Catabolic ornithine transcarbamylase of Halobacterium halobium (salinarium): purification, characterization, sequence determination, and evolution.
Halobacterium halobium (salinarium) is able to grow fermentatively via the arginine deiminase pathway, which is mediated by three enzymes and one membrane-bound arginine-ornithine antiporter. One of the enzymes, catabolic ornithine transcarbamylase (cOTCase), was purified from fermentatively grown cultures by gel filtration and ammonium sulfate-mediated hydrophobic chromatography. It consists of a single type of subunit with an apparent molecular mass of 41 kDa. As is common for proteins of halophilic Archaea, the cOTCase is unstable below 1 M salt. In contrast to the cOTCase from Pseudomonas aeruginosa, the halophilic enzyme exhibits Michaelis-Menten kinetics with both carbamylphosphate and ornithine as substrates with Km values of 0.4 and 8 mM, respectively. The N-terminal sequences of the protein and four peptides were determined, comprising about 30% of the polypeptide. The sequence information was used to clone and sequence the corresponding gene, argB. It codes for a polypeptide of 295 amino acids with a calculated molecular mass of 32 kDa and an amino acid composition which is typical of halophilic proteins. The native molecular mass was determined to be 200 kDa, and therefore the cOTCase is a hexamer of identical subunits. The deduced protein sequence was compared to the cOTCase of P. aeruginosa and 14 anabolic OTCases, and a phylogenetic tree was constructed. The halobacterial cOTCase is more distantly related to the cOTCase than to the anabolic OTCase of P. aeruginosa. It is found in a group with the anabolic OTCases of Bacillus subtilis, P. aeruginosa, and Mycobacterium bovis.
Ruepp
A
A
Max-Planck-Institut für Biochemie, Martinsried, Germany.
Müller
H N
HN
Lottspeich
F
F
Soppa
J
J
D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D001483:Base Sequence; D002221:Carbamyl Phosphate / Q000378:metabolism; D003001:Cloning, Molecular; D005285:Fermentation; D019617:Halobacterium salinarum / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000494:pharmacology; D009952:Ornithine / Q000378:metabolism; D009954:Ornithine Carbamoyltransferase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D010802:Phylogeny; D011189:Potassium Chloride / Q000494:pharmacology; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid
D009711:Nucleotides; D002221:Carbamyl Phosphate; D011189:Potassium Chloride; D009952:Ornithine; D009954:Ornithine Carbamoyltransferase