N-ACETYLGLUTAMATE-5-PHOSPHOTRANSFERASE OF THERMOPHILIC BACTERIUM BACILLUS-STEAROTHERMOPHILUS - NUCLEOTIDE-SEQUENCE AND ENZYME CHARACTERIZATION

A nucleotide sequence of the argB gene of strain Bacillus stearothermophilus NCIB 8224 was determined. The argB gene codes for N acetylglutamate-5-phosphotransferase of 258 amino acids with a molecular weight of 26918 D. This value is in good agreement with the SDS-PAG electrophoresis gata for identification of the heat stable B stearothermophilus argB product synthesized in mesophilic Escherichia coli host cells. The substrates MgATP and N-acetyl-L-glutamate efficiently protect the enzyme against temperature denaturation. Amino acid sequences of bacterial (B. stearothermophilus and E. coli) and yeast (Saccharomyces cerevisiae and S. pombe) N acetylglutamate-5-phosphotransferases share homologous conservative sites which can be responsible for MgATP binding and other structural and functional features of the enzymes of evolutionary distant microorganisms. Gel-filtration followed by K-phosphate buffer/N-acetyl-L-glutamate elution points out that the enzyme should have a molecular weight of 55000 D. This predicts a dimeric form of the enzyme in physiological conditions. N-acetylglutamate-5-phosphotransferase activity is not inhibited by arginine - the end product of the biosynthetic pathway. The enzyme synthesis is repressed 4-fold in B. stearothermophilus by adding arginine to a growth medium. On the contrary, in E. coli hosts independent of their argR status, bacillar enzyme synthesis is not influenced by arginine. The plasmid-cloned B. stearothermophilus argB gene is well expressed in heterologous host cells (N-acetylglutamate-5-phosphotransferase activity was more than 150 and 600-fold higher in comparison with the plasmidless F. coli and B. stearothermophilus hosts, respectively). This is a result of efficient utilization of bacillar transcriptional and translational signals, convenient codon usage of the argB gene in E. coli and the absence of any repressive action of arginine and E. coli ArgR repressor on mRNA synthesis.