Shiota Ayako, Inaba Satomi, Oda Masayuki
a Graduate School of Life and Environmental Sciences , Kyoto Prefectural University , Kyoto , Japan.
b Research & Utilization Division , Japan Synchrotron Radiation Research Institute , Hyogo , Japan.
Biosci Biotechnol Biochem. 2018 Oct;82(10):1702-1707. doi: 10.1080/09168451.2018.1486175. Epub 2018 Jun 18.
We overexpressed and purified 3α-hydroxysteroid dehydrogenase from Pseudomonas sp. B-0831 (Ps3αHSD) and its mutants where the active site residues known as the SYK triad, Ser114, Tyr153, and Lys157, were mutated. Ps3αHSD catalyzes the reaction by using a nucleotide cofactor. The NADH binding affinity of K157A mutant was much lower than that of the wild-type, mainly due to loss of a hydrogen bond. The decreased affinity would result in decreased k Compared to the wild-type, the mutants S114A and Y153F showed higher K and lower k values in both oxidation and reduction reactions. Simultaneous mutation of S114A and Y153F resulted in a significant decrease in k relative to the single mutant. These results are supported by the notion that Tyr153 is a catalytic base and Ser114 would be a substitute. Loss of hydrogen bonding with NADH upon the Y153F mutation resulted in increased enthalpy change, partially compensated by increased entropy change.
我们对来自假单胞菌属B - 0831（Ps3αHSD）的3α - 羟基类固醇脱氢酶及其突变体进行了过表达和纯化，这些突变体的活性位点残基即所谓的SYK三联体（Ser114、Tyr153和Lys157）发生了突变。Ps3αHSD通过使用核苷酸辅因子催化反应。K157A突变体对NADH的结合亲和力远低于野生型，主要是由于氢键的丧失。亲和力降低会导致k值降低。与野生型相比，突变体S114A和Y153F在氧化和还原反应中均表现出更高的K值和更低的k值。S114A和Y153F的同时突变导致相对于单突变体k值显著降低。这些结果支持了Tyr153是催化碱基而Ser114是替代物的观点。Y153F突变导致与NADH的氢键丧失，从而导致焓变增加，部分由熵变增加所补偿。
