THE CRYSTAL-STRUCTURE OF PYROCOCCUS-FURIOSUS ORNITHINE CARBAMOYLTRANSFERASE REVEALS A KEY ROLE FOR OLIGOMERIZATION IN ENZYME STABILITY AT EXTREMELY HIGH-TEMPERATURES

The Pyrococcus furiosus (PF) ornithine carbamoyltransferase (OTCase; E C 2.1.3.3) is an extremely heat-stable enzyme that maintains about 50% of its activity after heat treatment for 60 min at 100 degrees C. To understand the molecular basis of thermostability of this enzyme, we h ave determined its three-dimensional structure at a resolution of 2.7 Angstrom and compared it with the previously reported structures of OT Cases isolated from mesophilic bacteria. Most OTCases investigated up to now are homotrimeric and devoid of allosteric properties. A strikin g exception is the catabolic OTCase from Pseudomonas aeruginosa, which is allosterically regulated and built up of four trimers disposed in a tetrahedral manner, an architecture that actually underlies the allo stery of the enzyme. We now report that the thermostable PF OTCase (42 0 kDa) presents the same 23-point group symmetry. The enzyme displays Michaelis-Menten kinetics. A detailed comparison of the two enzymes su ggests that, in OTCases, not only allostery but also thermophily was a chieved through oligomerization of a trimer as a common catalytic moti f. Thermal stabilization of the PF OTCase dodecamer is mainly the resu lt of hydrophobic interfaces between trimers, at positions where allos teric binding sites have been identified in the allosteric enzyme. The present crystallographic analysis of PF OTCase provides a structural illustration that oligomerization can play a major role in extreme the rmal stabilization.