大肠杆菌磷酸甘油酸脱氢酶突变体的构建及抗反馈抑制效应

Construction and characterization of Escherichia coli D-3-phosphoglycerate dehydrogenase mutants with feedback-inhibition relief

摘要:

磷酸甘油酸脱氢酶 (D-3-phosphoglycerate dehydrogenase，PGDH，EC 1.1.1.95) 为L-丝氨酸合成途径的关键酶，其编码基因为serA，其活性受到合成产物L-丝氨酸的反馈抑制调控。为解除丝氨酸的反馈抑制，采用定点突变技术把编码PGDH酶344位组氨酸或346位天冬氨酸或364位天冬氨酸的密码子定点突变为丙氨酸密码子。改造后的serAFbr被连到表达载体pT7-7上，并转入大肠杆菌Escherichia coli BL21 (DE3) 中进行表达，破壁回收粗酶液，通过DEAE阴离子柱纯化PGDH突变体，并对其酶活性和IC50值进行了测定。结果，野生型PGDH酶IC50值为7 μmol/L，而PGDH双突变体N346A/H344A催化活性与野生型相近，在丝氨酸浓度为160 mmol/L时，其酶活仍保持未添加丝氨酸时酶活的96%，基本解除反馈抑制。

Abstract:

3-Phosphoglycerate dehydrogenase (PGDH, EC 1.1.1.95) is the key enzyme in L-serine biosynthesis and its coding gene is serA. PGDH is feedback inhibited by L-serine. In order to relieve the feedback-inhibition of PGDH by L-serine, H344 or D346 or D364 were chosen for site directed mutagenesis. The mutants were generated by the standard QuikChange mutagenesis, further subcloned into expression vector pT7-7 and transformed into Escherichia coli BL21 (DE3) cells. The recombinant cells were collected after cultured in LB media post induced by Isopropyl beta-D- thiogalactopyranoside. The enzymes were purified by anion exchange chromatography, and SDS-PAGE showed that the purified enzymes were homogenous. Enzyme characterization indicated that the mutant enzyme showed similar activity, optimal temperature, and optimal pH as that of the wild-type enzyme. Moreover, feedback inhibition study showed that the activity of the double mutant (N346A/H344A) could remain 96% in the presence of serine up to 160 mmol/L, whereas the activity of the wild-type enzyme remains only 50% in the presents of serine of 7 μmol/L, thus successfully relieving the feedback inhibition of PGDH with its activity remained.