Publications

• Functional genomics enables identification of genes of the arginine transaminase pathway in Pseudomonas aeruginosa.

  Yang Z., Lu C.D.

  Arginine utilization in Pseudomonas aeruginosa with multiple catabolic pathways represents one of the best examples of the metabolic versatility of this organism. To identify genes involved in arginine catabolism, we have employed DNA microarrays to analyze the transcriptional profiles of this org ... >> More

  Arginine utilization in Pseudomonas aeruginosa with multiple catabolic pathways represents one of the best examples of the metabolic versatility of this organism. To identify genes involved in arginine catabolism, we have employed DNA microarrays to analyze the transcriptional profiles of this organism in response to L-arginine. While most of the genes involved in arginine uptake, regulation, and metabolism have been identified as members of the ArgR (arginine-responsive regulatory protein) regulon in our previous study, they did not include any genes of the arginine dehydrogenase (ADH) pathway. In this study, 18 putative transcriptional units of 38 genes, including the two known genes of the ADH pathway, kauB and gbuA, were found to be inducible by exogenous L-arginine in the absence of ArgR. To identify the missing genes that encode enzymes for the initial steps of the ADH pathway, the potential physiological functions of those candidate genes in arginine utilization were studied by growth phenotype analysis of knockout mutants. Expression of these genes was induced by L-arginine in an aruF mutant strain devoid of a functional arginine succinyltransferase pathway, the major route of arginine utilization. Disruption of dadA, a putative catabolic alanine dehydrogenase-encoding gene, in the aruF mutant produced no growth on L-arginine, suggesting the involvement of L-alanine in arginine catabolism. This hypothesis was further supported by the detection of an L-arginine-inducible arginine:pyruvate transaminase activity in the aruF mutant. Knockout of aruH and aruI, which encode an arginine:pyruvate transaminase and a 2-ketoarginine decarboxylase in an operon, also abolished the ability of the aruF mutant to grow on L-arginine. The results of high-performance liquid chromatography analysis demonstrated consumption of 2-ketoarginine and suggested that generation of 4-guanidinobutyraldehyde occurred in the aruF mutant but not in the aruF aruI mutant. These results led us to propose the arginine transaminase pathway that removes the alpha-amino group of L-arginine via transamination instead of oxidative deamination by dehydrogenase or oxidase as originally proposed. In the same genetic locus, we also identified a two-component system, AruRS, for the regulation of arginine-responsive induction of the arginine transaminase pathway. This work depicted a wider network of arginine metabolism than we previously recognized. << Less

  J. Bacteriol. 189:3945-3953(2007) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Characterization of an arginine:pyruvate transaminase in arginine catabolism of Pseudomonas aeruginosa PAO1.

  Yang Z., Lu C.D.

  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. Her ... >> More

  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. << Less

  J. Bacteriol. 189:3954-3959(2007) [PubMed] [EuropePMC]