ASPARTATE-TRANSCARBAMYLASE FROM THE DEEP-SEA HYPERTHERMOPHILIC ARCHAEON PYROCOCCUS-ABYSSI - GENETIC ORGANIZATION, STRUCTURE, AND EXPRESSIONIN ESCHERICHIA-COLI

The genes coding for aspartate transcarbamylase (ATCase) in the deep-s ea hyperthermophilic archaeon Pyococcus abyssi were cloned by compleme ntation of a pyrB Escherichia coli mutant. The sequence revealed the e xistence of a pyrBI operon, coding for a catalytic chain and a regulat ory chain, as in Enterobacteriaceae. Comparison of primary sequences o f the polypeptides encoded by the pyrB and pyrI genes with those of ho mologous eubacterial and eukaryotic chains showed a high degree of con servation of the residues which in E. coli ATCase are involved in cata lysis and allosteric regulation. The regulatory chain shows more-exten sive divergence with respect to that of E. coli and other Enterobacter iaceae than the catalytic chain. Several substitutions suggest the exi stence in P. abyssi ATCase of additional hydrophobic interactions and ionic bonds which are probably involved in protein stabilization at hi gh temperatures. The catalytic chain presents a secondary structure si milar to that of the E. coli enzyme. Modeling of the tridimensional st ructure of this chain provides a folding close to that of the E. coli protein in spite of several significant differences. Conservation of n umerous pairs of residues involved in the interfaces between different chains or subunits in E. coli ATCase suggests that the P. abyssi enzy me has a quaternary structure similar to that of the E. coli enzyme. P . abyssi ATCase expressed in transgenic E. coli cells exhibited reduce d cooperativity for aspartate binding and sensitivity to allosteric ef fecters, as well as a decreased thermostability and barostability, sug gesting that in P. abyssi cells this enzyme is further stabilized thro ugh its association with other cellular components.