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• W1973940014 abstract "Oxalobacter formigenes is an obligate anaerobe that colonizes the human gastrointestinal tract and employs oxalate breakdown to generate ATP in a novel process involving the interplay of two coupled enzymes and a membrane-bound oxalate:formate antiporter. Formyl-CoA transferase is a critical enzyme in oxalate-dependent ATP synthesis and is the first Class III CoA-transferase for which a high resolution, three-dimensional structure has been determined (Ricagno, S., Jonsson, S., Richards, N., and Lindqvist, Y. (2003) EMBO J. 22, 3210–3219). We now report the first detailed kinetic characterizations of recombinant, wild type formyl-CoA transferase and a number of site-specific mutants, which suggest that catalysis proceeds via a series of anhydride intermediates. Further evidence for this mechanistic proposal is provided by the x-ray crystallographic observation of an acylenzyme intermediate that is formed when formyl-CoA transferase is incubated with oxalyl-CoA. The catalytic mechanism of formyl-CoA transferase is therefore established and is almost certainly employed by all other members of the Class III CoA-transferase family. Oxalobacter formigenes is an obligate anaerobe that colonizes the human gastrointestinal tract and employs oxalate breakdown to generate ATP in a novel process involving the interplay of two coupled enzymes and a membrane-bound oxalate:formate antiporter. Formyl-CoA transferase is a critical enzyme in oxalate-dependent ATP synthesis and is the first Class III CoA-transferase for which a high resolution, three-dimensional structure has been determined (Ricagno, S., Jonsson, S., Richards, N., and Lindqvist, Y. (2003) EMBO J. 22, 3210–3219). We now report the first detailed kinetic characterizations of recombinant, wild type formyl-CoA transferase and a number of site-specific mutants, which suggest that catalysis proceeds via a series of anhydride intermediates. Further evidence for this mechanistic proposal is provided by the x-ray crystallographic observation of an acylenzyme intermediate that is formed when formyl-CoA transferase is incubated with oxalyl-CoA. The catalytic mechanism of formyl-CoA transferase is therefore established and is almost certainly employed by all other members of the Class III CoA-transferase family. Oxalobacter formigenes is a Gram-negative, obligate anaerobe (1Allison M.J. Dawson K.A. Mayberry W.R. Foss J.G. Arch. Microbiol. 1985; 141: 1-7Crossref PubMed Scopus (318) Google Scholar, 2Dawson K.A. Allison M.J. Hartman P.A. Appl. Environ. Microbiol. 1980; 40: 833-839Crossref PubMed Google Scholar) that employs oxalate as a source of both energy and carbon for cellular biosynthesis (3Cornick N.A. Allison M.J. Appl. Environ. Microbiol. 1996; 62: 3011-3013Crossref PubMed Google Scholar). This microorganism, which colonizes the gastrointestinal tract of humans and other mammals, is of biological interest for two reasons. First, recent work has demonstrated an intriguing correlation between the absence of O. formigenes in humans and kidney stone formation because of elevated levels of oxalate in the blood (4Sidhu H. Hoppe B. Hesse A. Tenbrock K. Brömme S. Rietschel E. Peck A.B. Lancet. 1998; 352: 1026-1029Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 5Allison M.J. Cook H.M. Milne D.B. Gallagher S. Clayman R.V. J. Nutr. 1986; 116: 455-460Crossref PubMed Scopus (167) Google Scholar), an observation that has led to the somewhat controversial hypothesis that O. formigenes plays a key role in mediating mammalian oxalate homeostasis (6Sidhu H. Schmidt M.E. Cornelius J.E. Thamilsevan S. Khan S.R. Hesse A. Peck A.B. J. Am. Soc. Nephrol. 1999; 14: S334-S340Google Scholar). Second, ATP production appears to depend solely on the anaerobic conversion of oxalate to formate and CO2 (7Anatharam V. Allison M.J. Maloney P.C. J. Biol. Chem. 1989; 264: 7244-7250Abstract Full Text PDF PubMed Google Scholar). Carbohydrates cannot be used to replace oxalate as a growth substrate, perhaps implying that this organism lacks a functional glycolytic pathway (8Cornick N.A. Allison M.J. Can. J. Microbiol. 1996; 42: 1081-1086Crossref PubMed Scopus (26) Google Scholar).As reported for other bacteria (9Quayle J.R. Biochem. J. 1963; 89: 492-503Crossref PubMed Scopus (24) Google Scholar, 10Jakoby W.B. Ohmura E. Hayaishi O. J. Biol. Chem. 1956; 222: 435-446Abstract Full Text PDF PubMed Google Scholar, 11Zaitsev G.M. Govorukhina N.I. Laskovneva O.V. Trotsenko Y.A. Microbiology. 1993; 62: 378-382Google Scholar), cleavage of the oxalate C–C bond in O. formigenes is accomplished by the action of a thiamin-dependent, oxalyl-CoA decarboxylase (12Baetz A.L. Allison M.J. J. Bacteriol. 1989; 171: 2605-2608Crossref PubMed Google Scholar) encoded by the oxc gene (13Lung H.-Y. Baetz A.L. Peck A.B. J. Bacteriol. 1994; 176: 2468-2472Crossref PubMed Google Scholar) (Fig. 1). ATP-dependent formation of oxalyl-CoA is avoided by coupling decarboxylation to an acyl transfer reaction in which formyl-CoA 1 and oxalate 2 are converted to oxalyl-CoA 3 and formate 4 by formyl-CoA transferase (FRC) 1The abbreviations used are: FRC, formyl-CoA transferase; WT, wild type; HPLC, high performance liquid chromatography.1The abbreviations used are: FRC, formyl-CoA transferase; WT, wild type; HPLC, high performance liquid chromatography. (14Baetz A.L. Allison M.J. J. Bacteriol. 1990; 172: 3537-3540Crossref PubMed Google Scholar). The overall catalytic cycle therefore transforms oxalate into formate and CO2 (Fig. 1). The metabolic importance of this coupled enzyme system in O. formigenes is suggested by the fact that oxalyl-CoA decarboxylase and FRC constitute ∼20% of the total protein content in this microorganism (15Baetz A.L. Allison M.J. Syst. Appl. Microbiol. 1992; 15: 167-171Crossref Scopus (8) Google Scholar). In contrast to aerobic bacteria, such as Pseudomonas oxalaticus (16Quayle J.R. Keech D.B. Biochem. J. 1959; 72: 631-637Crossref PubMed Scopus (34) Google Scholar), which employ formate dehydrogenase to oxidize formate to CO2 with concomitant production of NADH (17Jormakka M. Tornroth S. Byrne B. Iwata S. Science. 2002; 295: 1863-1868Crossref PubMed Scopus (396) Google Scholar), O. formigenes uses a membrane-bound, formate:oxalate antiporter (18Hirai T. Heymann J.A.W. Shi D. Sarker R. Maloney P.C. Subramaniam S. Nat. Struct. Biol. 2002; 9: 597-600PubMed Google Scholar, 19Maloney P.C. Anantharam V. Allison M.J. J. Biol. Chem. 1992; 267: 10531-10546Abstract Full Text PDF PubMed Google Scholar, 20Ruan Z.-S. Anantharam V. Crawford I.T. Ambudkar S.V. Rhee S.Y. Allison M.J. Maloney P.C. J. Biol. Chem. 1992; 267: 10537-10543Abstract Full Text PDF PubMed Google Scholar), encoded by the oxlT gene (21Abe K. Ruan Z.-S. Maloney P.C. J. Biol. Chem. 1996; 271: 6789-6793Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), to create the electrochemical gradient necessary for ATP synthesis (8Cornick N.A. Allison M.J. Can. J. Microbiol. 1996; 42: 1081-1086Crossref PubMed Scopus (26) Google Scholar, 20Ruan Z.-S. Anantharam V. Crawford I.T. Ambudkar S.V. Rhee S.Y. Allison M.J. Maloney P.C. J. Biol. Chem. 1992; 267: 10537-10543Abstract Full Text PDF PubMed Google Scholar). We also note that the genome sequence of Escherichia coli (22Blattner F.R. Plunkett III, G. Bloch C.A. Perna N.T. Burland V. Riley M. Collado-Vides J. Glasner J.D. Rode C.K. Mayhew G.F. Gregor J. Davis N.W. Kirkpatrick H.A. Goeden M.A. Rose D.J. Mau B. Shao Y. Science. 1997; 277: 1453-1474Crossref PubMed Scopus (5973) Google Scholar) reveals the existence of open reading frames encoding proteins that are homologous to both FRC and oxalyl-CoA decarboxylase. This is an intriguing observation given that the ability of this microorganism to metabolize oxalate has not been reported.There are several aspects of FRC that are of considerable biochemical and mechanistic interest. First, primary sequence analysis has placed FRC within a new family (Class III) of CoA-transferases (23Heider J. FEBS Lett. 2001; 509: 345-349Crossref PubMed Scopus (107) Google Scholar), which also includes enzymes that are involved in the anaerobic metabolism of carnitine (24Elssner T. Engemann C. Baumgart K. Kleber H.-P. Biochemistry. 2001; 40: 11140-11148Crossref PubMed Scopus (58) Google Scholar), toluene catabolism (25Leutwein C. Heider J. Arch. Microbiol. 2002; 178: 517-524Crossref PubMed Scopus (33) Google Scholar, 26Leutwein C. Heider J. J. Bacteriol. 2001; 183: 4288-4295Crossref PubMed Scopus (62) Google Scholar), Strickland fermentation (27Dickert S. Pierik A.J. Linder D. Buckel W. Eur. J. Biochem. 2000; 267: 3874-3884Crossref PubMed Scopus (53) Google Scholar), and (putatively) bile acid transformation (28Ye H.-Q. Mallonee H. Wells J.E. Björkhem I. Hylemon P.B. J. Lipid Res. 1999; 40: 17-23Abstract Full Text Full Text PDF PubMed Google Scholar). All of these enzymes have similar masses and are active as homo- or heterodimers (23Heider J. FEBS Lett. 2001; 509: 345-349Crossref PubMed Scopus (107) Google Scholar). The catalytic mechanism employed by the Class III CoA-transferases has yet not been experimentally defined, although it has been the subject of considerable speculation (23Heider J. FEBS Lett. 2001; 509: 345-349Crossref PubMed Scopus (107) Google Scholar, 29Ricagno S. Jonsson S. Richards N. Lindqvist Y. EMBO J. 2003; 22: 3210-3219Crossref PubMed Scopus (42) Google Scholar, 30Gruez A. Roig-Zamboni V. Valencia C. Campanacci V. Cambillau C. J. Biol. Chem. 2003; 278: 34582-34586Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Our recent x-ray crystallographic studies have shown that FRC exists as an interlocked dimer (Fig. 2A) (29Ricagno S. Jonsson S. Richards N. Lindqvist Y. EMBO J. 2003; 22: 3210-3219Crossref PubMed Scopus (42) Google Scholar), raising significant questions concerning the folding pathways that might lead to such a structure. In this regard, we note that the protein encoded by the YfdW gene in E. coli, which is clearly homologous to FRC, has been shown independently to possess an identical interlocked structure (30Gruez A. Roig-Zamboni V. Valencia C. Campanacci V. Cambillau C. J. Biol. Chem. 2003; 278: 34582-34586Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 31Gagos A. Gorman J. Shapiro L. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 507-511Crossref PubMed Scopus (13) Google Scholar). The ability of the YfdW gene product to catalyze coenzyme A transfer, however, has not yet been reported.Fig. 2Crystal structure of the interlocked FRC dimer complexed to Co-A.A, for clarity, the protein monomers are colored red and green and represented by molecular ribbons. The bound co-factor is shown as a space-filling model, which is colored using the following scheme: carbon, black; nitrogen, blue; oxygen, red; sulfur, yellow; phosphorus, purple. B, active site residues located within 4 Å of the thiol moiety of bound CoA. The letter designation (A or B) in the numbering scheme indicates the FRC monomer in which the residue is located. This image was generated using VMD (58Humphrey A. Dalke A. Schulten K. J. Mol. Graph. 1996; 14: 33-38Crossref PubMed Scopus (36920) Google Scholar) and POV-Ray (Persistence of Vision Development Team).View Large Image Figure ViewerDownload (PPT)We now report the first detailed kinetic characterization of recombinant, wild type (WT) FRC, and a number of site-specific mutants, which suggest that catalysis proceeds via anhydride intermediates as proposed for Class I CoA-transferases (32White H. Jencks W.P. J. Biol. Chem. 1976; 251: 1688-1699Abstract Full Text PDF PubMed Google Scholar). Further evidence for this mechanistic proposal is provided by the x-ray crystallographic observation of an acylenzyme intermediate that is formed when FRC is incubated with oxalyl-CoA. These studies therefore establish the catalytic mechanism of FRC, which is almost certainly employed by all other members of the Class III CoA-transferase family.EXPERIMENTAL PROCEDURESMaterials—All of the materials were of the highest purity available and, unless stated otherwise, were obtained from Fisher or Sigma-Aldrich. Protein concentrations were determined by the Lowry method (33Lowry O.H. Rosebrough S.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) based on a standard curve constructed using known amounts of bovine serum albumin. DNA sequencing was performed by the DNA Sequencing Core of the Interdisciplinary Center for Biotechnology Research at the University of Florida. The BL21(DE3) E. coli expression strain transformed with pET-9a plasmid carrying the gene for WT FRC was supplied by Dr. Harmeet Sidhu (Ixion Biotechnology, Inc., Alachua, FL). PCR primers were obtained from either GenoMechanix LLC (Gainesville, FL) (D169A and D169E) or Integrated DNA Technologies, Inc. (Coralville, IA) (D169S).FRC Expression and Purification—WT formyl CoA transferase (WT FRC) from O. formigenes was overexpressed in E. coli using literature procedures (34Sidhu H. Ogden S.D. Lung H.-Y. Luttge B.G. Baetz A.L. Peck A.B. J. Bacteriol. 1997; 179: 3378-3381Crossref PubMed Google Scholar) and purified using anion exchange and affinity chromatography. Hence, the lysate supernatant from 4–6 liters of culture was loaded on a 120-ml DEAE Fast Flow column equilibrated with buffer A (25 mm sodium phosphate, 1 mm dithiothreitol, pH 6.2), and FRC was obtained by stepping to a concentration of 35% buffer B (25 mm sodium phosphate, 1.0 m NaCl, 1 mm dithiothreitol, pH 6.2). Fractions containing FRC were then loaded on a 20-ml Blue-FF affinity column equilibrated with buffer A, and the column was washed with 1:1 buffer A/buffer B. Recombinant FRC was then obtained by eluting with buffer C (25 mm glycine, 1 mm dithiothreitol, 20% isopropanol, pH 9.0). After buffer exchange by passage through a 135-ml G-25 desalting column, equilibrated with buffer A, the solution containing FRC was injected on a 60-ml Q-Sepharose HP column equilibrated with buffer A. Purified FRC was then obtained by stepping to 20% buffer B followed by a linear increase in buffer B to a final concentration of 35%, with the desired enzyme eluting near the middle of this gradient. Glycerol was added to the combined FRC-containing fractions to a final concentration of 10%, given that the purified protein has a tendency to precipitate. The purity at each chromatographic step was verified by SDS-PAGE with Coomassie Blue staining. The flow rates used in these experiments were 5, 4, 10, and 5 ml/min for the DEAE, Blue-FF, G-25, and Q-Sepharose columns, respectively.Expression and Purification of the D169A, D169E, and D169S FRC Mutants—Mutagenic primers were designed using Gene Runner version 3.05 (Hastings Software). The pET-9a plasmid containing the WT FRC sequence was purified using the Wizard Plus Minipreps® DNA purification system (Promega) and used as a template for PCR with mutagenetic primers using the QuikChange® site-directed mutagenesis kit (Stratagene). The desired point mutations were verified by DNA sequencing of the inserts in the resulting pET-9a plasmids isolated from transformed XL-1 or XL-10 Gold supercompetent cells (Stratagene). Subsequent transformation of BL21(DE3) competent cells (Novagen) permitted overexpression and purification of the D169A, D169E, and D169S FRC mutants as described for recombinant, WT FRC.Synthesis of Formyl-CoA and Oxalyl-CoA—Formyl-CoA of very high purity was prepared by modification of literature procedures (35Sly W.S. Stadtman E.R. J. Biol. Chem. 1963; 238: 2632-2638Abstract Full Text PDF PubMed Google Scholar, 36Bax P.C. Stevens W. Recl. Trav. Chim. 1970; 89: 265-269Crossref Scopus (13) Google Scholar) (Fig. 3). Thus, formic acid (5.8 ml, 150 mmol) was added dropwise to acetic anhydride (7.1 ml, 75 mmol), and the resulting mixture was heated at 45 °C for 2.5 h to give a solution of the mixed anhydride (formylating reagent) (37Stevens W. Vanes A. Recl. Trav. Chim. 1964; 83: 863-872Crossref Scopus (28) Google Scholar). After cooling to room temperature, pyridine (61 μl, 0.75 mmol) was added to the solution immediately followed by thiophenol (5.1 ml, 50 mmol), and the resulting mixture stirred at room temperature for 24 h. Unreacted anhydrides, formic acid, and acetic acid were then removed by distillation under reduced pressure (20 mm Hg) at 50 °C, until the total volume was ∼6 ml. This oily material was then washed with cold, deionized water, and the organic layer was dried (MgSO4). The desired formyl thioester 5 was then obtained by vacuum distillation as a clear oil: 3.1 g, 45%; boiling point 115–117 °C, 23 mm Hg (literature valve 101 °C, 15 mm Hg (36Bax P.C. Stevens W. Recl. Trav. Chim. 1970; 89: 265-269Crossref Scopus (13) Google Scholar)). This material was then reacted with the sodium salt of coenzyme A using literature protocols to give formyl-CoA 1 (35Sly W.S. Stadtman E.R. J. Biol. Chem. 1963; 238: 2632-2638Abstract Full Text PDF PubMed Google Scholar).Fig. 3Improved synthetic preparation of formyl-CoA.View Large Image Figure ViewerDownload (PPT)Oxalyl-CoA 3 was prepared by reaction of coenzyme A with thiocresoxalic acid using literature procedures (38Quayle J.R. Biochem. J. 1963; 87: 368-373Crossref PubMed Scopus (25) Google Scholar). Formyl-CoA 1 and oxalyl-CoA 3 were further purified by reverse-phase HPLC on a preparative C18 column (Dynamax 60A C18, 250 × 21.4 mm). In our standard procedure, the column was equilibrated with 90% mobile phase A (10 mm NaH2PO4, pH 4.5) and 10% mobile phase B (phase A containing 20% CH3CN) running at 8 ml/min. After injection of each sample, the amount of B was increased to 40% using a linear gradient over 20 min. The absorbance of the eluent was monitored at 260 nm, and fractions containing the pure CoA derivative were combined and lyophilized to give the desired compounds as white solids, which could be stored at -80 °C without significant amounts of decomposition.Enzymatic Assay—Wild type, recombinant FRC was assayed by measuring the initial rate of oxalyl-CoA formation. The assay mixture contained 60 mm potassium phosphate, pH 6.7, FRC (90 ng), appropriate concentrations of substrates (formyl-CoA and oxalate), and, in the case of the product inhibition experiments, oxalyl-CoA or formate (total volume, 200 μl). The reaction was started by the addition of formyl-CoA after incubating the other components at 30 °C for about 30 s. Aliquots of the reaction mixture (90 μl) were typically taken after 60 and 90 s and quenched with 10% AcOH (10 μl) before being analyzed by reverse-phase HPLC using a modification of previous procedures for analyzing CoA derivatives (39Demoz A. Garras A. Asiedu D.K. Netteland B. Berge R.K. J. Chromatogr. B. 1995; 667: 148-152Crossref PubMed Scopus (60) Google Scholar). Thus, aliquots (75 μl) of the quenched reaction mixture were injected onto a C18 analytical column (Dynamax Microsorb 60–8 C18, 250 × 4.6 mm) that had been equilibrated using 86% buffer A (25 mm NaOAc, pH 4.5) and 14% buffer B (buffer A containing 20% CH3CN) running at 1.0 ml/min. Immediately after injection the proportion of buffer B was increased to 30% over a 210-s time period, followed by a step to 100% buffer B that was continued for 90 s before re-equilibrating with 86:14 buffer A:buffer B. The eluant was monitored at 260 nm. Under these conditions, oxalate eluted close to the void volume of the column (2.6 min), oxalyl-CoA eluted after 6.3 min, and free CoA and formyl-CoA eluted last from the column (9.0 min). The amount of oxalyl-CoA in the aliquots was determined by integration of the oxalyl-CoA peaks in the HPLC chromatograms and comparison with known amounts of authentic material. These measurements were calibrated using independent determinations of formyl-CoA concentration using a hydroxylamine-based colorimetric assay (35Sly W.S. Stadtman E.R. J. Biol. Chem. 1963; 238: 2632-2638Abstract Full Text PDF PubMed Google Scholar) and oxalate concentration in hydrolyzed and nonhydrolyzed samples of oxalyl-CoA with a standard detection kit (Sigma). No formation of oxalyl-CoA was detected in control experiments when the enzyme or either substrate was omitted or boiled enzyme was used. The limit of detection of this HPLC-based assay is 0.05 μm of oxalyl-CoA when 75-μl aliquots are injected onto the column.The specific activities of the D169A, D169E, and D169S FRC mutants were assayed using an identical procedure except that reaction mixtures were incubated for up to 60 min prior to quenching with AcOH. In addition, given the much lower activity of the FRC mutants, the amount of protein in each assay was increased to 2 μg, and the initial concentrations of oxalate and formyl-CoA were 100 mm and 200 μm, respectively.Analysis of Kinetic Data—Steady state kinetic constants (Km and Vmax) were determined by fitting the data from the initial rate studies using weighted hyperbolic regression analysis (Hyper, version 1.1). Data analysis was performed using standard equations (40Segel I.H. Enzyme Kinetics. Wiley-Interscience, New York.1975: 273-329Google Scholar).Determination of the Equilibrium Constant for FRC-catalyzed CoA Transfer—The equilibrium constant for the FRC-catalyzed reaction was determined by incubating the recombinant, WT enzyme (18 μg) with 73 μm formyl-CoA, 50 μm potassium oxalate in 60 mm potassium phosphate, pH 6.7 (total volume, 200 μl). The solution also contained 13 μm formate and 13 μm free CoA, present in the initial sample of formyl-CoA used in the experiment. This mixture was then incubated at 22 °C for 90 min. During this time, aliquots (45 μl) were withdrawn after 10, 27, and 52 min and quenched with 10% AcOH (5 μl). The concentration of oxalyl-CoA, free CoA (and therefore formate), and formyl-CoA in each sample was measured by reverse-phase HPLC as described above. Under these conditions, equilibrium was achieved after 27 min, giving Keq = 32 ± 3.Size Exclusion Chromatography Measurements—A BIOSEP SEC-S2000 column (300 × 7.8 mm with 75 × 7.8-mm guard column) was calibrated using lysozyme (14.4 kDa), carbonic anhydrase (29.0 kDa), peroxidase (44.0 kDa), bovine serum albumin (66.0 kDa), alcohol dehydrogenase (150 kDa), and β-amylase (200 kDa). The void volume was measured by injecting blue dextran. A sample of recombinant, WT O. formigenes FRC (72 μg) was prepared by filtration through a 50-kDa cut-off spin column, and the retentate resuspended in 100 mm potassium phosphate, pH 6.6 (total volume, 75 μl). An aliquot of this solution was then injected onto the BIOSEP SEC-S2000 column, giving a single peak with a retention time corresponding to a molecular mass of 81 kDa.Crystallization and Structure Determination of the D169A, D169E, and D169S FRC Mutants—All three Asp169 mutants were dialyzed and subsequently crystallized as their complexes with coenzyme A using identical conditions to those described previously for WT FRC (41Ricagno S. Jonsson S. Richards N. Lindqvist Y. Acta Crystallogr. Sect. D Biol. Crystallogr. 2003; 59: 1276-1277Crossref PubMed Scopus (4) Google Scholar). Diffraction data for the Ala and Glu mutants were collected at beam line X11 (Deutsches Electronen Synchrotron, Hamburg, Germany) equipped with a Mar research 165-mm CCD detector; diffraction data for the Ser mutant were collected at I711 (MaxLab) equipped with a Mar research 165-mm CCD detector. All of the data were processed with MOSFLM (42Leslie A.G.W. MOSFLM, version 5.40. MRC Laboratory of Molecular Biology, Cambridge, UK1997Google Scholar) and scaled with SCALA (43Number Collaborative Computational Project Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19707) Google Scholar). The crystals of the three mutants all belonged to space group I4 with the following unit cell parameters: D169E: a = b = 150.0 Å, c = 99.6 Å; D169A: a = b = 150.0 Å, c = 99.6 Å; D169S: a = b = 151.8 Å, c = 100.2 Å. All of the asymmetric units for these complexes contain a dimer.The structures were solved by difference Fourier using the FRC-CoA complex (29Ricagno S. Jonsson S. Richards N. Lindqvist Y. EMBO J. 2003; 22: 3210-3219Crossref PubMed Scopus (42) Google Scholar) as a model and were refined by REFMAC5 (44Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13781) Google Scholar) using maximum likelihood residual, anisotropic scaling, bulk solvent correction, and atomic displacement parameter refinement as used in the “translation, libration, screw rotation” method with each monomer as a rigid group. 5% of the reflections were excluded for subsequent use in monitoring Rfree. Noncrystallographic symmetries were not employed during the refinement. Water molecules were added by ARP-WARP (45Perrakis A. Sixma T.K. Wilson K.S. Lamzin V.S. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 448-455Crossref PubMed Scopus (479) Google Scholar), and the O (46Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13004) Google Scholar) software package was used for model building. The quality of the models was assessed by PROCHECK (47Laskowski R.A. Moss D.S. Thornton J.M. J. Mol. Biol. 1993; 231: 1049-1067Crossref PubMed Scopus (1077) Google Scholar), and structure comparisons were made in TOP (48Lu G. J. Appl. Crystallogr. 2000; 33: 176-183Crossref Scopus (193) Google Scholar) using default parameters. Statistics for data collection and the refined models are listed in Table I.Table IStructure determination statisticsD169AD169SD169 EFRC/oxalyl-CoAData collectionMethodX11 DESYI711 MAXX11 DESYI711 MAXResolution (Å)20.0–2.1320.0–2.3020.0–2.1020.0–2.70Total observations259,721215,424336,692180,867Unique observations61,07150,343644,44228,117Completeness (%)aNumbers in parentheses are calculated for the highest resolution shell97.2 (81.9)99.6 (99.6)100 (100)99.1 (100.0)RedundancyaNumbers in parentheses are calculated for the highest resolution shell4.3 (3.3)4.3 (4.3)5.2 (4.7)6.4 (6.7)Average I/σaNumbers in parentheses are calculated for the highest resolution shell13.7 (2.2)17.0 (3.6)12.7 (2.1)15.6 (4.6)Rsym (%)aNumbers in parentheses are calculated for the highest resolution shell9.1 (41.7)6.1 (33.7)11.4 (64.0)11.8 (33.2)RefinementNumber of atoms7172715871656865Protein6616661866246627CoA98989696Water455442445142B-factors Wilson plot31.350.332.764.6Mean B-factor model34.140.239.034.4R factor (%)aNumbers in parentheses are calculated for the highest resolution shell17.917.818.820.3Rfree (%)aNumbers in parentheses are calculated for the highest resolution shell20.021.622.528.0Ramachandran plotMost allowed region (%)91.891.090.890.7Additional allowed (%)7.88.68.69.0Generously allowed (%)0.30.10.30.1Disallowed region (%)0.10.30.30.1a Numbers in parentheses are calculated for the highest resolution shell Open table in a new tab Crystallization and Structure Determination of the FRC/Oxalyl-CoA Complex—FRC was dialyzed as described previously (29Ricagno S. Jonsson S. Richards N. Lindqvist Y. EMBO J. 2003; 22: 3210-3219Crossref PubMed Scopus (42) Google Scholar, 41Ricagno S. Jonsson S. Richards N. Lindqvist Y. Acta Crystallogr. Sect. D Biol. Crystallogr. 2003; 59: 1276-1277Crossref PubMed Scopus (4) Google Scholar) and crystallized with 20 mm oxalyl-CoA in 100 mm sodium cacodylate, pH 6.5, 0.2 m Mg(OAc)2, and polyethylene glycol 8000 at 4 °C using the hanging drop technique. Silicone oil was added to the crystallization drop as cryoprotectant, and crystals were flash frozen. Diffraction data were collected at 110 K at beamline I711 (MaxLab) on fresh crystals (11 days) to minimize uncatalyzed hydrolysis of oxalyl-CoA. All of the data were processed with MOSFLM (42Leslie A.G.W. MOSFLM, version 5.40. MRC Laboratory of Molecular Biology, Cambridge, UK1997Google Scholar) and scaled with SCALA (43Number Collaborative Computational Project Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19707) Google Scholar). The crystals were tetragonal and belonged to the space group p43212 (a = b = 100.2 Å, c = 196.9 Å).Structure determination was achieved by difference Fourier using the structure of the Q17A FRC mutant 2S. Ricagno, unpublished results. as a model. Refinement was carried out using REFMAC5 (44Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13781) Google Scholar), as described above for the Asp169 FRC mutants but using noncrystallographic symmetry, and water molecules were added by ARP-WARP (45Perrakis A. Sixma T.K. Wilson K.S. Lamzin V.S. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 448-455Crossref PubMed Scopus (479) Google Scholar). The software programs O (46Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13004) Google Scholar) and Coot (49Emsley P. Cowtan K. 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