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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{triangledown}

Zhe Yang and Chung-Dar Lu*

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 Vmax and kcat values 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 {Delta}1-piperideine-2-carboxylate as its transamination product. These results implied that AruH may have a broader physiological function in amino acid catabolism.

* Corresponding author. Mailing address: Department of Biology, Georgia State University, 24 Peachtree Center Avenue NE, Atlanta, GA 30303. Phone: (404) 651-2531. Fax: (404) 651-2509. E-mail: biocdl{at}langate.gsu.edu

{triangledown} Published ahead of print on 6 April 2007.

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.



This article has been cited by other articles:

  • Li, C., Lu, C.-D. (2009). Arginine racemization by coupled catabolic and anabolic dehydrogenases. Proc. Natl. Acad. Sci. USA 106: 906-911 [Abstract] [Full Text]  
  • Yang, Z., Lu, C.-D. (2007). Functional Genomics Enables Identification of Genes of the Arginine Transaminase Pathway in Pseudomonas aeruginosa. J. Bacteriol. 189: 3945-3953 [Abstract] [Full Text]  


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