Catabolic ornithine carbamoyltransferase of Pseudomonas aeruginosa

Abstract

Ornithine carbamoyltransferases (OTCases) catalyse the formation of citrulline and phosphate from ornithine and carbamoylphosphate by a thermodynamically favoured reaction. In vivo, catabolic OTCase of Pseudomonas aeruginosa promotes the reverse reaction, the phosphorolysis of citrulline. Although the enzyme is assayed in vitro in the direction of citrulline synthesis, the enzyme cannot perform this reaction in vivo due to poor affinity for carbamoylphosphate and high cooperativity towards this substrate. Furthermore, the dodecameric catabolic OTCase is an allosteric enzyme; the enzyme is stimulated by nucleoside monophosphates and inhibited by polyamines (e.g. spermidine). A previous study showed that a modification of the C-terminus of the catabolic OTCase alters the homotropic cooperativity of the enzyme. We have now investigated the importance of the C-terminus for homotropic and heterotropic cooperativity by site-directed mutagenesis. Deletion of the C-terminal Ile335 residue strongly reduced cooperativity for carbamoylphosphate and sensitivity to spermidine. These properties were essentially restored when the two C-temiinal amino acids (Asp334 and Ile335) were removed by deletion. However, in this variant enzyme, AMP failed to abolish carbamoylphosphate cooperativity completely, whereas the wild-type enzyme was rendered virtually non-cooperative by AMP. An extension of catabolic ornithine carbamoyltransferase by 15 amino acid residues interfered with both homotropic and heterotropic interactions and lowered the maximal velocity. All variant enzymes had the same dodecameric structure as the wild type and differed only slightly in affinity for the second substrate ornithine. A structural model of the dodecamer, at 0.3-nm resolution, suggests that the C-terminus could be involved in trimerltrimer interaction. We propose that modifications at the C-terminus alter the trimerhrimer interface and, in addition, removes the salt bridge His5-Ile335 within a monomer. These changes profoundly and indirectly modify the allosteric transition and consequently the interactions of the dodecamer with carbamoylphosphate and effectors.

The arginine deiminase pathway in Rhizobium etli: DNA sequence analysis and functional study of the arcABC genes

“…All these features of the bacteroid OTCase are comparable to the properties of the P. aeruginosa cOTCase (52,53). The H. salinarium cOTCase, on the other hand, does not bind carbamoyl phosphate cooperatively but exhibits a Michaelis-Menten kinetic for both carbamoyl phosphate and ornithine, and the native enzyme functions as a hexamer.…”

“…The Pseudomonas cOTCase is unable to perform the anabolic reaction due to the high cooperativity and the poor affinity with respect to carbamoyl phosphate. However, when specific amino acid residues in the P. aeruginosa cOTCase, namely glutamate-106, methionine-322, and isoleucine-336, are changed, the protein can act as an anabolic OTCase in vivo, resulting from either reduced cooperativity or the absence of cooperativity with respect to carbamoyl phosphate (4,35,53). The glutamate-106 residue is also present in the R. etli protein but is absent in all but one anabolic OTCase.…”

The crystal structure of Pyrococcus furiosus ornithine carbamoyltransferase reveals a key role for oligomerization in enzyme stability at extremely high temperatures

“…Besides anabolic OTCases involved in the arginine biosynthetic pathway, a catabolic OTCase converting citrulline into ornithine and carbamoylphosphate is found when the arginine deiminase pathway is present, such as in Pseudomonas aeruginosa (PA) where the enzyme has been investigated in much detail (10)(11)(12). This catabolic OTCase displays marked cooperativity toward CP and is allosterically activated by nucleoside monophosphates or inorganic phosphate, and inhibited by polyamines such as spermidine or putrescine.…”

“…(1998) inhibitors (11). However the exact role in effector binding and͞or signal transmission of this interface needs to be further investigated.…”

Catabolic ornithine transcarbamylase of Halobacterium halobium (salinarium): purification, characterization, sequence determination, and evolution

“…(i) Replacement of glutamic acid 106 (position 144) with glycine or alanine converts the cOTCase to the anabolic enzyme, the K m value is lowered about 40 or 70 times, respectively, the saturation curve becomes hyperbolic, and the enzyme is active in the anabolic direction in vivo (4). (ii) Deletion of C-terminal isoleucine 335 (position 386) strongly reduces cooperativity (53). In contrast, the cOTCase of H. salinarium exhibits Michaelis-Menten kinetics with both carbamylphosphate and ornithine as substrates.…”