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

A nucleotide sequence of the argB gene of strain Bacillus stearothermo philus NCIB 8224 was determined. The argB gene codes for N acetylgluta mate-5-phosphotransferase of 258 amino acids with a molecular weight o f 26918 D. This value is in good agreement with the SDS-PAG electropho resis gata for identification of the heat stable B stearothermophilus argB product synthesized in mesophilic Escherichia coli host cells. Th e substrates MgATP and N-acetyl-L-glutamate efficiently protect the en zyme against temperature denaturation. Amino acid sequences of bacteri al (B. stearothermophilus and E. coli) and yeast (Saccharomyces cerevi siae and S. pombe) N acetylglutamate-5-phosphotransferases share homol ogous conservative sites which can be responsible for MgATP binding an d other structural and functional features of the enzymes of evolution ary distant microorganisms. Gel-filtration followed by K-phosphate buf fer/N-acetyl-L-glutamate elution points out that the enzyme should hav e a molecular weight of 55000 D. This predicts a dimeric form of the e nzyme in physiological conditions. N-acetylglutamate-5-phosphotransfer ase activity is not inhibited by arginine - the end product of the bio synthetic pathway. The enzyme synthesis is repressed 4-fold in B. stea rothermophilus by adding arginine to a growth medium. On the contrary, in E. coli hosts independent of their argR status, bacillar enzyme sy nthesis is not influenced by arginine. The plasmid-cloned B. stearothe rmophilus argB gene is well expressed in heterologous host cells (N-ac etylglutamate-5-phosphotransferase activity was more than 150 and 600- fold higher in comparison with the plasmidless F. coli and B. stearoth ermophilus hosts, respectively). This is a result of efficient utiliza tion of bacillar transcriptional and translational signals, convenient codon usage of the argB gene in E. coli and the absence of any repres sive action of arginine and E. coli ArgR repressor on mRNA synthesis.