Sürmeli Yusuf, Durmuş Naciye, Şanlı-Mohamed Gülşah
Department of Agricultural Biotechnology, Tekirdağ Namık Kemal University, 59030 Tekirdağ, Turkey.
Department of Molecular Biology and Genetics, İstanbul Technical University, 34485 İstanbul, Turkey.
ACS Omega. 2024 Jul 22;9(30):32931-32941. doi: 10.1021/acsomega.4c03818. eCollection 2024 Jul 30.
This study conducted an analysis of two biochemically characterized thermostable esterases, Est2 and Est3, from strains. To achieve this, the amino acid sequences of Est2 and Est3 were examined to assess their biophysicochemical properties, evolutionary connections, and sequence similarities. Three-dimensional models were constructed and validated through diverse bioinformatics tools. Molecular dynamics (MD) simulation was employed on a NP-C2 ligand to explore interactions between enzymes and ligand. Biophysicochemical property analysis indicated that aliphatic indices and theoretical values of enzymes were between 82-83 and 55-65 °C, respectively. Molecular phylogeny placed Est2 and Est3 within Family XIII, alongside other esterases. DeepMSA2 revealed that Est2, Est3, and homologous sequences shared 12 conserved residues in their core domain (L39, D50, G53, G55, S57, G92, S94, G96, P108, P184, D193, and H223). BANΔIT analysis indicated that Est2 and Est3 had a significantly more rigid cap domain compared to Est30. Salt bridge analysis revealed that E150-R136, E124-K165, E137-R141, and E154-K157 salt bridges made Est2 and Est3 more stable compared to Est30. MD simulation indicated that Est3 exhibited greater fluctuations in the N-terminal region including conserved F25, cap domain, and C-terminal region, notably including H223, suggesting that these regions might influence esterase catalysis. The common residues in the ligand-binding sites of Est2-Est3 were determined as F25 and L167. The analysis of root mean square fluctuation (RMSF) revealed that region 1, encompassing F25 within the β2-α1 loop of Est3, exhibited higher fluctuations compared to those of Est2. Overall, this study might provide valuable insights for future investigations aimed at improving esterase thermostability and catalytic efficiency, critical industrial traits, through targeted amino acid modifications within the N-terminal region, cap domain, and C-terminal region using rational protein engineering techniques.
本研究对来自菌株的两种经生化特性鉴定的耐热酯酶Est2和Est3进行了分析。为此，对Est2和Est3的氨基酸序列进行了检查，以评估它们的生物物理化学性质、进化联系和序列相似性。通过各种生物信息学工具构建并验证了三维模型。对一种NP-C2配体进行了分子动力学（MD）模拟，以探索酶与配体之间的相互作用。生物物理化学性质分析表明，酶的脂肪族指数和理论值分别在82-83和55-65℃之间。分子系统发育分析将Est2和Est3置于第十三家族中，与其他酯酶在一起。DeepMSA2显示，Est2、Est3和同源序列在其核心结构域（L39、D50、G53、G55、S57、G92、S94、G96、P108、P184、D193和H223）共有12个保守残基。BANΔIT分析表明，与Est30相比，Est2和Est3的帽状结构域明显更刚性。盐桥分析表明，与Est30相比，E150-R136、E124-K165、E137-R141和E154-K157盐桥使Est2和Est3更稳定。MD模拟表明，Est3在包括保守的F25、帽状结构域和C末端区域在内的N末端区域表现出更大的波动，特别是包括H223，这表明这些区域可能影响酯酶催化作用。Est2-Est3配体结合位点的共同残基确定为F25和L167。均方根波动（RMSF）分析表明，与Est2相比，Est3的β2-α1环内包含F25的区域1表现出更高的波动。总体而言，本研究可能为未来旨在通过使用合理的蛋白质工程技术在N末端区域、帽状结构域和C末端区域进行靶向氨基酸修饰来提高酯酶耐热性和催化效率（关键工业特性）的研究提供有价值的见解。
