flavin-adenine-dinucleotide and 5-hydroxyisourate

HpxO is a flavin-dependent urate oxidase that catalyzes the hydroxylation of uric acid to 5-hydroxyisourate and functions in a novel pathway for purine catabolism found in Klebsiella pneumoniae. We have determined the structures of HpxO with and without uric acid at 2.0 and 2.2 Å, respectively. We have also determined the structure of the R204Q variant at 2.0 Å resolution in the absence of uric acid. The variant structure is very similar to that of wild-type HpxO except for the conformation of Arg103, which interacts with FAD in the variant but not in the wild-type structure. Interestingly, the R204Q variant results in the uncoupling of nicotinamide adenine dinucleotide oxidation from uric acid hydroxylation. This suggests that Arg204 facilitates the deprotonation of uric acid, activating it for the oxygen transfer. On the basis of these data, a mechanism for this reaction consisting of a nucleophilic attack of the urate anion on the flavin hydroperoxide resulting in the formation of 5-hydroxyisourate is proposed.

Topics: Amino Acid Substitution; Bacterial Proteins; Biocatalysis; Catalytic Domain; Crystallography, X-Ray; Flavin-Adenine Dinucleotide; Hydroxylation; Kinetics; Klebsiella pneumoniae; Ligands; Models, Molecular; Molecular Conformation; Mutagenesis, Site-Directed; Mutant Proteins; NAD; Oxidation-Reduction; Recombinant Proteins; Urate Oxidase; Uric Acid

The HpxO enzyme from Klebsiella pneumoniae was recently proposed, on the basis of genetic studies, to catalyze the hydroxylation of uric acid to 5-hydroxyisourate as part of the purine catabolic pathway. Its primary sequence suggests that the HpxO catalytic activity depends on a flavin cofactor (FAD), contrasting with all previously studied urate oxidase enzymes, which have no cofactor requirement. Here we demonstrate biochemically that HpxO is an FAD-dependent urate oxidase. Our data are consistent with the proposal that HpxO-bound flavin hydroperoxide is the hydroxylating species. These results confirm the existence of a novel mechanistic paradigm in purine catabolism.

Topics: Flavin-Adenine Dinucleotide; Hydroxylation; Klebsiella pneumoniae; Purines; Urate Oxidase; Uric Acid