Biochemical and structural characterization of a ureidoglycine aminotransferase in the Klebsiella pneumoniae uric acid catabolic pathway

Abstract

Many plants, fungi, and bacteria catabolize allantoin as a mechanism for nitrogen assimilation. Recent reports have shown that in plants and some bacteria the product of hydrolysis of allantoin by allantoinase is the unstable intermediate ureidoglycine. While this molecule can spontaneously decay, genetic analysis of some bacterial genomes indicates that an aminotransferase may be present in the pathway. Here we present evidence that Klebsiella pneumoniae HpxJ is an aminotransferase that preferentially converts ureidoglycine and an alpha-keto acid into oxalurate and the corresponding amino acid. We determined the crystal structure of HpxJ, allowing us to present an explanation for substrate specificity.

Figure 1

Characterization of K. pneumoniae HpxJ. A) HPLC traces of the amino products of the HpxJ aminotransfer reactions. From top to bottom: control reaction run with HpxJ in the absence of ureidoglycine, control reaction run in the absence of HpxJ with all other components present, the reaction with HpxJ and ureidoglycine in the absence of exogenous amino acceptor, and the HpxJ catalyzed reaction run in the presence of ureidoglycine and pyruvate. B) HPLC traces of the keto-acid products of the HpxJ aminotransfer reaction. From top to bottom: control in the absence of HpxJ, reaction with HpxJ and ureidoglycine in the absence of exogenous amino acceptor, transfer reaction in the presence of pyruvate, and the transfer reaction in the presence of oxaloacetate. Note that any unreacted allantoate in the reaction is hydrolyzed to glyoxylate during the derivatization work-up. C) UV absorbance spectra of native HpxJ (solid line) and reduced HpxJ (dashed line). D) Pre-steady state kinetics of the first half reaction catalyzed by HpxJ and rates from a fit to an exponential function. Note that the alanine and ureidoglycine traces have been shifted down the y-axis for clarity.

Figure 2

The active site of HpxJ. A) The structure of the PLP binding site of HpxJ showing a molecule of PLP covalently bound to lysine 200. F_O - F_C density is shown contoured at 3σ around the PLP and lysine residue. B) Superposition of the active site of HpxJ (green) and A. aegypti alanine glyoxylate aminotransferase (grey). Ureidoglycine is shown docked in the active site (yellow, ball and stick).

Scheme 1

Uric acid catabolic pathway in Klebsiella pneumoniae. The enzyme names are taken from the K. pneumoniae degradative pathway. The dashed line indicates an alternative route that operates in other organisms.