Construction of Engineered Water-soluble PQQ Glucose Dehydrogenase with Improved Substrate Specificity
Construction of Engineered Water-soluble PQQ Glucose Dehydrogenase with Improved Substrate Specificity
复制标题
具有改进的底物特异性的工程化水溶性 PQQ 葡萄糖脱氢酶的构建
DOI：
10.1080/1024242021000058694
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发表时间：
2002
影响因子：
1.8
通讯作者：
H. Yoshida
中科院分区：
文献类型：
--
作者：
K. Sode;S. Igarashi;A. Morimoto;H. Yoshida
这是第一项缩小以吡咯喹啉醌为辅基的水溶性葡萄糖脱氢酶（PQQGDH-B）底物特异性的研究。我们在酶的环6BC区域引入氨基酸取代，弥补了活性位点裂口，不与底物直接相互作用，构建了一系列定点突变体。在这些突变体中，Asn452Thr 显示出最窄的底物特异性，同时保留了与野生型酶相似的催化效率、热稳定性和 EDTA 耐受性。突变型酶与乳糖的相对活性低于野生型酶。发现突变酶的底物特异性谱的改变主要是由于底物的 Km 值比葡萄糖的增加。 Asn452Thr 和野生型酶的预测 3D 结构表明，在 6BC 环区域与乳糖之间的相互作用中观察到氨基酸取代的最显着影响。
This was the first study that achieved a narrowing of the substrate specificity of water soluble glucose dehydrogenase harboring pyrroloquinoline quinone as their prosthetic group, PQQGDH-B. We conducted the introduction of amino acid substitutions into the loop 6BC region of the enzyme, which made up the active site cleft without directly interacting with the substrate, and constructed a series of site directed mutants. Among these mutants, Asn452Thr showed the least narrowed substrate specificity while retaining a similar catalytic efficiency, thermal stability and EDTA tolerance as the wild-type enzyme. The relative activities of mutant enzyme with lactose were lower than that of the wild-type enzyme. The altered substrate specificity profile of the mutant enzyme was found to be mainly due to increase in Km value for substrate than glucose. The predicted 3D structures of Asn452Thr and the wild-type enzyme indicated that the most significant impact of the amino acid substitution was observed in the interaction between the 6BC loop region with lactose.