Göthel S F, Schmid R, Wipat A, Carter N M, Emmerson P T, Harwood C R, Marahiel M A
Philipps-Universität Marburg, Biochemie, Fachbereich Chemie, Marburg, Germany.
Eur J Biochem. 1997 Feb 15;244(1):59-65. doi: 10.1111/j.1432-1033.1997.00059.x.
Two major families of peptidylprolyl cis-trans-isomerases, the cyclophilins and the structurally unrelated FK506-binding proteins (FKBPs), have been identified as cellular factors involved in protein folding in vitro. Here we report on the biochemical characterization of a second prolyl isomerase of Bacillus subtilis that was purified from a cyclophilin-negative (ppiB null) mutant and was shown to be the trigger factor (TigBS). N-terminal sequencing of 27 amino acid residues of the purified protein revealed 100% identity to the deduced sequence encoded by the tig gene, sequenced as a part of the B. subtilis genome project. The tigBS gene, located at 246 degrees on the genetic map upstream of the clpX and lonA,B genes, encodes an acidic protein (pI 4.3) of 47.5 kDa. Purified and recombinant TigBS-His proteins share the same substrate specificity and catalytic activity (Kcat/K(m) of 1.5 microM-1 s-1); both are inhibited by the macrolide FK506 with IC50 the range of 500 nM. We also demonstrate that the prolyl isomerase activity of TigBS is mediated by an internal domain of about 13 kDa (homologous to FKPB12) that represents the catalytic core of the trigger factor.
肽基脯氨酰顺反异构酶有两大主要家族，即亲环蛋白和结构不相关的FK506结合蛋白（FKBPs），它们已被确定为体外参与蛋白质折叠的细胞因子。在此，我们报告了从亲环蛋白阴性（ppiB缺失）突变体中纯化得到的枯草芽孢杆菌第二种脯氨酰异构酶的生化特性，该酶被证明是触发因子（TigBS）。对纯化蛋白27个氨基酸残基进行N端测序，结果显示其与tig基因推导序列100%相同，tig基因是作为枯草芽孢杆菌基因组计划的一部分进行测序的。tigBS基因位于遗传图谱上clpX和lonA、B基因上游246度处，编码一种47.5 kDa的酸性蛋白（pI 4.3）。纯化的和重组的TigBS-His蛋白具有相同的底物特异性和催化活性（Kcat/K(m)为1.5 μM-1 s-1）；二者均被大环内酯类药物FK506抑制，IC50在500 nM范围内。我们还证明，TigBS的脯氨酰异构酶活性由一个约13 kDa的内部结构域介导（与FKPB12同源），该结构域代表触发因子的催化核心。
