Ornithine carbamoyltransferase from the extreme thermophile Thermus thermophilus--analysis of the gene and characterisation of the protein.
The ornithine carbamoyltransferase (OTC) gene from Thermus thermophilus was cloned from a lambda-ZAP genomic library. An ORF of 903 bp was found coding for a protein of Mr 33,200. The coding region has a very high overall G+C content of 68.0%. T. thermophilus OTC displays 38-48% amino acid identity with other OTC, the most closely related proteins being OTC from the archaeon Pyrococcus furiosus and from Bacillus subtilis. The enzyme was expressed in Escherichia coli and purified to homogeneity using a thermoshock followed by affinity chromatography on delta-N-phosphonoacetyl-L-ornithine-Sepharose. The native enzyme has an Mr of about 110,000, suggesting a trimeric structure, as for most anabolic OTC from various organisms. T. thermophilus OTC exhibits Michaelis-Menten kinetics for carbamoyl phosphate and ornithine with a Km(app) of 0.10 mM for both substrates. The pH optimum was dependent on ornithine concentration with an optimum at pH 8 for ornithine concentrations around Km values. Higher concentrations shift the optimum towards lower pH. The optimal temperature was above 65 degrees C and the activation energy 39.1 kJ/mol. The enzyme is highly thermostable. In the presence of its substrates the half-life time was several hours at 85 degrees C. Ionic and hydrophobic interactions contribute to the stability. The expression of T. thermophilus OTC was negatively regulated by arginine.
Sanchez
R
R
Laboratorium voor erfelijkheidsleer en Microbiologie, Vrije Universiteit Brussel, Brussels, Belgium.
Baetens
M
M
Van de Casteele
M
M
Roovers
M
M
Legrain
C
C
Glansdorff
N
N
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004795:Enzyme Stability; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D009954:Ornithine Carbamoyltransferase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D016963:Thermus thermophilus / Q000201:enzymology*
D009954:Ornithine Carbamoyltransferase