Mechulam Y, Schmitt E, Panvert M, Schmitter J M, Lapadat-Tapolsky M, Meinnel T, Dessen P, Blanquet S, Fayat G
Laboratoire de Biochimie, Unité de Recherche Associée no. 240 du Centre National de la Recherche Scientifique, Palaiseau, France.
Nucleic Acids Res. 1991 Jul 11;19(13):3673-81. doi: 10.1093/nar/19.13.3673.
The metS gene encoding homodimeric methionyl-tRNA synthetase from Bacillus stearothermophilus has been cloned and a 2880 base pair sequence solved. Comparison of the deduced enzyme protomer sequence (Mr 74,355) with that of the E. coli methionyl-tRNA synthetase protomer (Mr 76,124) revealed a relatively low level (32%) of identities, although both enzymes have very similar biochemical properties (Kalogerakos, T., Dessen, P., Fayat, G. and Blanquet, S. (1980) Biochemistry 19, 3712-3723). However, all the sequence patterns whose functional significance have been probed in the case of the E. coli enzyme are found in the thermostable enzyme sequence. In particular, a stretch of 16 amino acids corresponding to the CAU anticodon binding site in the E. coli synthetase structure is highly conserved in the metS sequence. The metS product could be expressed in E. coli and purified. It showed structure-function relationships identical to those of the enzyme extracted from B. stearothermophilus cells. In particular, the patterns of mild proteolysis were the same. Subtilisin converted the native dimer into a fully active monomeric species (62 kDa), while trypsin digestion yielded an inactive form because of an additional cleavage of the 62 kDa polypeptide into two subfragments capable however of remaining firmly associated. The subtilisin cleavage site was mapped on the enzyme polypeptide, and a gene encoding the active monomer was constructed and expressed in E. coli. Finally, trypsin attack was demonstrated to cleave a peptidic bond within the KMSKS sequence common to E. coli and B. stearothermophilus methionyl-tRNA synthetases. This sequence has been shown, in the case of the E. coli enzyme, to have an essential role for the catalysis of methionyl-adenylate formation.
编码嗜热脂肪芽孢杆菌同型二聚体甲硫氨酰 - tRNA合成酶的metS基因已被克隆，并解析出其2880个碱基对的序列。将推导的酶原序列（分子量74,355）与大肠杆菌甲硫氨酰 - tRNA合成酶原序列（分子量76,124）进行比较，发现两者的同源性相对较低（32%），尽管这两种酶具有非常相似的生化特性（Kalogerakos, T., Dessen, P., Fayat, G.和Blanquet, S. (1980) Biochemistry 19, 3712 - 3723）。然而，在大肠杆菌酶中已探究其功能意义的所有序列模式都能在这种耐热酶的序列中找到。特别地，在大肠杆菌合成酶结构中对应于CAU反密码子结合位点的一段16个氨基酸的序列在metS序列中高度保守。metS产物能够在大肠杆菌中表达并纯化。它表现出与从嗜热脂肪芽孢杆菌细胞中提取的酶相同的结构 - 功能关系。特别地，温和蛋白酶解的模式是相同的。枯草杆菌蛋白酶将天然二聚体转化为完全有活性的单体形式（62 kDa），而胰蛋白酶消化产生无活性形式，因为62 kDa多肽进一步裂解为两个亚片段，不过这两个亚片段仍能紧密结合。枯草杆菌蛋白酶的切割位点在酶多肽上定位，并且构建了编码活性单体的基因并在大肠杆菌中表达。最后，证明胰蛋白酶攻击能切割大肠杆菌和嗜热脂肪芽孢杆菌甲硫氨酰 - tRNA合成酶共有的KMSKS序列内的一个肽键。在大肠杆菌酶的情况下，该序列已被证明对甲硫氨酰 - 腺苷酸形成的催化起着重要作用。
