Steve Ealick's Research Group
Klebsiella pneumoniae HpxO
3RP6 complexed with FAD
3RP7 complexed with FAD and uric acid
3RP8 R204Q mutant complexed with FAD
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. HpxO is particularly interesting since it belongs to the Class A flavin-dependent monooxygenase superfamily and is the first example of an FAD-dependent urate oxidase. As such, it represents a novel paradigm in purine metabolism. We have already determined the structures of and characterized the activity of these enzymes from the same pathway: HpxA, HpxQ, HpxJ, and HpxT.
HpxO is structurally similar to other flavin-dependent monooxygenases and consists of three domains. The N-terminal domain forms most of the interations with the FAD molecule. A second domain forms part of the uric acid binding pocket while the C-terminal domain connects the other two domains. Its biologically relevant form is as a monomer and it has been shown to also exist as a monomer in solution.
The FAD binding site has residues that are well conserved among members of the Class A flavin-dependent monooxygenase superfamily. The substrate binding site is lined with aromatic residues to enable π-π stacking interactions with uric acid; however there are also a number of charged residues available to form hydrogen bonds.
O'Leary SE, Hicks KA, Ealick SE, and Begley TP. Biochemical Characterization of the HpxO Enzyme from Klebsiella pneumoniae, a Novel FAD Dependent Urate Oxidase. Biochemistry 48:3033-3035 (2009).
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).