ORNITHINE CARBAMOYLTRANSFERASE FROM THE EXTREME THERMOPHILE THERMUS-THERMOPHILUS - ANALYSIS OF THE GENE AND CHARACTERIZATION OF THE PROTEIN

The ornithine carbamoyltransferase (OTC) gene from Thermus thermophilu s was cloned from a lambda-ZAP genomic library. An ORF of 903 bp was f ound coding for a protein of M-r 33 200. The coding region has a very high overall G + C content of 68.0%. T. thermophilus OTC displays 38-4 8% amino acid identity with other OTC, the most closely related protei ns being OTC from the archaeon Pyrococcus furiosus and from Bacillus s ubtilis. 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 M-r of about 110000, suggesting a trimeric structure, as for most a nabolic OTC from various organisms. T. thermophilus OTC exhibits Micha elis-Menten kinetics for carbamoyl phosphate and ornithine with a K-m( app) of 0.10 mM for both substrates. The pH optimum was dependent on o rnithine concentration with an optimum at pH 8 for ornithine concentra tions around K-m values. Higher concentrations shift the optimum towar ds lower pH. The temperature was above 65 degrees C and the activation energy 39.1 kJ/mol. The enzyme is highly thermostable. In the presenc e of its substrates the half-life time was several hours at 85 degrees C. Ionic and hydrophobic interactions contribute to the stability. Th e expression of T. thermophilus OTC was negatively regulated by argini ne.