台湾家白蚁内切葡聚糖酶活性中心氨基酸的饱和突变

Saturation mutagenesis of three amino acid positions consisting of the active site of an endoglucanase from termiteCoptotermes formosanus

摘要:

对内切葡聚糖酶的功能改进一直是纤维素酶研究领域的焦点。本研究对台湾家白蚁内切葡聚糖酶(CfEG)的活性位点做了饱和突变。首先, 以PDB数据库中高山象白蚁内切葡聚糖酶(NtEG)的三维结构(PDB id=1ks8)为模板, 对CfEG进行三维结构同源建模, 二者序列一致性高达79%。位于CfEG活性中心的D53、D56、E411, 分别与NtEG的催化残基 D54、D57、E412重合。用简并引物对CfEG的假定活性位点D53、D56、E411进行定点饱和突变。在位点D53、D56各筛选到羧甲基纤维素酶活有一定提高的突变子D53E、D56C, 其中D56C的 Km值减小为原始酶的三分之一 。双突变子D53L/D56I的比活比原始酶提高了近2倍, 同时Km值减小至原始酶的一半。而E411的饱和突变子库均没有活性, 进一步将其替换为近似氨基酸的E411D、E411Q定点突变子也丧失了酶活。由突变结果可推断, 位点E411为该酶行使功能的必需残基。

Abstract:

Functional improvement to one component of the cellulase, endo-b-1, 4-glucanase, has been a focus of the recent research in this area. We report here the saturation mutagenesis of the active site of an endoglucanase (CfEG) from termite Coptotermes formosanus. First, three dimensional structure of CfEG was built via homology modeling by using a close-related (79% homology in sequence) endo-b-1,4-glucanase (NtEG, PDB id=1ks8) from higher termite Nasutitermes takasagoensis as a template. Second, we identified three corresponding amino acid positions at the active site of CfEG by structural superposition onto NtEG. These three putative amino acids for the active site of CfEG, i.e., Asp53, Asp56 and Glu411, were subjected to saturation mutagenesis using degenerate primers. Among the mutants, Asp53Glu and Asp56Cys showed somewhow higher activities than the wildtype, with the latter having more than 3-fold decrease in Km. Double mutation Asp53Leu/Asp56Ile showed nearly 2-fold increase in specific activity and in the same time 2-fold decrease in Km. Saturation mutagenesis to the position Glu411 produced no active mutant, even changing Glu411 explicitly into its similar amino acids such as Glu411Asp and Glu411Gln could not result in any active mutant. These imply that position Glu411 could be extremely important and therefore indispensable for CfEG functionality.