Aspartate carbamoyltransferase from the thermoacidophilic archaeon Sulfolobus acidocaldarius - Cloning, sequence analysis, enzyme purification and characterization

The genes coding for aspartate carbamoyltransferase (ATCase) in the extreme ly thermophilic archaeon Sulfolobus acidocaldarius have been cloned by comp lementation of a pyr-BI deletion mutant of Escherichia coli. Sequencing rev ealed the existence of an enterobacterial-like pyrBI operon encoding a cata lytic chain of 299 amino acids (34 kDa) and a regulatory chain of 170 amino acids (17.9 kDa). The deduced amino acid sequences of the pyrB and pyrI ge nes showed 27.6-50% identity with archaeal and enterobacterial ATCases. The recombinant S. acidocaldarius ATCase was purified to homogeneity, allowing the first detailed studies of an ATCase isolated from a thermophilic organ ism. The recombinant enzyme displayed the same properties as the ATCase syn thesized in the native host. It is highly thermostable and exhibits Michael ian saturation kinetics for carbamoylphosphate (CP) and positive homotropic cooperative interactions for the binding of L-aspartate. Moreover, it is a ctivated by nucleoside triphosphates whereas the catalytic subunits alone a re inhibited. The holoenzyme purified from recombinant E. coli cells or pre sent in crude extract of the native host have an M-r of 340 000 as estimate d by gel filtration, suggesting that it has a quaternary structure similar to that of E. coli ATCase, Only monomers could be found in extracts of reco mbinant E. coli or Saccharomyces cerevisiae cells expressing the pyrB gene alone. In the presence of CP these monomers assembled into trimers. The sta bility of S. acidocaldarius ATCase and the allosteric properties of the enz yme are discussed in function of a modeling study.