Carbamoylphosphate Synthetase from Pseudomonas aeruginosa

Abstract

Carbamoylphosphate synthetase from Pseudomonas ueruginosa is subject to repression by pyrimidines and significant derepression by limitation of arginine or pyrimidines. Carbamoylphosphate synthetase was purified to homogeneity from a derepressed strain of P. aeruginosa. The molecular weight of the enzyme was estimated to be 168 000 by sucrose gradient ultracentrifugation. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed that the enzyme is composed of two non-identical subunits with molecular weights of 122000 and 44000. Cross-linking the enzyme prior to electrophoresis yielded an additional band corresponding to a molecular weight of 165000, showing that the enzyme is composed of one of each subunit. The enzyme utilized either glutamine ( K , 0.15 mM) or NH3 (K, 17 mM) and requires free Mg2+ for maximal activity with the optimal level between 4 mM and 10 mM. Hill plots of MgATP saturation data yielded coefficients of 1.2 and 1.4 at pH 8.0 and 8.5, respectively. A Hill equation was derived on the assumptions that MgATP binds at the same time to two distinct sub-sites as was shown to be the case for carbamoylphosphate synthetase from Escherichia coli and that these sub-sites are strictly non-interacting. The resulting theoretical Hill coefficients correspond very closely to the experimental coefficients. Carbamoylphosphate synthetase activity was subject to activation by ornithine and N-acetylornithine and feedback inhibition by UMP. Carbamoylphosphate synthetase from P. aerugino.ra does not associate under all conditions examined, establishing that self-association does not play a role in regulation of enzyme activity as suggested by other workers for the enzyme from E. coli.Carbamoylphosphate is an intermediate in the biosynthesis of arginine and pyrimidines. In enteric bacteria [l -51 it is synthesized by a single enzyme, carbamoylphosphate synthetase, which catalyzes the following reaction :Ammonia can replace glutamine as a nitrogen donor in the reaction but the affinity for ammonia is much less than that for glutamine; the latter substrate is considered the physiological nitrogen donor. The enteric enzymes [l -51 are all subject to cumulative repression by arginine and a pyrimidine compound and to feedback control: U M P inhibits activity and ornithine stimulates it. The combined effects provide an efficient mechanism for controlling the supply of carbamoylphosphate for arginine and pyrimidine biosynthesis.Examination of carbamoylphosphate metabolism in Pseudomonas aeruginosa is of particular interest in view of the significant differences that distinguish arginine metabolism in this organism from that in enteric bacteria [6,7]. These differences include the ability of P. ueruginosu and other fluorescent pseudomonads to degrade arginine via the arginine deiminase pathway (Fig. 1). This pathway involves the activity of carbamate kinase which catalyzes the phosphorylation of ADP by carbamoylphosphate [7] according to the following reaction :The possible presence of two enzyme...

The PitA protein contributes to colistin susceptibility in Pseudomonas aeruginosa

“…A second potential mechanism for modification of LPS was revealed by the finding that five of the experimentally evolved mutants had mutations in PA5005 or carB . The CarB enzyme synthesises carbamoyl phosphate [ 40 ] and the enzyme encoded by the PA5005 gene (provisionally named WapO) is likely responsible for the addition of carbamoyl groups to the inner core of LPS [ 39 ]. It therefore seems likely that absence of carbamoyl groups from the LPS, due to mutations in PA5005 or carB , reduces the affinity of the LPS for colistin.…”

“…A second potential mechanism for modification of LPS was revealed by the finding that five of the experimentally evolved mutants had mutations in PA5005 or carB. The CarB enzyme synthesises carbamoyl phosphate [40] and the enzyme encoded by the PA5005 gene…”

Disruption of the carA gene in Pseudomonas syringae results in reduced fitness and alters motility

“…However the regulation of the pyrimidine pathway in Pseudomonas is strongly influenced by pyrimidine and purine nucleotide effectors [46]. For example, the activity of the carbamoyl-phosphate synthase is inhibited by UMP and activated by ornithine and N-acetylornithine [47] and carAB expression is subject to pyrimidine control via an attenuation mechanism. Therefore it is possible, that under the conditions we examined expression, the DC3000 cells are experiencing a high requirement for pyrimidines and expression of P32 and carA is not repressed upon supplementation of arginine.…”