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

Department of Biology, Georgia State University, Atlanta GA 30303

^* To whom correspondence should be addressed. Email: biocdl{at}langate.gsu.edu.

	Abstract

The arginine transaminase (ATA) pathway represents one of the multiple pathways for L-arginine catabolism in P. aeruginosa. The AruH protein was proposed to catalyze the first step in the ATA pathway, converting 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 over-expressed in E. coli and its product was purified to homogeneity. HPLC and MS analyses were employed to detect the presence of transamination products 2-ketoarginine and L-alanine, therefore demonstrating the proposed biochemical reaction catalyzed by AruH. The enzymatic properties and kinetics parameters of the 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 being Arg > Lys > Met > Leu > Orn > Gln. Taking L-arginine and pyruvate as the substrates, Lineweaver-Burk plots of the data revealed a series of parallel lines characteristic of ping-pong kinetics mechanism, with calculated Vmax and kcat of 54.6 ± 2.5 µmol/min/mg and 38.6 ± 1.8 s^-1. The apparent Km and catalytic efficiency (kcat/Km) 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 ¹-piperideine-2-carboxylate as its transamination product. These results implied that AruH may have a broader physiological function in amino acid catabolism.