Isolation of Klebsiella pneumoniae HpxO. The recombinant enzyme with an N-terminal His₆-tag was overexpressed in Escherichia coli BL21(DE3) using standard protocols. The enzyme was purified by Ni²⁺-nitrilotriacetic acid chromatography. (A) Purine catabolic pathway from hypoxanthine to allantoin. HpxO catalyzes hydroxylation of urate 3 to the unstable 5-hydroxyisourate 4, which is converted both enzymatically and non-enzymatically via 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline (OHCU) 5 to allantoin 6; (B) SDS-PAGE analysis (12% acrylamide) of the purified enzyme. The molecular weight is ~45 kDa including the affinity tag; (C) UV-visible spectrum of HpxO, with absorbance maxima at 454, 381, 273 and <220 nm; (D) Reverse-phase (C₁₈) HPLC analysis of the small molecule released from HpxO on heat-denaturation. Absorbance detection was at 250 nm. The mobile phase consisted of a gradient from 0–65% methanol in potassium phosphate buffer. A minor impurity (R_T = 15.9 min) present in the commercial sample of FAD (R_T = 14.7 min) was also found in the HpxO-derived sample; the increased proportion of this impurity in the HpxO sample was attributed to the effects of the heat denaturation; (E) UV-Vis spectrum of the HPLC purified FAD isolated from heat-denatured HpxO.

Reverse-phase (C₁₈) HPLC analysis of the urate oxidase activity of HpxO. All reactions were performed in 0.1 M potassium phosphate, pH 8.0 at room temperature (~22°C). Reaction components, where present, were at the following concentrations: Uric acid, 0.1 mM; NADPH, 0.25 mM; HpxO, 0.13 μM; commercially-available urate oxidase (Sigma-Aldrich) from Candida sp., 2.2 units/mL (one unit is defined as the quantity of enzyme which oxidizes 1.0 μmol of uric acid to allantoin at pH 8.5 and at 25 °C); (A) HpxO control reaction omitting urate; (B) Control omitting NADPH; (C) Control omitting HpxO; (D) NADPH-independent reaction of urate catalyzed by urate oxidase from Candida sp.; (E) HpxO reaction including urate, NADPH and HpxO; (F) Proposed mechanism for HpxO-catalyzed formation of 5-HIU.