title: Structural and mechanistic studies of HpxO, a novel flavin adenine dinucleotide-dependent urate oxidase from Klebsiella pneumoniae.
abstract: 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.
authors.affiliation: Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853, USA.
authors.forename: Katherine A
authors.initials: KA
authors.lastname: Hicks
authors.forename: Seán E
authors.initials: SE
authors.lastname: O'Leary
authors.forename: Tadhg P
authors.initials: TP
authors.lastname: Begley
authors.forename: Steven E
authors.initials: SE
authors.lastname: Ealick
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D055162:Biocatalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry / Q000378:metabolism*; D006900:Hydroxylation; D007700:Kinetics; D007711:Klebsiella pneumoniae / Q000201:enzymology*; D008024:Ligands; D008958:Models, Molecular; D008968:Molecular Conformation; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009243:NAD / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014503:Urate Oxidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014527:Uric Acid / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism
chemical_list: C510200:5-hydroxyisourate; D001426:Bacterial Proteins; D008024:Ligands; D050505:Mutant Proteins; D011994:Recombinant Proteins; D009243:NAD; D005182:Flavin-Adenine Dinucleotide; D014527:Uric Acid; D014503:Urate Oxidase