嗜热脂肪地芽孢杆菌
苹果酸脱氢酶
嗜热菌
柠檬酸合酶
突变体
生物化学
热稳定性
化学
生物
酶
基因
作者
Yuya Shimozawa,Tomoki Himiyama,T. Nakamura,Yoshiaki Nishiya
出处
期刊:Protein Engineering Design & Selection
[Oxford University Press]
日期:2021-01-01
卷期号:34
被引量:2
标识
DOI:10.1093/protein/gzab026
摘要
Abstract Malate dehydrogenase (MDH) catalyzes the reversible reduction of nicotinamide adenine dinucleotide from oxaloacetate to L-malate. MDH from moderate thermophilic Geobacillus stearothermophilus (gs-MDH) has high thermal stability and substrate specificity and is used as a diagnostic reagent. In this study, gs-MDH was engineered to increase its catalytic activity at low temperatures. Based on sequential and structural comparison with lactate dehydrogenase from G. stearothermophilus, we selected G218 as a mutation site to increase the loop flexibility pivotal for MDH catalysis. The G218 mutants showed significantly higher specific activities than the wild type at low temperatures and maintained thermal stability. The crystal structure of the G218Y mutant, which had the highest catalytic efficiency among all the G218 mutants, suggested that the flexibility of the mobile loop was successfully increased by the bulky side chain. Therefore, this study demonstrated the low-temperature adaptation of MDH by facilitating conformational changes during catalysis.
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