Sequence analysis and expression of the arginine-deiminase and carbamate-kinase genes of Pseudomonas aeruginosa

Abstract

The arcABC operon of Pseudomonas aeruginosa encodes arginine deiminase, catabolic ornithine carbamoyltransferase and carbamate kinase, respectively. We have determined the nucleotide sequences of the arcA and arcC genes. The arcA open reading frame specifies a polypeptide of 46.3 kDa. The same molecular mass was obtained for the subunit of purified arginine deiminase after electrophoresis under denaturing conditions. The N-terminal amino acid sequence of arginine deiminase was in agreement with the corresponding nucleotide sequence. The native arginine deiminase had an estimated molecular mass of 175-180 kDa, suggesting a tetrametric structure. The enzyme was activated by Mg2+ or Mn2+ and strongly inhibited by Zn2+. The apparent Km for L-arginine was 0.04 mM in the presence of Mg2+ and 0.47 mM without Mg2+. The arcC open reading frame codes for a 33-kDa protein, confirming the molecular mass previously reported for the subunit of carbamate kinase. The translation-initiation site of arcC was determined by deletion mapping. Two regions of dyad symmetry found between arcA and arcC might stabilize the putative arcABC transcript in the upstream (arcA) region; this might contribute to the high level of arcA expression as compared to the moderate level of arcC expression. Carbamate kinase had 37% sequence similarity (and 13.5% identity) with the C-terminal part of carbamoyl-phosphate synthetase (large subunit) from Escherichia coli. Arginine deiminase had no apparent similarity with argininosuccinate lyase. Thus, the arcA and arcC genes do not appear to be closely related to arginine biosynthetic genes, whereas it had previously been shown that the arcB gene has a high degree of identity with the arginine biosynthetic argF genes of P. aeruginosa and E. coli.