Aspartate carbamoyltransferase from the thermoacidophilic archaeon Sulfolobus acidocaldarius. Cloning, sequence analysis, enzyme purification and characterization.
The genes coding for aspartate carbamoyltransferase (ATCase) in the extremely thermophilic archaeon Sulfolobus acidocaldarius have been cloned by complementation of a pyrBI deletion mutant of Escherichia coli. Sequencing revealed the existence of an enterobacterial-like pyrBI operon encoding a catalytic 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 genes 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 organism. The recombinant enzyme displayed the same properties as the ATCase synthesized in the native host. It is highly thermostable and exhibits Michaelian saturation kinetics for carbamoylphosphate (CP) and positive homotropic cooperative interactions for the binding of L-aspartate. Moreover, it is activated by nucleoside triphosphates whereas the catalytic subunits alone are inhibited. The holoenzyme purified from recombinant E. coli cells or present in crude extract of the native host have an Mr of 340 000 as estimated 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 recombinant E. coli or Saccharomyces cerevisiae cells expressing the pyrB gene alone. In the presence of CP these monomers assembled into trimers. The stability of S. acidocaldarius ATCase and the allosteric properties of the enzyme are discussed in function of a modeling study.
Durbecq
V
V
Laboratoire de Microbiologie, Université de Libre de Bruxelles, Belgium.
Thia-Toong
T L
TL
Charlier
D
D
Villeret
V
V
Roovers
M
M
Wattiez
R
R
Legrain
C
C
Glansdorff
N
N
D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D001221:Aspartate Carbamoyltransferase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D006358:Hot Temperature; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017386:Sequence Homology, Amino Acid; D016945:Sulfolobus acidocaldarius / Q000201:enzymology*
D017931:DNA Primers; D001221:Aspartate Carbamoyltransferase