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.

Figure 1

The purine degradation pathway in K. pneumoniae.

Figure 2

Structure of HpxO. (A) Stereoview ribbon diagram of HpxO with FAD and uric acid bound and secondary structural elements labeled. The α-helices are shown in blue and the β-strands are shown in green with loops colored yellow. Ligands are shown in stick representation: FAD is colored red and uric acid is labeled as “UA” and is colored orange. (B) Topology diagram of HpxO. The color scheme is the same as Figure 2A.

Figure 3

Stereoview diagram of FAD binding site in HpxO with uric acid bound. The carbon residues in HpxO and uric acid are shown in green using the ball and stick representation and the interactions with the protein indicated using black dashed lines. Water molecules are shown as red nonbonded spheres. FAD adopts the in conformation and is shown with yellow carbon atoms. Clear electron density is not seen for the side chain of Ile42, which labeled as I42*.

Figure 4

Space filling model of HpxO showing the substrate tunnel leading to the active site. FAD and uric acid are red and in ball and stick. Water molecules are shown as red spheres. The surface of HpxO is colored green and the secondary structure elements are shown as cartoons.

Figure 5

Uric acid binding site. (A) Stereoview diagram of the active site of HpxO with FAD and uric acid bound. Water molecules are shown as nonbonded red spheres. Hydrogen bonds between uric acid and HpxO are shown as black, dashed lines. (B) Composite omit density is shown around the uric acid at a contour level of 3σ in blue.

Figure 6

Stereoview comparison of the active sites of R204Q and wild type HpxO. The carbon residues in wild type HpxO structure are shown in green and are yellow in the R204Q variant structure. Residues and ligands are shown using the ball and stick representation using the same color scheme. Interactions with the protein are indicated by black dashed lines. In the wild type structure, there is a SeMet at position 208.

Figure 7

Proposed mechanism for the HpxO catalyzed reaction.