• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of jbacterPermissionsJournals.ASM.orgJournalJB ArticleJournal InfoAuthorsReviewers
J Bacteriol. Apr 2008; 190(7): 2556–2564.
Published online Feb 1, 2008. doi:  10.1128/JB.01823-07
PMCID: PMC2293189

Differential Substrate Specificity and Kinetic Behavior of Escherichia coli YfdW and Oxalobacter formigenes Formyl Coenzyme A Transferase[down-pointing small open triangle]

Abstract

The yfdXWUVE operon appears to encode proteins that enhance the ability of Escherichia coli MG1655 to survive under acidic conditions. Although the molecular mechanisms underlying this phenotypic behavior remain to be elucidated, findings from structural genomic studies have shown that the structure of YfdW, the protein encoded by the yfdW gene, is homologous to that of the enzyme that mediates oxalate catabolism in the obligate anaerobe Oxalobacter formigenes, O. formigenes formyl coenzyme A transferase (FRC). We now report the first detailed examination of the steady-state kinetic behavior and substrate specificity of recombinant, wild-type YfdW. Our studies confirm that YfdW is a formyl coenzyme A (formyl-CoA) transferase, and YfdW appears to be more stringent than the corresponding enzyme (FRC) in Oxalobacter in employing formyl-CoA and oxalate as substrates. We also report the effects of replacing Trp-48 in the FRC active site with the glutamine residue that occupies an equivalent position in the E. coli protein. The results of these experiments show that Trp-48 precludes oxalate binding to a site that mediates substrate inhibition for YfdW. In addition, the replacement of Trp-48 by Gln-48 yields an FRC variant for which oxalate-dependent substrate inhibition is modified to resemble that seen for YfdW. Our findings illustrate the utility of structural homology in assigning enzyme function and raise the question of whether oxalate catabolism takes place in E. coli upon the up-regulation of the yfdXWUVE operon under acidic conditions.

With the completion of genome sequences for several strains of Escherichia coli (6, 27, 45, 61), attention has turned to the annotation of proteins encoded by specific genes of unknown function (56). Deletion studies have shown that the yfdXWUVE operon, in which the yfdX gene is under the control of the EvgAS regulatory system (40), encodes proteins that enhance the ability of E. coli MG1655 to survive under acidic conditions (41). Although the molecular mechanisms underlying this phenotypic behavior remain to be elucidated, the proteins encoded by the yfdW and yfdU genes in this operon (YfdW and YfdU, respectively) are homologous to proteins present in the obligate anaerobe Oxalobacter formigenes (53), O. formigenes formyl coenzyme A transferase (FRC) (2, 30, 46) and oxalyl coenzyme A (oxalyl-CoA) decarboxylase (3, 5). We note that FRC and oxalyl-CoA decarboxylase are essential for the survival of Oxalobacter in that they mediate the conversion of oxalate into formate and CO2 in a coupled catalytic cycle (Fig. (Fig.1).1). In combination with an oxalate-formate antiporter (OxlT) (29, 60), this cycle is thought to maintain the electrochemical and pH gradients needed for ATP synthesis (1, 35). It has therefore been proposed previously that (i) YfdW catalyzes the conversion of oxalate into oxalyl-CoA by using formyl-CoA as a donor and (ii) the YfdU protein mediates oxalyl-CoA decarboxylation (26). High-resolution X-ray crystallography supports the likely functional similarity of FRC and YfdW in that the two proteins adopt the same unusual interlocked, catalytically active dimer (Fig. (Fig.2)2) (24, 26, 46) despite having only 61% sequence identity (Fig. (Fig.3).3). On the other hand, the ability of E. coli to metabolize oxalate into formate and CO2 does not seem to have been systematically characterized, and the relevance of this activity to survival under conditions of low pH remains to be established (20).

FIG. 1.
Coupled enzymes of oxalate catabolism in O. formigenes.
FIG. 2.
Superimposition of apo-YfdW (cyan) and apo-FRC (white) dimer structures. Coordinates were obtained from the Protein Data Bank files 1pt7 and 1p5h, respectively, and the figure was made using PyMOL (16; http://www.pymol.sourceforge.net).
FIG. 3.
Structure-based sequence alignment of class III CoA transferase family members. This alignment was generated by the superimposition of the crystal structures of O. formigenes FRC (Protein Data Bank accession number 1p5h), E. coli YfdW (Protein Data Bank ...

We now report the first detailed examination of the steady-state kinetic behavior and substrate specificity of recombinant, wild-type YfdW. Our studies confirm that YfdW is a formyl-CoA transferase, and YfdW appears to be more stringent than the corresponding enzyme (FRC) in Oxalobacter in employing formyl-CoA and oxalate as substrates. Given the difference in substrate specificity of the two homologous enzymes, even though the residues defining the active sites are highly conserved (Fig. (Fig.4),4), we have examined the effects of replacing Trp-48 in FRC with the glutamine residue that occupies an equivalent position in the E. coli sequence. The replacement of Trp-48 by Gln-48 yields an FRC variant for which oxalate-dependent substrate inhibition is modified to resemble that seen for YfdW. Our findings illustrate the utility of structural homology in assigning enzyme function and raise the question of whether oxalate catabolism takes place in E. coli upon the up-regulation of the yfdXWUVE operon under acidic conditions.

FIG. 4.
Comparison of the active-site residues in YfdW (cyan) and FRC (white). Conserved residues are indicated by a one-letter code for amino acids. For positions where amino acids differ, the first letter refers to the residue present in YfdW.

MATERIALS AND METHODS

Materials.

Unless otherwise stated, all chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO) or Fisher Scientific (Pittsburgh, PA) and were of the highest available purity. Recombinant, wild-type FRC was expressed and purified by following procedures described in the literature (46). Protein concentrations were determined using a modified Bradford assay (Pierce, Rockford, IL) (8) for which standard curves were constructed with bovine serum albumin as previously reported (30) or the Edelhoch method (22). PCR primers were obtained from Integrated DNA Technologies, Inc. (Coralville, IA), and DNA sequencing was performed by the DNA Sequencing Core of the Interdisciplinary Center for Biotechnology Research at the University of Florida, Gainesville. Formyl-CoA and oxalyl-CoA were prepared as described elsewhere (30).

Expression and purification of His-tagged YfdW.

The subcloning and expression of the yfdW gene have been described in detail elsewhere (54, 59). Briefly, the yfdW gene was PCR amplified from the genomic DNA of E. coli K-12 and subcloned into the pDest17 vector by using Gateway technology (Invitrogen, Carlsbad, CA). Protein production was carried out with the Tuner(DE3)pLysS strain of E. coli, and the His-tagged YfdW protein was purified by metal affinity chromatography and subsequent gel filtration on a Superdex 200 column, eluting with 5 mM HEPES buffer containing 150 mM NaCl, pH 7.5.

Expression and purification of His-tagged FRC.

The gene encoding FRC (50) was cloned into the pET-28b vector (Novagen, San Diego, CA) so as to introduce a His tag with a 10-amino-acid linker at the N terminus of the recombinant protein. BL21(DE3) competent cells were transformed with the resulting construct, and protein expression was induced by the addition of IPTG (isopropyl-β-d-thiogalactopyranoside) at an optical density at 600 nm of 0.6. After being harvested and pelleted by centrifugation at 5,000 × g for 15 min, the cells were resuspended in lysis buffer (50 mM potassium phosphate, pH 7.2, containing 300 mM NaCl, 10 mM imidazole, and 1 mM β-mercaptoethanol) and sonicated. Cell debris was removed by centrifugation at 10,000 × g for 15 min, and the supernatant was loaded onto a 0.5-ml Ni-nitrilotriacetic acid column (Novagen) equilibrated with lysis buffer at 4°C. The column was washed with lysis buffer containing 50 mM imidazole, and His-FRC was eluted (in five 0.5-ml volumes) with elution buffer (50 mM potassium phosphate, pH 7.2, containing 300 mM NaCl and 250 mM imidazole). Size exclusion chromatography on a Sephadex G-25 column (30 ml) equilibrated with storage buffer (25 mM sodium phosphate, pH 6.7, 300 mM NaCl, and 1 mM dithiothreitol) removed the imidazole, and the purified protein was stored at −80°C in 10% glycerol.

Expression and purification of FRC variants.

The expression and purification of wild-type FRC and the W48F and W48Q FRC variants lacking the N-terminal histidine tag were performed by following procedures described in the literature (30, 46).

Steady-state kinetic assays.

All kinetic measurements were performed using a high-performance liquid chromatography (HPLC)-based assay, as reported in previous studies of FRC (30, 46). For measurements of YfdW-catalyzed CoA transfer, assay mixtures consisted of YfdW (54 ng) and the carboxylic acid acceptor in 100 mM potassium phosphate, pH 6.7 (total volume, 100 μl). The concentrations of free CoA in all samples were normalized to those of free CoA present as a contaminant in the assay mixtures containing the largest amounts of formyl-CoA; i.e., free CoA was added to solutions in which formyl-CoA was at low initial concentrations so that all reaction mixtures contained identical concentrations of free CoA. After the incubation of each solution at 30°C, the reaction was initiated by the addition of formyl-CoA. An aliquot (90 μl) was taken after 60 s, and the reaction was quenched by the addition of aqueous acetic acid to a concentration of 20% (10 μl). In separate control experiments, a series of smaller aliquots were taken and product formation was observed to be linear over a period of 60 s. The amount of the appropriate thioester product was then determined by the injection of the samples onto a C18 analytical column (Dynamax Microsorb 60-8 C18 250-by-4.6-mm reverse-phase analytical column) equilibrated with 86% buffer A (25 mM sodium acetate, pH 4.5) and 14% buffer B (buffer A containing 20% CH3CN) at a flow rate of 1 ml/min. Immediately after injection, the proportion of buffer B was increased to 6% over 210 s and then to 100% for 90 s before the elution using 98% buffer A and 2% buffer B. CoA-containing species were observed by monitoring the absorbance at 260 nm, and their concentrations were determined by integrating the peak areas and comparing them with those for known amounts of authentic material. These measurements were calibrated using (i) independent determinations of formyl-CoA concentrations by a hydroxylamine-based colorimetric assay (51) and (ii) the oxalate concentrations in hydrolyzed and nonhydrolyzed samples of oxalyl-CoA as measured with a standard detection kit (Sigma). No formation of CoA ester products in control experiments was observed when the enzyme or either substrate was omitted from the mixture.

In kinetic assays of FRC, His-tagged FRC, and the two FRC variant enzymes, a similar HPLC-based procedure was followed, except that assay mixtures contained either FRC (41 ng), the W48F variant (41 ng), the W48Q variant (43 ng), or His-tagged FRC (46 ng). Reactions were quenched with 10% aqueous acetic acid (10 μl), and aliquots were eluted initially with 96% buffer A (25 mM sodium acetate, pH 4.5) and 4% buffer B (buffer A containing 40% CH3CN) at a flow rate of 1 ml/min. The amount of buffer B was then increased to 11% over 210 s and then to 100% for 90 s, after which it was returned to 4%.

Determination of steady-state kinetic constants.

Kinetic constants were obtained by curve fitting to the following equations for sequential bi-bi kinetics (equation 1), substrate inhibition (equation 2), competitive inhibition (equation 3), and mixed-type inhibition (equation 4) (12):

equation M1
(1)

equation M2
(2)

equation M3
(3)

equation M4
(4)

In these equations, ν is the velocity, Kia, KmA, and KmB represent the dissociation constant of the first substrate to bind to the enzyme (formyl-CoA) and the Km values for formyl-CoA (A) and oxalate (B), respectively, KiS is the substrate inhibition constant of oxalate, S is the substrate, and Kic and Kin are the inhibition constants for competitive and noncompetitive mechanisms, respectively. Patterns of intersecting lines in double-reciprocal plots (see the supplemental material) were used to ascertain the mode of inhibition and, hence, the correct equations for use in evaluating the inhibition constants (12, 49). Kic and Kin and the formyl-CoA Km were determined by fitting initial velocity plots of CoA inhibition directly with the Michaelis-Menten equation for competitive (equation 3) or mixed-type (equation 4) inhibition. Km values for oxalate and succinate and Kia could then be determined by fitting initial velocity plots using an ordered bi-bi equation when the concentration of the second substrate (oxalate or succinate) was varied (equation 1). In the case of YfdW, attaining saturating formyl-CoA concentrations proved to be impractical. In this case, apparent Km and Vmax values for either oxalate or succinate were obtained by fitting to the initial velocity plots at different fixed formyl-CoA concentrations and various concentrations of the appropriate acid acceptor. Linear fits to the replots of (Km/Vmax)app and (1/Vmax)app against [formyl-CoA] were then used to estimate Km and Kia (49). All curve fitting was performed with KaleidaGraph 3.5 (Synergy Software, Reading, PA).

Determination of the specific activity of FRC and YfdW with alternate substrates.

The specific activities of FRC with alternate substrates were determined by incubating 8.8 nM enzyme (83 ng of FRC in 200 μl) in 60 mM potassium phosphate, pH 6.7, a 125 mM concentration of the CoA acceptor, and an 80 μM concentration of the CoA donor at 30°C. In the case of YfdW, 11.2 nM (108 ng of YfdW in 200 μl) was used with a 75 mM concentration of the acceptor and a 350 μM concentration of the CoA donor. Specific activities for YfdW with succinate or oxalate were determined from initial velocity experiments. Substrates were regarded as having no activity when no products were detected in reactions that were run for 60 min.

Crystallization and structure determination for the W48F and W48Q FRC variants.

The crystallization of the FRC variants was performed by the vapor diffusion method in 24-well plates where hanging drops of 2 μl of protein solution and 2 μl of a well solution were set up to equilibrate against 1 ml of the well solution at 293 K. A protein solution containing 7.5 mg of the desired variant/ml in 50 mM MES [2-(N-morpholino)ethanesulfonic acid] buffer, pH 6.2, with the addition of 10% glycerol was used in screening for optimal conditions for crystallization. The W48Q variant of FRC was crystallized against a well solution of 1.35 M sodium citrate and 0.1 M HEPES buffer, pH 7.2 to 7.4, resulting in crystals of the monoclinic space group C2. These crystals were protected in a cryosolution of 3 parts of the well solution mixed with 1 part of 100% ethylene glycol before being flash frozen in liquid nitrogen. For the W48F FRC variant, a well solution of 1.9 M malic acid, pH 7.0, gave crystals belonging to the tetragonal space group I4. The crystallization drops containing the W48F variant were covered in silicon oil, through which the crystals were dragged before being flash frozen. X-ray data were collected in a nitrogen stream at the beamlines ID14 eh1 and ID23 eh2 at the European Synchrotron Research Facility, Grenoble, France. All crystallographic data were processed with MOSFLM (38), followed by SCALA of the CCP4 program suite (13). The structure of the apoenzyme (Protein Data Bank accession number 1p5h) (46) was used to retrieve the phases by molecular replacement using the program MOLREP (58). Refinement was carried out with REFMAC (43), and manual model building was performed in COOT (18), where water molecules were assigned and the structures were validated. The stereochemistry of the structures was checked with PROCHECK (36). Statistics for the FRC W48F and W48Q variants are given in Table Table11.

TABLE 1.
Data collection and refinement statistics for the W48F and W48Q FRC variantsa

Protein structure accession numbers.

Coordinates and structure factors for the FRC W48F and W48Q variants have been deposited in the Protein Data Bank with accession numbers 2VJP and 2VJQ, respectively.

RESULTS

Kinetic characterization of YfdW.

Our initial experiments examined whether E. coli YfdW could catalyze the synthesis of oxalyl-CoA from formyl-CoA and oxalate, as inferred on the basis of structural genomics (24, 26). Incubating formyl-CoA and oxalate with YfdW in phosphate buffer, pH 6.7, did indeed result in the appearance of oxalyl-CoA, and the amount of this product could be quantified by direct HPLC measurement (Fig. (Fig.5).5). The analysis of initial rate data by standard fitting methods (12) gave a value of 510 ± 30 μM for the apparent Km of oxalate, almost an order of magnitude less than the cognate parameter determined for this substrate in the FRC-catalyzed reaction (3.9 ± 0.3 mM) (46). The variation of oxalate concentrations at different fixed concentrations of formyl-CoA gave intersecting lines in the Lineweaver-Burk plot (see Fig. S1 in the supplemental material), suggesting an ordered bi-bi sequential kinetic mechanism, as reported previously for FRC (30) and other class III CoA transferases (19, 21, 28, 32, 33, 39). The finding that oxalate concentrations higher than 2.5 mM inhibited the activity of YfdW (Fig. (Fig.6,6, upper panel) was again in sharp contrast to the kinetic behavior of FRC, which is not inhibited by oxalate at concentrations in excess of 230 mM (30). The evaluation of steady-state kinetic parameters for formyl-CoA in the YfdW-catalyzed reaction was, however, complicated by the presence of 10% free CoA in this substrate as a result of the procedures used to remove a 2′-phosphorylated isomer of this compound, which exhibited a slightly longer retention time than formyl-CoA upon reverse-phase HPLC (Fig. (Fig.5).5). The contamination of the commercially available CoA used in the synthesis of formyl-CoA (30) has been reported previously in studies of enzymes for which malonyl-CoA (42) and β-hydroxybutyryl-CoA (9) are substrates. The extent to which free CoA inhibited YfdW activity was assessed using standard kinetic methods, and inspection of the double-reciprocal plot showed mixed-type inhibition of formyl-CoA (see Fig. S2 in the supplemental material). After fitting to the appropriate kinetic equation, values of Kic and Kin of 220 ± 21 and 210 ± 16 μM, respectively, were obtained for inhibition by free CoA, which then permitted the apparent Km of formyl-CoA to be estimated as 352 ± 4 μM. The turnover number, kcat, under these conditions could then be determined as 130 ± 17 s−1, which is considerably greater than that for FRC, 5.3 ± 0.1 s−1 (Table (Table2).2). Given the presence of a polyhistidine tag at the N terminus of YfdW, a similarly tagged variant of FRC was prepared and its steady-state kinetic parameters were measured using the HPLC-based end point assay (see Fig. S3 in the supplemental material). The results of these experiments showed that the observed difference in kcat values for YfdW and FRC (Table (Table2)2) cannot be attributed to this structural modification.

FIG. 5.
HPLC properties of the CoA-containing components in the assay mixture. Key: (1), oxalate; (2), oxalyl-CoA; (3), free CoA; (4), formyl-CoA. The arrows indicate the retention times of iso-CoA (≈11 min) and 2′-phosphoformyl-CoA (≈13 ...
FIG. 6.
Substrate inhibition of YfdW by oxalate. (Upper panel) Initial velocities (v) measured for YfdW as a function of the oxalate concentration at 73.3 μM formyl-CoA. The line is computed from a fit to the Michaelis-Menten equation modified for substrate ...
TABLE 2.
Steady-state parameters for the formyl-CoA and oxalate transferase activities of YfdW, FRC, and the Trp-48 FRC variantsa

The crystallographic observation of a YfdW-acetyl-CoA-oxalate ternary complex (26) suggested that YfdW may be inhibited by acetyl-CoA, and we therefore assayed the steady-state kinetic behavior of the enzyme in the presence of this compound (see Fig. S4 in the supplemental material). As in our earlier experiments, the concentration of free CoA was maintained at a fixed value (52 μM) as that of formyl-CoA was varied. Given that acetyl-CoA binds to the CoA site in the YfdW crystal structure (26), we assumed that acetyl-CoA and CoA were mutually exclusive inhibitors at a given active site. This property permitted the separation of their contributions to the overall rate equation (19), and acetyl-CoA proved to be a noncompetitive inhibitor of formyl-CoA, with a Kin value of 94 ± 2 μM. In contrast, acetyl-CoA is a competitive inhibitor of FRC with respect to formyl-CoA, exhibiting a Kic value of 56 ± 6 μM at a fixed CoA concentration of 1.5 μM (see Fig. S5 in the supplemental material).

Alternate substrate studies.

A variety of CoA donors and acceptors were incubated with the recombinant, tagged YfdW to elucidate the substrate specificity of the enzyme (see Table S1 in the supplemental material). YfdW showed high levels of substrate specificity, being unable to catalyze CoA transfer from formyl-CoA to acetate, maleate, or glutarate. Given that malonyl-CoA and succinyl-CoA are known metabolic intermediates in E. coli, however, we assayed whether either of these diacids could function as a substrate. In the case of malonate, YfdW exhibited very low specific activity (0.01%) with formyl-CoA as the donor. Control experiments were also performed to ensure that any malonyl-CoA formed did not undergo extensive uncatalyzed decarboxylation under the conditions. Acetyl-CoA formation was also below the detection limits of the HPLC-based assay when the enzyme was incubated with formyl-CoA and malonate. In contrast, when succinate was used as an acceptor, succinyl-CoA was formed, albeit with a low specific activity (4%) relative to that observed for oxalate with formyl-CoA. A complete determination of the steady-state kinetic parameters for the YfdW-catalyzed conversion of succinate into succinyl-CoA was therefore performed to evaluate the substrate specificity of the enzyme (see Fig. S6 in the supplemental material). These studies gave 80 ± 40 mM as the apparent Km of succinate and a turnover number of only 5.3 ± 0.4 s−1 when formyl-CoA was employed as a donor. In contrast to our observations on YfdW, succinate was an excellent substrate for FRC, the specificity constant being two orders of magnitude greater for this substrate than for oxalate with formyl-CoA as a donor (Table (Table3).3). In a similar manner, we observed that FRC could employ succinyl-CoA as an alternate CoA donor for the synthesis of formyl- and oxalyl-CoA (see Table S1 in the supplemental material). The specific activity of FRC with malonate and formyl-CoA as substrates was also substantially lower (0.1%) than that observed when oxalate was present as the CoA acceptor. No products were detected in the HPLC-based assay when malonyl-CoA was used as a substrate with either formate or oxalate.

TABLE 3.
Steady-state parameters for the formyl-CoA and succinate transferase activities of YfdW, FRC, and the Trp-48 FRC variantsa

Kinetic and structural characterization of the W48F and W48Q FRC variants.

The extent to which active-site residues must be modified in order to change the substrate specificities of enzymes remains an interesting problem in enzyme evolution (23, 44, 55), and its resolution has important implications for efforts to redesign biological catalysts for biotechnological applications (11, 31). The active sites of FRC and YfdW, however, are composed of conserved residues, making it difficult to understand (i) the observed differences in substrate specificity and (ii) the ability of oxalate to exhibit substrate inhibition only in the case of YfdW. Structural studies of FRC had, however, revealed the importance of a tetraglycine segment in stabilizing a putative reaction intermediate (30), and conformational changes in this loop appeared to be correlated with the orientation of the Trp-48 side chain in FRC (46). Superimposition of the crystal structures for the two CoA transferases showed that this tryptophan residue was replaced by glutamine in YfdW (Fig. (Fig.4).4). Moreover, for YfdW, an oxalate molecule was seen to bind to a closed conformation of this tetraglycine loop (corresponding to residues 246GGGGQ250 in YfdW), although the observed glutamine side chain rotamer was the same as that seen for Trp-48 in FRC when the cognate loop segment was in its open conformation (46). We therefore investigated whether site-specific mutagenesis of Trp-48 in FRC might yield variant enzymes exhibiting modified kinetic behavior that was similar to that determined for YfdW. Two variants in which Trp-48 was replaced by phenylalanine (W48F) and glutamine (W48Q) were prepared and characterized under steady-state conditions. Relatively little change in the specificity constants (kcat/Km) of the two FRC variant enzymes for formyl-CoA and oxalate compared with those of the wild-type enzyme was evident (Table (Table2).2). Perhaps more importantly, the W48Q FRC variant exhibited substrate inhibition with oxalate, having a Ki value of 74 mM (Fig. (Fig.6,6, lower panel), as observed for YfdW. In contrast, the W48F FRC variant was not inhibited by oxalate at concentrations of up to 154 mM, suggesting that hydrogen bonding to the Gln-48 side chain is an essential element for the interaction of this substrate with the site, as suggested by the YfdW-acetyl-CoA-oxalate crystal structure (26).

So as to understand the structural effects of changing the tryptophan residue in more detail, we obtained the crystal structures of the two FRC variants. Neither the W48Q nor the W48F FRC variant displayed any major structural changes compared to wild-type FRC, with the root mean square deviation of the Cα atoms being 0.2 to 0.3 Å2 and 0.6 to 0.7 Å2 relative to subunit A and subunit B of apo-FRC, respectively. In both variant enzymes, the tetraglycine loop (corresponding to residues 258GGGGQ262 in FRC) was seen to adopt a closed conformation (46). In wild-type FRC, a 90° reorientation of the Trp-48 side chain seems to be important in controlling the tetraglycine loop conformation. This flipping of the indole moiety is accompanied by the repositioning of Met-44 when the loop adopts its open conformation. For YfdW, in which a glutamine residue (Gln-48) replaces tryptophan, however, oxalate can bind to the tetraglycine loop in the closed conformation, even though Gln-48 adopts the rotamer conformation corresponding to that of Trp-48 in FRC when the cognate loop is open. A comparison of the YfdW and W48Q FRC variant structures showed that Gln-48 in W48Q, in the absence of oxalate, does not take the side chain rotamer conformation seen for the cognate residue in YfdW, presumably because of the proximal methionine residue (Met-44) (Fig. (Fig.7).7). For the W48Q FRC variant to bind oxalate in the site with the tetraglycine loop in a closed conformation, Gln-48 and Met-44 would both have to change the rotamer conformation. In YfdW, the methionine position is occupied by a smaller valine residue.

FIG. 7.
Active-site structure in the W48Q FRC variant. (A) Superimposition of apo-FRC with the tetraglycine loop in its open (white) and closed (green) conformations and the W48Q FRC variant (pink). (B) Superimposition of apo-YfdW (cyan) with the open conformation ...

DISCUSSION

These experiments clearly demonstrate that YfdW is a formyl-CoA:oxalate CoA transferase, as anticipated on the basis of its sequence and structural similarity to FRC (26). Although this may seem to be an obvious finding, recent studies have shown that assigning enzyme function on the basis of sequence similarity can often lead to misannotation in metabolic databases (47). Moreover, the location of the gene encoding a CoA transferase in an operon that confers resistance to acidic environments seems, at first sight, unexpected. A further interesting outcome of these biochemical studies concerns the high level of substrate specificity that is exhibited by YfdW. Thus, despite considerable efforts to identify other CoA acceptors and donors, only formyl-CoA and oxalate (and equivalently, oxalyl-CoA and formate) seem to be substrates for the enzyme. YfdW can therefore mediate oxalate catabolism in E. coli without affecting cellular succinyl-CoA levels. This observation stands in sharp contrast to the kinetic behavior of the Oxalobacter enzyme, for which succinate is a better CoA acceptor than oxalate when formyl-CoA is employed as a donor (Table (Table3).3). In light of the importance of oxalate as an energy source for O. formigenes (1), the ability of FRC to synthesize succinyl-CoA is unexpected because this enzyme-catalyzed reaction removes a molecule of formyl-CoA, thereby breaking the catalytic cycle (Fig. (Fig.1).1). On the other hand, this side activity of FRC may be one mechanism by which Oxalobacter can use oxalate in the biosynthesis of other carbon-containing compounds, given that succinyl-CoA is a key component of lysine biosynthesis and other biosynthetic pathways (25). The presence of succinate in the cytoplasm of Oxalobacter is suggested by studies that have shown the presence of succinate dehydrogenase, fumarase, and malate dehydrogenase, which can be employed to interconvert oxaloacetate and succinate in the latter part of the citric acid cycle (14, 15).

YfdW is inhibited by a variety of components, including acetyl-CoA, free CoA, and oxalate. On the basis of previous work on O. formigenes (30), we anticipated that CoA derivatives would compete with formyl-CoA for the free enzyme. Acetyl-CoA and free CoA are noncompetitive and mixed-type inhibitors, however, with respect to both formyl-CoA and oxalate. Hence, it seems that both these compounds can bind to YfdW-substrate complexes that are formed during catalytic turnover. The simplest explanation for such kinetic behavior is that the two active sites in the YfdW dimer can communicate so that only a single active site can catalyze the reaction at a given time (“half-sites” reactivity) (34). As a result, if CoA derivatives bind to a free CoA site in a YfdW-substrate complex (or a catalytic intermediate), then YfdW undergoes a conformational change that precludes the formation of critical intermediates (4, 30) or product release at the other site. A more interesting observation was that YfdW is inhibited by elevated levels of oxalate, a kinetic behavior that is not seen for FRC. This inhibition was hypothesized to arise from oxalate binding at a second nonproductive site defined (in part) by the Gln-48 side chain in YfdW. Such binding is precluded by the presence of a tryptophan residue in FRC, and the replacement of Trp-48 by glutamine to give the W48Q FRC variant yields an enzyme for which oxalate inhibition is observed. Hence, it seems that replacing the indole side chain by that of glutamine opens a hole in the FRC active site into which oxalate can bind in a nonproductive conformation. This mutation also results in altered conformational preferences of a tetraglycine loop that is known to be important for catalytic function (4, 30, 46), implying that altered active-site dynamic motions may play a role in modulating kinetic properties (7, 17). The high Ki determined for oxalate in YfdW inhibition seems to preclude any physiological importance for this behavior.

With the identification of YfdW as a formyl-CoA:oxalate CoA transferase, questions are raised concerning the extent and importance of oxalate-related metabolism in E. coli (37), especially because our work bolsters the assignment of YfdU as a thiamine-dependent oxalyl-CoA decarboxylase. Although E. coli has been implicated in the biomineralization processes leading to the formation of calcium oxalate crystals (10), recent measurements suggest that E. coli does not degrade oxalate in media containing this compound at a 5 mM concentration (57). The experiments, however, did not systematically vary the incubation conditions, and so it is possible that conditions under which E. coli can metabolize exogenous oxalate exist. On this point, we note that YhjX has been annotated as a possible formate-oxalate antiporter based on 25% sequence identity to O. formigenes OxlT, which has been extensively characterized previously (29, 60). More work is therefore needed to establish if E. coli can mediate oxalate degradation, especially in low-pH environments. It is interesting that the coupled action of YfdW and YfdU results in the consumption of a proton, as employed in the AR2 and AR3 mechanisms of acid resistance mediated by the pyridoxal phosphate-dependent enzymes glutamate decarboxylase and arginine decarboxylase (20). Thus, the results of the studies reported herein raise the question of whether oxalate catabolism in E. coli can take place upon the up-regulation of the yfdXWUVE operon under conditions of low pH.

Supplementary Material

[Supplemental material]

Acknowledgments

We acknowledge the access of beam time at the European Synchrotron Research Facility, Grenoble, France.

This work was supported (in part) by National Institutes of Health grant R01 DK61666 (N.G.J.R.) and by the Swedish Research Council-Scientific Council for Natural and Engineering Sciences (Y.L.). We also thank the University of Florida for the provision of alumni and Reugamer fellowships (C.G.T.).

Footnotes

[down-pointing small open triangle]Published ahead of print on 1 February 2008.

Supplemental material for this article may be found at http://jb.asm.org/.

REFERENCES

1. Anatharam, V., M. J. Allison, and P. C. Maloney. 1989. Oxalate:formate exchange. The basis for energy coupling in Oxalobacter. J. Biol. Chem. 2647244-7250. [PubMed]
2. Baetz, A. L., and M. J. Allison. 1990. Purification and characterization of formyl-coenzyme A transferase from Oxalobacter formigenes. J. Bacteriol. 1723537-3540. [PMC free article] [PubMed]
3. Baetz, A. L., and M. J. Allison. 1989. Purification and characterization of oxalyl-coenzyme A decarboxylase from Oxalobacter formigenes. J. Bacteriol. 1712605-2608. [PMC free article] [PubMed]
4. Berthold, C. L., C. G. Toyota, N. G. J. Richards, and Y. Lindqvist. 27 December 2007, posting date. Re-investigation of the catalytic mechanism of formyl-CoA transferase, a class III CoA-transferase. J. Biol. Chem. doi/.10.1074/jbc.M709353200 [PubMed] [Cross Ref]
5. Berthold, C. L., P. Moussatche, N. G. J. Richards, and Y. Lindqvist. 2005. Structural basis of activation of the thiamin-dependent enzyme oxalyl-CoA decarboxylase by adenosine diphosphate. J. Biol. Chem. 28041645-41654. [PubMed]
6. Blattner, F. R., G. Plunkett III, C. A. Bloch, N. T. Perna, V. Burland, M. Riley, J. Collado-Vides, J. D. Glasner, C. K. Rode, G. F. Mayhew, J. Gregor, N. W. Davis, H. A. Kirkpatrick, M. A. Goeden, D. J. Rose, B. Mau, and Y. Shao. 1997. The complete genome sequence of Escherichia coli K-12. Science 2771453-1474. [PubMed]
7. Boehr, D. D., D. McElheny, H. J. Dyson, and P. E. Wright. 2006. The dynamic energy landscape of dihydrofolate reductase catalysis. Science 3131638-1642. [PubMed]
8. Bradford, M. M. 1976. Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein-dye binding. Anal. Biochem. 72248-254. [PubMed]
9. Burns, K. L., L. T. Gelbaum, M. C. Sullards, D. E. Bostwick, and S. W. May. 2005. Iso-coenzyme A. J. Biol. Chem. 28016550-16558. [PubMed]
10. Chen, L., Y. Shen, R. Jia, A. Xie, B. Huang, X. Cheng, Q. Zhang, and R. Guo. 2007. The role of Escherichia coliform in the biomineralization of calcium oxalate crystals. Eur. J. Inorg. Chem. 20073201-3207.
11. Chica, R. A., N. Doucet, and J. N. Pelletier. 2005. Semi-rational approaches to engineering enzyme activity: combining the benefits of directed evolution and rational design. Curr. Opin. Biotechnol. 16378-384. [PubMed]
12. Cleland, W. W. 1979. Statistical analysis of enzyme kinetic data. Methods Enzymol. 62151-160.
13. Collaborative Computational Project, Number 4. 1994. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50760-763. [PubMed]
14. Cornick, N. A., and M. J. Allison. 1996. Anabolic incorporation of oxalate by Oxalobacter formigenes. Appl. Environ. Microbiol. 623011-3013. [PMC free article] [PubMed]
15. Cornick, N. A., and M. J. Allison. 1996. Assimilation of oxalate, acetate, and CO2 by Oxalobacter formigenes. Can. J. Microbiol. 421081-1086. [PubMed]
16. DeLano, W. L. 2002. The PyMOL molecular graphics system. DeLano Scientific LLC, Palo Alto, CA.
17. Eisenmesser, E. Z., O. Millet, W. Labeikovsky, D. M. Korzhnev, M. Wolf-Watz, D. A. Bosco, J. J. Skalicky, L. E. Kay, and D. Kern. 2005. Intrinsic dynamics of an enzyme underlies catalysis. Nature 438117-121. [PubMed]
18. Emsley, P., and K. Cowtan. 2004. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 602126-2132. [PubMed]
19. Engemann, C., T. Elssner, S. Pfeifer, C. Krumbholz, T. Maier, and H.-P. Kleber. 2005. Identification and functional characterization of genes and corresponding enzyme involved in carnitine metabolism of Proteus sp. Arch. Microbiol. 183176-189. [PubMed]
20. Foster, J. W. 2004. Escherichia coli acid resistance: tales of an amateur acidophile. Nat. Rev. Microbiol. 2898-907. [PubMed]
21. Friedmann, S., B. E. Alber, and G. Fuchs. 2006. Properties of succinyl-coenzyme A:D-citramalate-coenzyme A transferase and its role in the autotrophic 3-hydroxypropionate cycle of Chloroflexus aurantiacus. J. Bacteriol. 1886460-6468. [PMC free article] [PubMed]
22. Gill, S. C., and P. H. von Hippel. 1989. Calculation of protein extinction coefficients from amino acid sequence data. Anal. Biochem. 182319-326. [PubMed]
23. Glasner, M. E., J. A. Gerlt, and P. C. Babbitt. 2006. Evolution of enzyme superfamilies. Curr. Opin. Chem. Biol. 10492-497. [PubMed]
24. Gogos, A., J. Gorman, and L. Shapiro. 2004. Structure of Escherichia coli YfdW, a type III CoA transferase. Acta Crystallogr. D 60507-511. [PubMed]
25. Goto, S., Y. Okuno, M. Hattori, T. Nishioka, and M. Kanehisa. 2002. LIGAND: database of chemical compounds and reactions in biological pathways. Nucleic Acids Res. 30402-404. [PMC free article] [PubMed]
26. Gruez, A., V. Roig-Zamboni, C. Valencia, V. Campanacci, and C. Cambillau. 2003. The crystal structure of the Escherichia coli YfdW gene product reveals a new fold of two interlaced rings identifying a wide family of CoA transferases. J. Biol. Chem. 27834582-34586. [PubMed]
27. Hayashi, T., K. Makino, M. Ohnishi, K. Kurokawa, K. Ishii, K. Yokoyama, C. G. Han, E. Ohtsubo, K. Nakayama, T. Murata, M. Tanaka, T. Tobe, T. Iida, H. Takami, T. Honda, C. Sasakawa, N. Ogasawara, T. Yasunaga, S. Kuhara, T. Shiba, M. Hattori, and H. Shinagawa. 2001. Complete genome sequence of enterohaemorrhagic Escherichia coli O157:H7 and genomic comparison with a laboratory strain K-12. DNA Res. 811-22. [PubMed]
28. Heider, J. 2001. A new family of CoA transferases. FEBS Lett. 509345-349. [PubMed]
29. Hirai, T., J. A. W. Heymann, D. Shi, R. Sarker, P. C. Maloney, and S. Subramanian. 2002. Three-dimensional structure of a bacterial oxalate transporter. Nat. Struct. Biol. 9597-600. [PubMed]
30. Jonsson, S., S. Ricagno, Y. Lindqvist, and N. G. J. Richards. 2004. Kinetic and mechanistic characterization of the formyl-CoA transferase from Oxalobacter formigenes. J. Biol. Chem. 27936003-36012. [PubMed]
31. Kaur, J., and R. Sharma. 2006. Directed evolution: an approach to engineer enzymes. Crit. Rev. Biotechnol. 26165-199. [PubMed]
32. Kim, J., D. Darley, T. Selmer, and W. Buckel. 2006. Characterization of (R)-2-hydroxyisocaproate dehydrogenase and a family III coenzyme A transferase involved in reduction of l-leucine to isocaproate by Clostridium difficile. Appl. Environ. Microbiol. 726062-6069. [PMC free article] [PubMed]
33. Kim, J., D. Darley, T. Selmer, and W. Buckel. 2006. The involvement of coenzyme A esters in the dehydration of (R)-phenyllactate to (E)-cinnamate by Clostridium sporogenes. Eur. J. Biochem. 2673874-3884. [PubMed]
34. Koshland, D. E. 1996. The structural basis of negative cooperativity: receptors and enzymes. Curr. Opin. Struct. Biol. 6757-761. [PubMed]
35. Kuhner, C. A., P. A. Hartmann, and M. J. Allison. 1996. Generation of a proton motive force by the anaerobic oxalate-degrading bacterium Oxalobacter formigenes. Appl. Environ. Microbiol. 622494-2500. [PMC free article] [PubMed]
36. Laskowski, R. A., M. W. MacArthur, D. S. Moss, and J. M. Thornton. 1993. PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Crystallogr. 26283-291.
37. Lawrence, J. G., and H. Ochman. 1998. Molecular archaeology of the Escherichia coli genome. Proc. Natl. Acad. Sci. USA 959413-9417. [PMC free article] [PubMed]
38. Leslie, A. G. W. 1997. MOSFLM, version 5.40. MRC Laboratory of Molecular Biology, Cambridge, United Kingdom.
39. Leutwein, C., and J. Heider. 2001. Succinyl-CoA:(R)-benzylsuccinate CoA-transferase: an enzyme of the anaerobic toluene catabolic pathway in denitrifying bacteria. J. Bacteriol. 1834288-4295. [PMC free article] [PubMed]
40. Masuda, N., and G. M. Church. 2003. Regulatory network of acid resistance genes in Escherichia coli. Mol. Microbiol. 48699-712. [PubMed]
41. Masuda, N., and G. M. Church. 2002. Escherichia coli gene expression responsive to levels of the response regulator EvgA. J. Bacteriol. 1846225-6234. [PMC free article] [PubMed]
42. Minkler, P. E., V. E. Anderson, N. C. Maiti, J. Kerner, and C. L. Hoppel. 2004. Isolation and identification of two isomeric forms of malonyl-coenzyme A in commercial malonyl-coenzyme A. Anal. Biochem. 328203-209. [PubMed]
43. Murshudov, G. N., A. A. Vagin, and E. J. Dodson. 1997. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D 53240-255. [PubMed]
44. O'Brien, P. J., and D. Herschlag. 1999. Catalytic promiscuity and the evolution of new enzyme activities. Chem. Biol. 6R91-R105. [PubMed]
45. Perna, N. T., G. Plunkett III, V. Burland, B. Mau, J. D. Glasner, D. J. Rose, G. F. Mayhew, P. S. Evans, J. Gregor, H. A. Kirkpatrick, G. Posfai, J. Hackett, S. Klink, A. Boutin, Y. Shao, L. Miller, E. J. Grotbeck, N. W. Davis, A. Lim, E. T. Dimalanta, K. D. Potamousis, J. Apodaca, T. S. Anantharaman, J. Lin, G. Yen, D. C. Schwartz, R. A. Welch, and F. R. Blattner. 2001. Genome sequence of enterohaemorrhagic Escherichia coli O157:H7. Nature 409529-533. [PubMed]
46. Ricagno, S., S. Jonsson, N. G. Richards, and Y. Lindqvist. 2003. The crystal structure of formyl-CoA transferase reveals a CoA binding site at the interface of an interlocked dimer. EMBO J. 223210-3219. [PMC free article] [PubMed]
47. Rost, B. 2002. Enzyme function less conserved than anticipated. J. Mol. Biol. 318595-608. [PubMed]
48. Savolainen, K., P. Bhaumik, W. Schmitz, T. J. Kotti, E. Conzelmann, R. K. Wierenga, and J. K. Hiltunen. 2005. Alpha-methylacyl-CoA racemase from Mycobacterium tuberculosis: mutational and structural characterization of the active site and the fold. J. Biol. Chem. 28012611-12620. [PubMed]
49. Segel, I. H. 1993. Enzyme kinetics: behavior and analysis of rapid equilibrium and steady-state enzyme systems. Wiley-Interscience, New York, NY.
50. Sidhu, H., S. D. Ogden, H.-Y. Lung, B. G. Luttge, A. L. Baetz, and A. B. Peck. 1997. DNA sequencing and expression of the formyl coenzyme A transferase gene, frc, from Oxalobacter formigenes. J. Bacteriol. 1793378-3381. [PMC free article] [PubMed]
51. Sly, W. S., and E. R. Stadtman. 1963. Formate metabolism. 1. Formyl-coenzyme A, an intermediate in formate-dependent decomposition of acetyl phosphate in Clostridium kluyveri. J. Biol. Chem. 2382632-2638. [PubMed]
52. Stenmark, P., D. Gurmu, and P. Nordlund. 2004. Crystal structure of CaiB, a type III CoA transferase in carnitine metabolism. Biochemistry 4313996-14003. [PubMed]
53. Stewart, C. S., S. H. Duncan, and D. R. Cave. 2004. Oxalobacter formigenes and its role in oxalate metabolism in the human gut. FEMS Microbiol. Lett. 2301-7. [PubMed]
54. Sulzenbacher, G., A. Gruez, V. Roig-Zamboni, S. Spinelli, C. Valencia, F. Pagot, R. Vincentelli, C. Bignon, A. Salomoni, S. Grisel, D. Maurin, C. Huyghe, K. Johansson, A. Grassick, A. Roussel, Y. Bourne, S. Perrier, L. Miallau, P. Cantau, E. Blanc, M. Genevois, A. Grossi, A. Zenatti, V. Campanacci, and C. Cambillau. 2002. A medium-throughput crystallization approach. Acta Crystallogr. D 582109-2115. [PubMed]
55. Svedruzic, D., Y. Liu, L. A. Reinhardt, E. Wroclawska, W. W. Cleland, and N. G. J. Richards. 2007. Investigating the role of putative active site residues in the oxalate decarboxylase from Bacillus subtilis. Arch. Biochem. Biophys. 46436-47. [PMC free article] [PubMed]
56. Thomas, P. D., H. Y. Mi, and S. Lewis. 2007. Ontology annotation: mapping genomic regions to biological function. Curr. Opin. Chem. Biol. 114-11. [PubMed]
57. Turroni, S., B. Vitali, C. Bendazzoli, M. Candela, R. Gotti, F. Frederici, F. Pirovano, and P. Brigidi. 2007. Oxalate consumption by lactobacilli: evaluation of oxalyl-CoA decarboxylase and formyl-CoA transferase activity in Lactobacillus acidophilus. J. Appl. Microbiol. 1031600-1609. [PubMed]
58. Vagin, A., and A. Teplyakov. 1997. MOLREP: an automated program for molecular replacement. J. Appl. Crystallogr. 301022-1025.
59. Vincentelli, R., C. Bignon, A. Gruez, S. Canaan, G. Sulzenbacher, M. Tegoni, V. Campanacci, and C. Cambillau. 2003. Medium-scale structural genomics: strategies for protein expression and crystallization. Acc. Chem. Res. 36165-172. [PubMed]
60. Wang, X., R. I. Sarkar, and P. C. Maloney. 2006. Analysis of substrate-binding elements in OxlT, the oxalate:formate antiporter of Oxalobacter formigenes. Biochemistry 4510344-10350. [PMC free article] [PubMed]
61. Welch, R. A., V. Burland, G. Plunkett III, P. Redford, P. Roesch, D. Rasko, E. L. Buckles, S. R. Liou, A. Boutin, J. Hackett, D. Stroud, G. F. Mayhew, D. J. Rose, S. Zhou, D. C. Schwartz, N. T. Perna, H. L. Mobley, M. S. Donnenberg, and F. R. Blattner. 2002. Extensive mosaic structure revealed by the complete genome sequence of uropathogenic Escherichia coli. Proc. Natl. Acad. Sci. USA 9917020-17024. [PMC free article] [PubMed]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)
PubReader format: click here to try

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

  • Compound
    Compound
    PubChem Compound links
  • Gene
    Gene
    Gene links
  • GEO Profiles
    GEO Profiles
    Related GEO records
  • MedGen
    MedGen
    Related information in MedGen
  • Pathways + GO
    Pathways + GO
    Pathways, annotations and biological systems (BioSystems) that cite the current article.
  • Protein
    Protein
    Published protein sequences
  • PubMed
    PubMed
    PubMed citations for these articles
  • Structure
    Structure
    Published 3D structures
  • Substance
    Substance
    PubChem Substance links
  • Taxonomy
    Taxonomy
    Related taxonomy entry
  • Taxonomy Tree
    Taxonomy Tree

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...