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

Abstract

The arcABC operon of Pseudomonas aeruginosu encodes arginine deiminase, catabolic ornithine carbamoyltransferase and carbamate kinase, respectively. We have determined the nucleotide sequences of the arcA and urcC 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 Mgz+ or Mn2+ and strongly inhibited by Zn". The apparent K,,, for L-arginine was 0.04 mM in the presence of MgZf and 0.47 mM without Mgz+. 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 carbamoylphosphate synthetase (large subunit) from Escherichiu 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. c o kThe latter part of arginine biosynthesis and the catabolic arginine-deiminase pathway have a number of metabolites in common (Fig. 1). In Pseudomonas ueruginosa the enzymes which catalyze these reactions are all distinct [I, 21. A futile cycle is avoided by several control mechanisms. Carbamoylphosphate synthetase is derepressed by limitation of arginine or pyrimidines, activated by ornithine and inhibited by UMP [3]. The anabolic ornithine carbamoyltransferase is repressed by arginine and forms, with citrulline, a dead-end complex, which prevents the back reaction [4 -61. The three enzymes of the arginine deiminase pathway are coordinately induced by oxygen limitation and by arginine [7]. The catabolic ornithine carbamoyltransferase shows highly cooperative carbamoyl-phosphate binding and hence does not catalyze the anabolic reaction in vivo. AMP, CMP and inorganic phosphate are activators of the enzyme [8, 91 (V.S., unpublished results). Carbamate kinase is inhibited by ATP [lo]. Thus, conditions of energy depletion favour the functioning of the arginine deiminase pathway. Correspondence to D. Haas, Mikrobiologischcs Institut, ETHAhbreviution. X-gal, 5-bromo-4-chloro-3-indolyl-~-~-galactoside. Enzymes. Arginase (EC 3.5.3.1); arginine deiminase (EC 3.5.3.6 The gene...

Positive FNR-like control of anaerobic arginine degradation and nitrate respiration in Pseudomonas aeruginosa

“…The structural genes of the arcDABC operon (Fig. 3a) have been completely sequenced (5,6,30). Here we report the nucleotide sequence of the region upstream of arcD (Fig.…”

Kinetic Analysis of Pseudomonas aeruginosa Arginine Deiminase Mutants and Alternate Substrates Provides Insight into Structural Determinants of Function

“…Chromatographic materials, buffers, 2-benzyl-2-thiopseudourea, L-arginine, L-citrulline, and agmatine were purchased from Sigma, molecular biological materials were from Invitrogen, [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]-L-arginine (specific activity ) 50 mCi/mmol) was from Moravek Biochemicals, Inc., N ω -amino-L-arginine was from Alexis Biochemicals, and N ω -methyl-L-arginine was from Bachem California Inc. N-Amino-L-citrulline (12) was obtained as a gift from Dr. Bruce King at Wake Forest University.…”

Genetics of Acid Adaptation in Oral Streptococci

“…By the nomenclature developed for Pseudomonas aeruginosa (Baur et al, 1989), arcA encodes the AD gene itself; arcB encodes the ornithine transcarbamylase; and arcC, the carbamate kinase. Perhaps surprisingly, the S. sanguis ADS genes were not well-expressed in E. coli, which is uncommon for streptococcal genes, leading to speculation that the genes are positively regulated in their native host and not so in E. coli.…”