BIOCHEMICAL-CHARACTERIZATION OF ORNITHINE CARBAMOYLTRANSFERASE FROM PYROCOCCUS-FURIOSUS

Ornithine carbamoyltransferase (OTCase) was purified to homogeneity fr om the hyperthermophilic archaeon Pyrococcus furiosus. The enzyme is a 400 +/- 20-kDa polymer of a 35-kDa subunit, in keeping with the corre sponding gene sequence [Roovers, M., Hethke, C., Legrain. C., Thomm, M . & Glansdorff, N. (1997) Isolation of the gene encoding Pyrococcus fu riosus ornithine cabamoyltransferase and study of its expression profi le in vivo and in vitro. Eur. J. Biochem. 247, 1038-1045]. In contrast with the dodecameric catabolic OTCase of Pseudomonas aeruginosa, P. f uriosus OTCase exhibits no substrate cooperativity. In keeping with ot her data discussed in the text, this suggests that the enzyme serves a n anabolic function. Half-life estimates for the purified enzyme range d over 21-65 min at 100 degrees C according to the experimental condit ions and reached several hours in the presence of ornithine and phosph ate. The stability was not markedly influenced by the protein concentr ation. Whereas comparative examination of OTCase sequences did not poi nt to any outstanding feature possibly related to thermophily, modelli ng the enzyme on the X-ray structure of P. aeruginosa OTCase (constitu ted by four trimers assembled in a tetrahedral manner) suggests that t he molecule is stabilized, at least in part, by a set of hydrophobic i nteractions at the interfaces between the trimers. The comparison betw een P. aeruginosa and P. furiosus OTCases suggests that two different properties, allostery and thermostability, have been engineered starti ng from a similar quaternary structure of high internal symmetry. Reco mbinant P. furiosus OTCase synthesised by Escherichia coli proved less stable than the native enzyme. In Saccharomyces cerevisiae, however, an enzyme apparently identical to the native one could be obtained.