Ornithine Carbamoyltransferase from the Extreme Thermophile Thermus Thermophilus Analysis of the Gene and Characterisation of the Protein

Abstract

The ornithine carbamoyltransferase (OTC) gene from Thermus thermophilus was cloned from a ).-ZAP genomic library. An ORF of 903 bp was found coding for a protein of M , 33200. The coding region has a very high overall G + C content of 68.0%. 7: thermophilus OTC displays 38-48% amino acid identity with other OTC, the most closely related proteins being OTC from the archaeon Pyrococcus jiiriosus and from Bacillus subtilis. The enzyme was expressed in Escherichia coli and purified to homogeneity using a thermoshock followed by affinity chromatography on d-N-phosphonoacetyl-L-ornithineSepharose. The native enzyme has an M, of about 110000, suggesting a trimeric structure, as for most anabolic OTC from various organisms. 7: thermophilus OTC exhibits Michaelis-Menten kinetics for carbamoyl phosphate and ornithine with a K:Yf" 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 K,, values. Higher concentrations shift the optimum towards lower pH. The optimal temperature was above 65°C and the activation energy 39.1 W/mol. The enzyme is highly thermostable. In the presence of its substrates the half-life time was several hours at 85 "C. Ionic and hydrophobic interactions contribute to the stability. The expression of 7: thermophilus OTC was negatively regulated by arginine.

Metabolic Enzymes from Psychrophilic Bacteria: Challenge of Adaptation to Low Temperatures in Ornithine Carbamoyltransferase from Moritella abyssi

“…The optimal pHs for the reaction at 20°C were found to be between 9.0 and 10.0 (data not shown), whereas E. coli and T. thermophilus OTCases display broad optimal activity at about pH 8.0 (31,45). As expected for an anabolic OTCase (9), the reaction catalyzed by OTCase Mab displayed no allosteric kinetics.…”

“…Cold-active enzymes are frequently thermolabile, a fact which is understandable, since they have to be flexible enough to be active at a low energy cost (14). OTCase Mab is indeed considerably less resistant to thermal inactivation (Table 3) and has a weaker conformational stability (Table 4) than its E. coli or thermophilic counterparts (32,33,45). Psychrophilic enzymes counteract the inhibitory effect of low temperatures on activity by reducing the temperature dependence of the reaction rate (low activation enthalpy).…”

Organization and Expression of a Thermus thermophilus Arginine Cluster: Presence of Unidentified Open Reading Frames and Absence of a Shine-Dalgarno Sequence

“…One may argue that the product of coregulated ORFs could stabilize enzymes encoded by the same polycystronic mRNA (13). However, in Thermus, the argF and argJ gene products appear intrinsically stable (1,27). Another possibility would be that the unknown ORF product is involved in the formation of multienzyme complexes channeling arginine metabolic precursors (17,32).…”

“…The intensity was strongly reduced with RNA extracted from cells grown in the presence of arginine. Previous experiments already indicated that arginine represses OTCase synthesis in Thermus (27,31). The region thus appears to contain four consecutive transcription units: ORF6, ORF7, ORF8, argF; ORF1, argC, argJ; ORF4; and ORF5.…”

“…Chromosomal DNA partially digested with Sau3A was used to construct a -ZAP genomic library (Stratagene), which was screened as described by Sanchez et al (27). Primer extension experiments were performed according to the method described by Kholti et al (15) except that hybridization experiments were performed overnight at 45°C.…”