Characterization of an Arginine:Pyruvate Transaminase in Arginine Catabolism of Pseudomonas aeruginosa PAO1

ABSTRACT The arginine transaminase (ATA) pathway represents one of the multiple pathways for l -arginine catabolism in Pseudomonas aeruginosa . The AruH protein was proposed to catalyze the first step in the ATA pathway, converting the substrates l -arginine and pyruvate into 2-ketoarginine and l -alanine. Here we report the initial biochemical characterization of this enzyme. The aruH gene was overexpressed in Escherichia coli , and its product was purified to homogeneity. High-performance liquid chromatography and mass spectrometry (MS) analyses were employed to detect the presence of the transamination products 2-ketoarginine and l -alanine, thus demonstrating the proposed biochemical reaction catalyzed by AruH. The enzymatic properties and kinetic parameters of dimeric recombinant AruH were determined by a coupled reaction with NAD + and l -alanine dehydrogenase. The optimal activity of AruH was found at pH 9.0, and it has a novel substrate specificity with an order of preference of Arg > Lys > Met > Leu > Orn > Gln. With l -arginine and pyruvate as the substrates, Lineweaver-Burk plots of the data revealed a series of parallel lines characteristic of a ping-pong kinetic mechanism with calculated V max and k cat values of 54.6 ± 2.5 μmol/min/mg and 38.6 ± 1.8 s −1 . The apparent K m and catalytic efficiency ( k cat / K m ) were 1.6 ± 0.1 mM and 24.1 mM −1 s −1 for pyruvate and 13.9 ± 0.8 mM and 2.8 mM −1 s −1 for l -arginine. When l -lysine was used as the substrate, MS analysis suggested Δ 1 -piperideine-2-carboxylate as its transamination product. These results implied that AruH may have a broader physiological function in amino acid catabolism.