Expression and site-directed mutagenesis of the phosphatidylcholine-preferring phospholipase C of Bacillus cereus: probing the role of the active site Glu146.
Expression and site-directed mutagenesis of the phosphatidylcholine-preferring phospholipase C of Bacillus cereus: probing the role of the active site Glu146.
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蜡状芽孢杆菌磷脂酰胆碱偏好性磷脂酶 C 的表达和定点诱变：探讨活性位点 Glu146 的作用。
DOI：
10.1021/bi961316f
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Hergenrother,PJ
中科院分区：
文献类型：
--
作者：
Martin,SF;Spaller,MR;Hergenrother,PJ
制备了一系列蜡状芽孢杆菌（PLCBc）的磷脂酰胆碱偏好性磷脂酶C的位点特异性突变体，其中146位的谷氨酸残基被谷氨酰胺、天冬氨酸、组氨酸和亮氨酸取代，以阐明Glu 146在催化中可能发挥的作用。首先在大肠杆菌中开发了天然酶的表达系统，以提供PLCB c，其通过在其N-末端的插入因子Xa蛋白酶识别序列与麦芽糖结合蛋白（MBP）融合。该MBP− PLCB c融合蛋白以50 - 70 mg/L培养物的水平分离; MBP− PLCB c融合蛋白的选择性胰蛋白酶消化，随后通过色谱纯化产生ca的重组PLCB cat水平。10 mg/L。然后使用PLCB基因（plc）上的聚合酶链反应（PCR）诱变，将Glu 146密码子替换为谷氨酰胺（E146 Q）、天冬氨酸（E146 D）、组氨酸（E146 H）和亮氨酸（E146 L）的密码子。E146 Q突变体的催化效率是天然PLCBc的1.6%，而其他突变体各自具有野生型的0.2 - 0.3%的活性。E146 Q和天然PLCB的kcat/Kmvs pH曲线均具有上升的酸性分支，表明Glu 146在水解反应中不充当总碱。通过圆二色性测定，所有突变体蛋白含有较少的螺旋结构，并且在较低的温度下经历变性，其顺序为：野生型> E146 Q> E146 D> E146 H> E146 L。原子吸收分析表明，突变蛋白也表现出较低的Zn 2+含量比野生型。因此，PLCB中的Glu 146残基稳定了酶的二级和三级结构，并作为Zn 2的关键配体，但它似乎没有任何特定的催化作用。
A series of site-specific mutants of the phosphatidylcholine-preferring phospholipase C fromBacillus cereus(PLCBc) was prepared in which the glutamic acid residue at position 146 was replaced with glutamine, aspartic acid, histidine, and leucine to elucidate what role Glu146 might play in catalysis. An expression system for the native enzyme inEscherichia coliwas first developed to provide PLCBcthat was fused via an intervening factor Xa protease recognition sequence at its N-terminus to maltose binding protein (MBP). This MBP−PLCBcfusion protein was isolated at levels of 50−70 mg/L of culture; selective trypsin digestion of the MBP−PLCBcfusion protein followed by chromatographic purification yielded recombinant PLCBcat levels of ca. 10 mg/L. Polymerase chain reaction (PCR) mutagenesis on the PLCBcgene (plc) was then used to replace the Glu146 codon with those for glutamine (E146Q), aspartic acid (E146D), histidine (E146H), and leucine (E146L). The catalytic efficiency of the E146Q mutant was 1.6% that of native PLCBc, while the other mutants each possessed activities of 0.2−0.3% of the wild type. Thekcat/Kmvs pH profiles for both E146Q and native PLCBchave ascending acidic limbs, suggesting that Glu146 does not serve as the general base in the hydrolysis reaction. As measured by circular dichroism, all of the mutant proteins contained less helical structure and underwent denaturation at lower temperatures than the wild type in the order: wild type > E146Q > E146D ≈ E146H ≈ E146L. Atomic absorption analyses indicated that the mutant proteins also exhibited lower Zn2+content than the wild type. Thus, the Glu146 residue in PLCBcstabilizes the secondary and tertiary structure of the enzyme and serves as a critical ligand for Zn2, but it does not appear to have any specific catalytic role.