Steve Ealick's Research Group


Abstract:

Hicks KA, O'Leary SE, Begley TP, and Ealick SE. Structural and Mechanistic Studies of HpxO, a Novel FAD-dependent Urate Oxidase from Klebsiella pneumoniae. Biochemistry 52:477-487 (2013).

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 Å and 2.2 Å, respectively. We have also determined the structure of the R204Q mutant at 2.0 Å resolution in the absence of uric acid. The mutant structure is very similar to wild type HpxO except for the conformation of Arg103, which interacts with FAD in the mutant but not in the wild type structure. Interestingly, the R204Q mutation results in the uncoupling of NADH oxidation from uric acid hydroxylation. This suggests that Arg204 facilitates the deprotonation of uric acid activating it for the oxygen transfer. Based on these data, a mechanism for this reaction is proposed consisting of a nucleophilic attack of the urate anion on the flavin hydroperoxide resulting in the formation of 5-hydroxyisourate.

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