Microbial urate catabolism: characterization of HpyO , a non‐homologous isofunctional isoform of the flavoprotein urate hydroxylase HpxO

Summary In aerobic cells, urate is oxidized to 5‐hydroxyisourate by two distinct enzymes: a coenzyme‐independent urate oxidase ( EC 1.7.3.3) found in eukaryotes and bacteria like B acillus subtilis and a prokaryotic flavoprotein urate hydroxylase ( HpxO ) originally found in some K lebsiella species. More cases of analogous or non‐homologous isofunctional enzymes ( NISE ) for urate catabolism have been hypothesized by inspecting bacterial genomes. Here, we used a functional complementation approach in which a candidate gene for urate oxidation is integrated by homologous recombination in the A cinetobacter baylyi ADP 1 genome at the locus of its original hpxO gene. Catabolism of urate was restored in A . baylyi ADP 1 expressing a FAD ‐dependent protein from X anthomonas campestris , representing a new urate hydroxylase family that we called HpyO . This enzyme was kinetically characterized and compared with other HpxO enzymes. In contrast to the latter, HpyO is a typical Michaelian enzyme. This work provides the first experimental evidences for the function of HpyO in bacterial urate catabolism and establishes it as a NISE of HpxO .