Increasing loop flexibility affords low-temperature adaptation of a moderate thermophilic malate dehydrogenase from Geobacillus stearothermophilus.
Increasing loop flexibility affords low-temperature adaptation of a moderate thermophilic malate dehydrogenase from Geobacillus stearothermophilus.
复制标题
增加环的灵活性使得来自嗜热脂肪地芽孢杆菌的中等嗜热苹果酸脱氢酶能够适应低温。
DOI：
10.1093/protein/gzab026
复制
发表时间：
2021
影响因子：
2.4
通讯作者：
Yoshiaki Nishiya
中科院分区：
文献类型：
--
作者：
Yuya Shimozawa;Tomoki Himiyama;Tsutomu Nakamura;Yoshiaki Nishiya
苹果酸脱氢酶催化烟酰胺腺嘌呤二核苷酸从草酰乙酸酯可逆还原为L-苹果酸。来源于中等嗜热脂肪杆菌的MDH(GS-MDH)具有很高的热稳定性和底物特异性，被用作诊断试剂。在本研究中，GS-MDH被设计成在低温下提高其催化活性。通过与G。乳酸脱氢酶序列和结构的比较。我们选择G218作为突变位点，以增加MDH催化过程中的环灵活性。G218突变体在低温下表现出明显高于野生型的比活力，并保持了热稳定性。G218Y突变体在所有G218突变体中具有最高的催化效率，其晶体结构表明，巨大的侧链成功地增加了移动环的灵活性。因此，本研究证明了MDH的低温适应性是通过促进催化过程中的构象变化来实现的。
Malate dehydrogenase (MDH) catalyzes the reversible reduction of nicotinamide adenine dinucleotide from oxaloacetate to L-malate. MDH from moderate thermophilicGeobacillus 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 fromG. 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.