Characterization of an arginine:pyruvate transaminase in arginine catabolism of Pseudomonas aeruginosa PAO1.
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 micrromol/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 Delta(1)-piperideine-2-carboxylate as its transamination product. These results implied that AruH may have a broader physiological function in amino acid catabolism.
Yang
Zhe
Z
Department of Biology, Georgia State University, Atlanta, GA 30303, USA.
Lu
Chung-Dar
CD
D000596:Amino Acids / Q000378:metabolism; D001120:Arginine / Q000031:analogs & derivatives / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000378:metabolism*; D011773:Pyruvates / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D013379:Substrate Specificity; D013696:Temperature; D000637:Transaminases / Q000235:genetics / Q000378:metabolism*
C093466:2-ketoarginine; D000596:Amino Acids; D001426:Bacterial Proteins; D011773:Pyruvates; D011994:Recombinant Proteins; D001120:Arginine; D000637:Transaminases