Differential Substrate Specificity and Kinetic Behavior in Escherichia coli YfdW and Oxalobacter formigenes Formyl-CoA Transferase

Contribution from the Department of Chemistry, University of Florida, Gainesville, FL 32611, USA, AFMB-UMR 6098, CNRS-Universités Aix-Marseille I & II, Campus de Luminy, Case 932, 163 Avenue de Luminy 13288 Marseille Cedex 09, France, and Department of Medical Biochemistry and Biophysics, Molecular Structural Biology, Karolinska Institutet, S-17177 Stockholm, Sweden

^* To whom correspondence should be addressed. Email: richards{at}qtp.ufl.edu.

	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 behaviour remain to be elucidated, structural genomic studies have shown that the structure of YfdW, the protein encoded by the yfdW gene is homologous to the formyl-CoA transferase (FRC) that mediates oxalate catabolism in the obligate anaerobe Oxalobacter formigenes. 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, which 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 substituting Trp-48 in the FRC active site by the glutamine residue that occupies an equivalent position in the Escherichia coli protein. These experiments show that Trp-48 precludes oxalate binding to a site that mediates substrate inhibition in YfdW. In addition, replacement of Trp-48 by Gln-48 yields an FRC variant in 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 Escherichia coli upon up-regulation of the yfdXWUVE operon under acidic conditions.