大肠杆菌PGDH末端缺失突变体的构建及抗反馈抑制效应分析

为了获得具有抗反馈抑制性质的大肠杆菌磷酸甘油酸脱氢酶（PGDH, d-3-phosphoglycerate dehydrogenase, EC 1.1.1.95），通过对其碱基序列和蛋白质结构分析，用PCR突变法构建突变酶M1（缺失第410位氨基酸）、M2（缺失407~410位氨基酸）、M3（缺失337~410位氨基酸）。M0(野生型)及各突变型基因与pET22b(+)载体连接后，表达融合蛋白。在非变性条件下，由NTA-Ni镍离子螯合亲和层析柱纯化野生型和突变体的酶蛋白。酶活性测定结果表明，M1、M2蛋白酶均保持了原有的野生型磷酸甘油酸脱氢酶活性，且部分解除了终产物L-丝氨酸的反馈抑制作用；M3蛋白酶完全解除了终产物的反馈抑制作用，但酶本身的催化活性略有降低（为野生型的83%）。M0、M1、M2菌株PGDH与L-丝氨酸结合的Ki值分别约为7 μmol/L、20 μmol/L、50 μmol/L，说明该酶C-末端1~4个氨基酸残基对L-丝氨酸和调控区的结合有重要影响。

Construction and Characterization of E.coli PGDH Mutants with Feedback-inhibition Resistance

In order to obtain E.coli PGDH mutants with feedback-inhibition resistance, PCR mutagenesis was used to construct three mutants on the basis of homology of protein structure and the related PGDH amino acid sequences: M1(serA mutant with aa410 deleted), M2 (serA mutant with aa407~410 deleted), M3( serA mutant with aa337~410 deleted). The three mutant genes and wild type gene were inserted into prokaryotic fusion-expression vector pET22b(+). The expressed products were purified by NTA-Ni affinity chromatography resin in native condition. Catalytic activities of M3 retained only 83% of the wild type, but the activities of M1 and M2 were retained. M3 showed the complete feedback-resistance, M1 and M2 also showed resistance to some extent. Ki value of M0, M1, M2 were about 7 μmol/L, 20 μmol/L, 50 μmol/L, respectively. This result means that PGDH C-terminal residues 1~4 have a profound effect on the interaction between L-serine and R regulatory region.