be involved; however, a boundwatermolecule is suitably positionedfor the �-carbon to attack and abstract a proton. This water molecule isanchoredbyhydrogenbonds to the sidechainsofTyr-120andGlu-121andto the carbonyl of Ile-34, with the proceeding Pro-35 in cis-conformation,

FIGURE 5. Electron density (2 Fo � Fc) contoured at 1. 0 �. A, electron density showingpresence of the thiazolon derivative of ThDP and the Mg2� ion. B, electron density show-ing the presence of ADP.

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which is unique to OXC. A bound water molecule in the same position isfound in zPDC (49), where it forms hydrogen bonds withAsp-27, His-114,and Thr-72 (zPDC numbering). The yPDC double mutant D28A/E91D(yPDC numbering) is not able to release acetaldehyde from the intermedi-ate (65), which suggests that the same water molecule is involved in thisprotonation event in PDC. In BFD, the N�2 of His-70 is close to the sameposition as this water molecule but is suggested to be involved in protona-tion of the carbonyl oxygen of the intermediate before decarboxylation, i.e.

2-�-mandelyl-ThDP, and for deprotonation of the hydroxyl group in the2-�-hydroxybenzyl-ThDP intermediate before product formation. (66).In the last step before the second product, formyl-CoA, leaves, the

hydroxyl group of the � carbon has to return the proton. The acceptoris most probably the same group that donated the proton, the 4�-iminogroup. In the suggested mechanism, none of the residues in the activesite (except Glu-56) is strictly needed for catalysis. This is in accordancewith results on yPDC for which mutations of any single active site res-

FIGURE 6. The catalytic site of OXC. The oxalyl-CoA substrate covalently attached to the C2 posi-tion of ThDP has been modeled. Only the sulfuratom of CoA is shown. Pink-colored residues arefrom the PYR domain of one subunit, and greenresidues are from the PP domain of the other sub-unit. WAT, water.

FIGURE 7. Reaction mechanism for OXC. The nature of B1 is not known and might not be needed, B2 is the 4�imino group of ThDP, and B3 is a water molecule.

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idue never led tomore than a 100–1000-fold reduction in kcat or kcat/Km

(67).ADPBinding andOXCActivation—These structural studies confirm

that OXC is homologous to AHAS and, therefore, belongs to the familyof ThDP-dependent enzymes that are related to pyruvate oxidase (43).As described above, most enzymes in this family possess a FAD-bindingsite (68), the exception being some variants of acetolactate synthase (69)in which the cognate structural cavity is filled by amino acid side chains(38). Although FAD plays a role in electron transfer in the POX-cata-lyzed formation of acetyl-CoA from pyruvate (21), this co-factorappears merely to stabilize the structure of other enzymes, such asAHAS (70) and glyoxylate carboligase (71), in this evolutionary family.We anticipated thatOXCwould also contain this vestigial FAD-bindingsite, and the absence of bound flavin in the purified protein, therefore,suggested (given the structural similarity of the ADP-like, distal regions

of FAD and CoA) that this site had been co-opted during OXC evolu-tion (72–74) to function as a binding pocket for the CoA moiety of thesubstrate. Therefore the presence of strong electron density in theOXCR domain at a region cognate to the binding site of the non-catalyticFAD in AHAS (22) was interesting, especially because a surplus of CoAwas included in the crystallization mixture. A more detailed examina-tion, however, showed that the density could only be interpreted as anADP molecule bound within the Rossmann fold (Fig. 5B) at the samesite as the ADP part of the non-catalytic FAD in AHAS (22) (Fig. 4A).Thus, ADP is buried in a cleft between the Pyr domain and the R domainof the same subunit, with the latter contributingmost of the interactions(Fig. 8). The adenine ring is wedged between two isoleucine side chains;the ribose hydroxyl groups make hydrogen bonds to the side chains ofAsp-306 and Arg-160, with the pyrophosphate bound by water mole-cules and the positively charged side chains of Arg-282 and Lys-222.Our original hypothesis that the site could bind CoA was investi-

gated. Modeling oxalyl-CoA into the ADP-binding site does permit thethioester of the extended substrate to be located adjacent to the C2position of ThDP without further adjustment. However, these studiesalso showed that the 3�-ribose phosphate of the CoA moiety can beaccommodated only after substantial conformational rearrangement ofthe Arg-16, Arg-282, and Asp-306 side chains (Fig. 8).Given the presence of ADP in the OXC crystal structure despite the

fact that no ADP was added during enzyme purification or crystalliza-tion, we assumed that this ligand must have been supplied by the E. colicells during expression. We, therefore, tested the effects of this smallmolecule on the kinetics of OXC-catalyzed decarboxylation (Fig. 9). Inthese experiments, which were carried out in the presence of saturatingoxalyl-CoA, endogenousADP reproducibly increased the specific activ-ity of the recombinant enzyme at micromolar concentrations, whereasATP had no effect on the enzyme activity. Because millimolar levels ofADP are estimated to be present in E. coli cells, we therefore believe thatrecombinant OXC is saturated with ADP before purification. This also

FIGURE 8. ADP binding to OXC. Stereoview ofinteracting residues and ordered water molecules.

FIGURE 9. OXC activation by ADP. The enzyme was assayed in 500 �M oxalyl-CoA in thepresence of 5, 20, 60, and 150 �M ADP (filled circles) or ATP (empty circles). Reactions werecarried out in triplicate and quenched after 30 s at 30 °C.

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explains the observation that OXC exhibits high specific activity in celllysates (Supplemental Table S1) that is attenuated by subsequent puri-fication and storage,3 presumably due to the release of ADP from theprotein. Although the mechanism by which ADP stimulates activityremains to be established, it is probable that ADP binding stabilizes theactive conformation of the enzyme. The stimulation of decarboxylaseactivity by ADP may have physiological importance since oxalyl-CoAdecarboxylation is an essential step in ATP generation inO. formigenes.If ADP bindswithin the vestigial FAD-binding site of the enzyme, this

would imply that there is a second site for oxalyl-CoA. Unfortunately,despite several trials we have not been able to obtain co-crystals exhib-iting electron density corresponding to this substrate analogue. Theprecise location of binding site for oxalyl-CoA, therefore, remains elu-sive, although we note that there is a large cavity formed by the Rdomain and the PP domain of the same subunit that might accommo-date the substrate (Fig. 4). This cavity contains several lysine and argi-nine side chains that could participate in binding of the phosphategroups, and binding of oxalyl-CoA in this site would not prevent eitherthe mobile loop or the conformationally flexible C-terminal residues toclose down on the active site during catalysis. Importantly, this cleft issmaller in AHAS, where the isoalloxazine ring of FAD and an extra loopof seven residues (beginning atAHAS residue 264) partly fill the cognatecavity.

Implications of the Structure for Half-site Reactivity in OXC—Recentexperiments on the ThDP-dependent E1 component of pyruvate dehy-drogenase have suggested that there is communication between the twoactive sites in the homodimer (75, 76).More specifically, the existence ofa network of hydrogen bonding interactions has been identified as a“proton wire” that mediates the shuttling of a proton between activesites, giving rise to “half-site” reactivity (75). Taken together with find-ings on other ThDP-dependent enzymes (77, 78), such as differentialkinetics of co-factor binding to the two sites (79) and co-crystal struc-tures in which substrate analogs are observed to bind in only one of thepossible active sites (80, 81), this work on pyruvate dehydrogenase raisesquestions concerning the generality of this molecular mechanism inmediating active site communication. There is no evidence, however,for the existence of such a proton wire in the crystal structure of theOXC homodimer. In pyruvate dehydrogenase the active sites are linkedby an acidic solvated tunnel with six glutamic acid residues, two asparticacid residues, and amagnesium ion. The corresponding solvated tunnelin OXC includes six ionizable residues, two glutamic acids, and fourhistidine residues, but no hydrogen bond network connects the activesites.

Acknowledgments—We gratefully acknowledge access to synchrotron radia-tion at beamline ID23–1, European Synchrotron Radiation Facility, Grenoble.We also thank Dr. Harmeet Sidhu (Ixion Biotechnology, Inc.) for providing theplasmids used to express recombinant, wild typeOXCandDr. JianqiangWangfor preparing the thiocresol precursor to oxalyl-CoA.

REFERENCES1. Williams, H. E., and Smith, L. H., Jr. (1968) Am. J. Med. 45, 715–7352. James, L. F. (1972) Clin. Toxicol. 5, 231–2433. Rodby, R. A., Tyszka, T. S., and Williams, J. W. (1991) Am. J. Med. 90, 498–5044. Hodgkinson, A. (1977) Oxalic Acid in Biology and Medicine, Academic Press, Inc.,

London, UK5. Hatch, M., Freel, R. W., and Vaziri, N. D. (1994) Pfluegers Arch. Eur. J. Physiol. 426,

101–1096. Allison, M. J., Cook, H. M., Milne, D. B., Gallagher, S., and Clayman, R. V. (1986) J.

Nutr. 116, 455–4607. Allison, M. J., Dawson, K. A., Mayberry, W. R., and Foss, J. G. (1985) Arch. Microbiol.

141, 1–7

8. Stewart, C. S., Duncan, S. H., and Cave, D. R. (2004) FEMS Microbiol. Lett. 230, 1–79. Hatch, M., and Freel, R. W. (2005) Urol. Res. 33, 1–1610. Sidhu, H., Hoppe, B., Hesse, A., Tenbrock, K., Bromme, S., Reitschel, E., and Peck,

A. B. (1998) Lancet 352, 1026–102911. Sidhu,H., Schmidt,M. E., Cornelius, J. G., Thamilselvan, S., Khan, S. R., Hesse, A., and

Peck, A. B. (1999) J. Am. Soc. Nephrol. 10, Suppl. 14, 334–34012. Baetz, A. L., and Allison, M. J. (1990) J. Bacteriol. 172, 3537–354013. Ricagno, S., Jonsson, S., Richards, N. G., and Lindqvist, Y. (2003) EMBO J. 22,

3210–321914. Jonsson, S., Ricagno, S., Lindqvist, Y., and Richards, N. G. J. (2004) J. Biol. Chem. 279,

36003–3601215. Baetz, A. L., and Allison, M. J. (1989) J. Bacteriol. 171, 2605–260816. Baetz, A. L., and Allison, M. J. (1992) Syst. Appl. Microbiol. 15, 167–17117. Ruan, Z. S., Anantharam, V., Crawford, I. T., Ambudkar, S. V., Rhee, S. Y., Allison,

M. J., and Maloney, P. C. (1992) J. Biol. Chem. 267, 10537–1054318. Hirai, T., Heymann, J. A. W., Shi, D., Sarker, R., Maloney, P. C., and Subramanian, S.

(2002) Nat. Struct. Biol. 9, 597–60019. Anantharam, V., Allison, M. J., and Maloney, P. C. (1989) J. Biol. Chem. 264,

7244–725020. Lung, H. Y., Baetz, A. L., and Peck, A. B. (1994) J. Bacteriol. 176, 2468–247221. Muller, Y. A., and Schulz, G. E. (1993) Science 259, 965–96722. Pang, S. S., Duggleby, R. G., and Guddat, L. W. (2002) J. Mol. Biol. 317, 249–26223. Berthold, C. L., Sidhu, H., Ricagno, S., Richards, N. G., and Lindqvist, Y. (August 16,

2005) Biochim. Biophys. Acta. 10.1016/j.bbapap.2005.08.01624. Leslie, A. G.W. (1992) Joint CCP4� ESF-EAMCBNewsl. Protein Crystallogr.Vol. 26,

Daresbury Laboratory, Warrington, UK25. Collaborative Computational Project, Number 4. (1994) Acta Crystallogr. Sect. D 50,

760–76326. French, G. S., and Wilson, K. S. (1978) Acta Crystallogr. A 34, 517–52327. Rees, D. C. (1980) Acta Crystallogr. A 36, 578–58128. Yeates, T. O. (1997)Methods Enzymol. 276, 344–35829. Vagin, A., and Teplyakov, A. (1997) J. Appl. Crystallogr. 30, 1022–102530. Brunger, A. T., Adams, P. D., Clore, G.M., DeLano,W. L., Gros, P., Grosse-Kunstleve,

R. W., Jiang, J. S., Kuszewski, J., Nilges, M., Pannu, N. S., Read, R. J., Rice, L. M.,Simonson, T., and Warren, G. L. (1998) Acta Crystallogr. Sect. D 54, 905–921

31. Jones, T. A., Zou, J. Y., Cowan, S. W., and Kjeldgaard, M. (1991) Acta Crystallogr. A47, 110–119

32. Schuttelkopf, A. W., and van Aalten, D. M. (2004) Acta Crystallogr. Sect. D 60,1355–1363

33. Emsley, P., and Cowtan, K. (2004) Acta Crystallogr. Sect. D 60, 2126–213234. Sheldrick, G. M., and Schneider, T. R. (1997)Methods Enzymol. 277, 319–34335. Laskowski, R. A.,Moss, D. S., and Thornton, J.M. (1993) J. Mol. Biol. 231, 1049–106736. Laskowski, R. A., MacArthur, M.W., Moss, D. S., and Thornton, J. M. (1993) J. Appl.

Crystallogr. 26, 283–29137. Quayle, J. R. (1963) Biochem. J. 87, 368–37338. Pang, S. S., Duggleby, R. G., Schowen, R. L., and Guddat, L. W. (2004) J. Biol. Chem.

279, 2242–225339. Segel, I. H. (1993) Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and

Steady State Enzyme Systems, John Wiley & Sons, Inc., New York40. Benner, S. A., Chamberlin, S. G., Liberles, D. A., Govindarajan, S., and Knecht, L.

(2000) Res. Microbiol. 151, 97–10641. Singh, B. K., and Shaner, D. L. (1995) Plant Cell 7, 935–94442. LaRossa, R. A., and Schloss, J. V. (1984) J. Biol. Chem. 259, 8753–875743. Chang, Y. Y., and Cronan, J. E., Jr. (1988) J. Bacteriol. 170, 3937–394544. Chang, Y. Y., Wang, A. Y., Cronan, J. E., Jr. (1993) J. Biol. Chem. 268, 3911–391945. Lindqvist, Y., Schneider, G., Ermler, U., and Sundstrom, M. (1992) EMBO J. 11,

2373–237946. Arjunan, P., Nemeria, N., Brunskill, A., Chandrasekhar, K., Sax,M., Yan, Y., Jordan, F.,

Guest, J. R., and Furey, W. (2002) Biochemistry 41, 5213–522147. Aevarsson, A., Chuang, J. L., Wynn, R. M., Turley, S., Chuang, D. T., and Hol, W. G.

(2000) Structure Fold. Des. 8, 277–29148. Hasson, M. S., Muscate, A., McLeish, M. J., Polovnikova, L. S., Gerlt, J. A., Kenyon,

G. L., Petsko, G. A., and Ringe, D. (1998) Biochemistry 37, 9918–993049. Dobritzsch, D., Konig, S., Schneider, G., and Lu, G. (1998) J. Biol. Chem. 273,

20196–2020450. Arjunan, P., Umland, T., Dyda, F., Swaminathan, S., Furey, W., Sax, M., Farrenkopf,

B., Gao, Y., Zhang, D., and Jordan, F. (1996) J. Mol. Biol. 256, 590–60051. Muller, Y. A., Lindqvist, Y., Furey, W., Schulz, G. E., Jordan, F., and Schneider, G.

(1993) Structure 1, 95–10352. Pang, S. S., Guddat, L. W., and Duggleby, R. G. (2003) J. Biol. Chem. 278, 7639–764453. Pang, S. S., Guddat, L. W., and Duggleby, R. G. (2004) Acta Crystallogr. Sect. D 60,

153–15554. McCourt, J. A., Pang, S. S., Guddat, L.W., andDuggleby, R. G. (2005)Biochemistry 44,

2330–233855. Kim, J., Beak, D. G., Kim, Y. T., Choi, J. D., and Yoon, M. Y. (2004) Biochem. J. 384,

Crystal Structure of Oxalyl-CoA Decarboxylase

DECEMBER 16, 2005 • VOLUME 280 • NUMBER 50 JOURNAL OF BIOLOGICAL CHEMISTRY 41653

at University of F

lorida on August 9, 2006

ww

w.jbc.org

Dow

nloaded from

59–6856. Nilsson, U., Lindqvist, Y., Kluger, R., and Schneider, G. (1993) FEBS Lett. 326,

145–14857. Lindqvist, Y., and Schneider, G. (1993) Curr. Opin. Struct. Biol. 3, 896–90158. Hawkins, C. F., Borges, A., and Perham, R. N. (1989) FEBS Lett. 255, 77–8259. Kern, D., Kern, G., Neef, H., Tittmann, K., Killenberg-Jabs,M.,Wikner, C., Schneider,

G., and Hubner, G. (1997) Science 275, 67–7060. Schellenberger, A. (1998) Biochim. Biophys. Acta 1385, 177–18661. Jordan, F. (2003) Nat. Prod. Rep. 20, 184–20162. Schneider, G., and Lindqvist, Y. (1993) Bioorg. Chem. 21, 109–11763. Schneider, G., and Lindqvist, Y. (1998) Biochim. Biophys. Acta 1385, 387–39864. Hu, Q., and Kluger, R. (2004) J. Am. Chem. Soc. 126, 68–6965. Jordan, F., Liu, M., Sergienko, E., Zhang, Z., Brunskill, A., Arjunan, P., and Furey, W.

(2004)Thiamine: CatalyticMechanisms inNormal andDisease States (Jordan, F., andPatel, M., eds) pp. 173–215, Marcel Dekker, Inc., New York

66. Polovnikova, E. S., McLeish, M. J., Sergienko, E. A., Burgner, J. T., Anderson, N. L.,Bera, A. K., Jordan, F., Kenyon, G. L., and Hasson, M. S. (2003) Biochemistry 42,1820–1830

67. Jordan, F., and Nemeria, N. S. (2005) Bioorg. Chem. 33, 190–215

68. Bornemann, S. (2002) Nat. Prod. Rep. 19, 761–77269. Stormer, F. C. (1967) J. Biol. Chem. 242, 1756–175970. Duggleby, R. G., and Pang, S. S. (2000) J. Biochem. Mol. Biol. 33, 1–3671. Cromartie, T. H., and Walsh, C. T. (1976) J. Biol. Chem. 251, 329–33372. Gerlt, J. A., Babbit, P. C., and Rayment, I. (2005) Arch. Biochem. Biophys. 433, 59–7073. Bartlett, G. J., Borkakoti, N., and Thornton, J. M. (2003) J. Mol. Biol. 331, 829–86074. Gerlt, J. A., and Babbitt, P. C. (2001) Annu. Rev. Biochem. 70, 209–24675. Frank, R. A. W., Titman, C. F., Pratap, J. V., Luisi, B. F., and Perham, R. N. (2004)

Science 306, 872–87676. Nemeria, N., Tittmann, K., Joseph, E., Zhou, L., Vazquez-Coll, M. B., Ajunan, P.,

Hubner, G., Furey, W., and Jordan, F. (2005) J. Biol. Chem. 280, 21473–2148277. Sergienko, E. A., and Jordan, F. (2001) Biochemistry 40, 7382–740378. Sergienko, E. A., Wang, J., Polovnikova, L., Hasson, M. S., McLeish, M. J., Kenyon,

G. L., and Jordan, F. (2000) Biochemistry 39, 13862–1386979. Horn, F., and Biswanger, H. (1983) J. Biol. Chem. 258, 6912–691980. Nakai, T., Nakagawa, N., Maoka, N., Masui, R., Kuramitsu, S., and Kamiya, N. (2004)

J. Mol. Biol. 337, 1011–103381. Lu, G., Dobritzsch, D., Baumann, S., Schneider, G., Konig, S. (2000) Eur. J. Biochem.

267, 861–868

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