Differential Substrate Specificity and Kinetic Behavior of Escherichia coli YfdW and Oxalobacter formigenes Formyl Coenzyme A Transferase

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 CO 2 in a coupled catalytic cycle (Fig. 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...

YfdW and YfdU Are Required for Oxalate-Induced Acid Tolerance in Escherichia coli K-12

“…Without YfdU-catalyzed decarboxylation, hydrolysis of the oxalyl-CoA thioester would result in a reduction of the formyl-CoA pool and no net removal of oxalate. High specificity for formyl-CoA and oxalate makes it unlikely that the hydrolysis of succinyl-CoA (or other CoA derivatives) is significant in the yfdU mutant (44,45).…”

“…YfdW from E. coli is a formyl-CoA transferase (44), and YfdU is an oxalyl-CoA decarboxylase (45,46). YfdW and YfdU are homologous to FRC and OXC, two essential metabolic proteins from the obligate anaerobe Oxalobacter formigenes (47).…”

Identification and characterization of two bile acid coenzyme A transferases from Clostridium scindens, a bile acid 7α-dehydroxylating intestinal bacterium

“…Type III CoA transferases, such as the crotonoyl-CoA ( caiB ) and yfdW gene products from E. coli (19)(20)(21) as well as the formyl-CoA transferase (FRC) from Oxalobacter formigenes ( 22 ), share a conserved Asp 169 residue (D169 formyl CoA) involved in catalytic formation of oxo-acid mixed anhydrides as well as covalent CoA thioesters. The baiF and baiK gene products share this conserved residue ( Fig.…”

Use of the frc gene as a molecular marker to characterize oxalate-oxidizing bacterial abundance and diversity structure in soil

“…Recently, Toyota et al (2008) confirmed experimentally that the gene YfdW from E. coli was a formyl-CoA transferase. Oxalyl-CoA decarboxylase gene oxc (synonym yfdU) is also present in E. coli K12, so this organism could at least be an oxalate-oxidizing bacterium.…”

“…These genes were first identified in the anaerobe O. formigenes but occur in aerobes as well (Hokama et al, 2005;Toyota et al, 2008).…”