Primary structure, partial purification and regulation of key enzymes of the acetyl cycle of arginine biosynthesis in Bacillus stearothermophilus: dual function of ornithine acetyltransferase.
A 3.4 kb EcoRI fragment, cloned in E. coli, that carries part of a cluster of genes encoding arginine biosynthetic functions of the thermophilic bacterium Bacillus stearothermophilus, was sequenced on both strands. The sequence consists of a truncated argC gene, an argJ region encoding a polypeptide with both N-acetylglutamate synthase and ornithine acetyltransferase activities, the argB gene and the N-terminal part of argD. The argB gene encodes a 258-amino-acid polypeptide with a deduced M(r) of 26918. A very high and thermostable N-acetylglutamate 5-phosphotransferase activity was detected in extracts of E. coli arg B mutants transformed with the 3.4 kb fragment on a plasmid. A polypeptide band of M(r) 27,000 was detected by SDS-PAGE of heat-treated extract from such a strain. Both N-acetylglutamate synthase and ornithine acetyltransferase are encoded by the same 1290 bp open reading frame. The deduced sequence of 410 amino acids corresponds to a peptide of M(r) 43,349. The subcloned B. stearothermophilus argJ can complement a double argA argE E. coli mutant to prototrophy. Gel-filtration of a heat-treated extract of the complemented double mutant E. coli host showed that N-acetylglutamate synthase and ornithine acetyltransferase activities co-elute in a single peak corresponding to M(r) 110,000. Both activities were also heat-inactivated at the same temperature and strongly inhibited by ornithine. These results suggest that both activities can be ascribed to a single protein.
Sakanyan
V
V
Pharmagen, Yerevan, Republic of Armenia.
Charlier
D
D
Legrain
C
C
Kochikyan
A
A
Mett
I
I
Piérard
A
A
Glansdorff
N
N
D000123:Acetyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D051046:Amino-Acid N-Acetyltransferase; D001120:Arginine / Q000096:biosynthesis*; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D001411:Geobacillus stearothermophilus / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight
D004269:DNA, Bacterial; D001120:Arginine; D000123:Acetyltransferases; D051046:Amino-Acid N-Acetyltransferase; C020646:glutamate acetyltransferase