Sakanian V A, Legreĭn Kh, Charlier D, Kochikian A V
Genetika. 1993 Apr;29(4):556-70.
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 55,000 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, bacillary 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 E. coli and B. stearothermophilus hosts, respectively). This is a result of efficient utilization of bacillary 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.
测定了嗜热脂肪芽孢杆菌NCIB 8224菌株argB基因的核苷酸序列。argB基因编码一种含258个氨基酸、分子量为26918 D的N - 乙酰谷氨酸 - 5 - 磷酸转移酶。该数值与用于鉴定在嗜温大肠杆菌宿主细胞中合成的嗜热脂肪芽孢杆菌热稳定argB产物的SDS - PAG电泳数据高度吻合。底物MgATP和N - 乙酰 - L - 谷氨酸能有效保护该酶免受热变性影响。细菌（嗜热脂肪芽孢杆菌和大肠杆菌）及酵母（酿酒酵母和粟酒裂殖酵母）的N - 乙酰谷氨酸 - 5 - 磷酸转移酶的氨基酸序列具有同源保守位点，这些位点可能负责MgATP结合以及进化关系较远的微生物中该酶的其他结构和功能特征。经凝胶过滤，随后用K - 磷酸盐缓冲液/N - 乙酰 - L - 谷氨酸洗脱表明，该酶的分子量应为55000 D。这预示该酶在生理条件下呈二聚体形式。生物合成途径的终产物精氨酸不会抑制N - 乙酰谷氨酸 - 5 - 磷酸转移酶的活性。在嗜热脂肪芽孢杆菌中，向生长培养基中添加精氨酸会使该酶的合成受到4倍的抑制。相反，在大肠杆菌宿主中，无论其argR状态如何，杆菌酶的合成均不受精氨酸影响。质粒克隆的嗜热脂肪芽孢杆菌argB基因在异源宿主细胞中表达良好（与无质粒的大肠杆菌和嗜热脂肪芽孢杆菌宿主相比，N - 乙酰谷氨酸 - 5 - 磷酸转移酶活性分别高出150倍和600倍以上）。这是由于杆菌转录和翻译信号的有效利用、argB基因在大肠杆菌中便利的密码子使用以及精氨酸和大肠杆菌ArgR阻遏物对mRNA合成无任何抑制作用的结果。
