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Journal of Bacteriology, June 2007, p. 3954-3959, Vol. 189, No. 11
0021-9193/07/$08.00+0     doi:10.1128/JB.00262-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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

Department of Biology, Georgia State University, Atlanta, Georgia 30303

Received 15 February 2007/ Accepted 26 March 2007

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 ¹-piperideine-2-carboxylate as its transamination product. These results implied that AruH may have a broader physiological function in amino acid catabolism.

Published ahead of print on 6 April 2007.