腾冲嗜热厌氧菌丙氨酸消旋酶底物通道氨基酸位点的功能

Function of amino acid sites in the substrate entryway of alanine racemase TtAlr from Thermoanaerobacter tengcongensis

摘要:

[目的]通过定点突变探究腾冲嗜热厌氧菌MB4中生物合成型丙氨酸消旋酶TtAlr底物通道内氨基酸位点A172和S173的功能。[方法]利用定点突变PCR技术构建突变体，通过亲和层析法纯化酶蛋白，采用D-氨基酸氧化酶偶联法检测各突变蛋白的活性及其稳定性。[结果]通过定点突变PCR成功得到8个突变体，酶学特性分析发现，A172位点突变为丝氨酸（S）后酶蛋白的相对活性有所提升，但含有该位点突变的酶蛋白稳定性均大幅下降；S173位点突变为天门冬氨酸（D）后导致突变体蛋白的最适反应温度提升了15℃，半衰期大幅延长，但相对活性明显下降。[结论]丙氨酸消旋酶TtAlr底物通道内A172和S173位点均是影响酶蛋白催化活性和稳定性的关键位点。

Abstract:

[Objective] In order to study the function of amino acid sites A172 and S173 in the substrate entryway of alanine racemase TtAlr from Thermoanaerobacter tengcongensis MB4. [Methods] The mutant vectors were constructed by site-directed mutagenesis PCR using plasmid pET-TtAlr as the template and expressed in E. coli BL21(DE3). The enzymes were purified by affinity chromatography. D-amino acid oxidase coupling method was used to detect enzyme activity and stability of each mutant and wild type TtAlr proteins. [Results] Both TtAlr and mutant proteins were expressed and purified successfully. Results of enzymatic properties show that A172 site mutation could improve the catalytic activity of TtAlr, but the stability of enzyme proteins decreased significantly. Likewise, S173 site mutation could increase the optimal temperature of TtAlr, prolonged the half-life of the enzyme and improved its stability, but the catalytic activity of the enzyme decreased significantly. [Conclusion] A172 and S173 amino acids residues in the substrate entryway of alanine racemase TtAlr were the key sites that played a major role in the catalytic activity and stability of the enzyme protein.