Liu Xiaoyu, Zhou Mingyang, Sun Rui, Xing Shu, Wu Tao, He Hailun, Chen Jianbin, Bielicki John Kevin
School of Chemistry and Chemical Engineering, Qilu University of Technology (Shandong Academy of Sciences), Jinan, China.
State Key Laboratory of Medical Genetics, School of Life Sciences, Central South University, Changsha, China.
Front Microbiol. 2022 May 17;13:855658. doi: 10.3389/fmicb.2022.855658. eCollection 2022.
Studies of microorganisms from extreme environments can sometimes reveal novel proteins with unique properties. Here, we identified a novel esterase gene () from an Antarctic bacterium. The protein was expressed and purified for biochemical characterizations. Site-mutation variants including S94A, D205A, and H233A were constructed to explore the structure-function relationship of the catalytic triad of Est33, and we found mutating Ser, Asp, and His residues lead to a complete loss of enzyme activity. In addition, the catalytic Ser located in a conserved pentapeptide motif GVSWG. Phylogenetic analysis showed that Est33 and its closely related homologs belonged to an independent group apart from other known family members, indicating that Est33 represented a new family of esterase. The Est33 enzyme was found to be a cold-active esterase retaining 25%-100% activity from 10°C to 30°C and to have optimal catalytic activity toward -nitrophenol acetate (30°C and pH7.5). The serine modifying reagent phenylmethylsulfonyl fluoride inhibited the activity of Est33 by 77.34%, while thiol reagents such as dithiol threitol (DTT) activated the enzyme by 3-fold. Metal chelating reagents EDTA had no effects, indicating that Est33 is not a metalloenzyme. Collectively, these results indicate that Est33 constitutes the first member of a novel esterase family XXI that has been identified.
对来自极端环境的微生物进行研究有时可揭示具有独特性质的新型蛋白质。在此，我们从一种南极细菌中鉴定出一个新型酯酶基因（）。该蛋白质经表达和纯化后进行生化特性分析。构建了包括S94A、D205A和H233A在内的位点突变变体，以探究Est33催化三联体的结构 - 功能关系，我们发现突变丝氨酸、天冬氨酸和组氨酸残基会导致酶活性完全丧失。此外，催化性丝氨酸位于保守的五肽基序GVSWG中。系统发育分析表明，Est33及其密切相关的同源物属于一个独立于其他已知家族成员的组，这表明Est33代表了一个新的酯酶家族。发现Est33酶是一种冷活性酯酶，在10°C至30°C范围内保留25% - 100%的活性，并且对乙酸对硝基苯酚具有最佳催化活性（30°C和pH7.5）。丝氨酸修饰试剂苯甲基磺酰氟抑制Est33的活性达77.34%，而诸如二硫苏糖醇（DTT）等硫醇试剂使该酶活性提高3倍。金属螯合剂EDTA没有影响，表明Est33不是金属酶。总体而言，这些结果表明Est33构成了已被鉴定的新型酯酶家族XXI的首个成员。
