Esterases and lipases hydrolyze short-chain esters and long-chain triglycerides, respectively, and therefore play essential roles in the synthesis and decomposition of ester bonds in the pharmaceutical and food industries. Many SGNH family esterases share high similarity in sequences. However, they have distinct enzymatic activities toward the same substrates. Due to a lack of structural information, the detailed catalytic mechanisms of these esterases remain barely investigated. In this study, we identified two SGNH family esterases, CrmE10 and AlinE4, from marine bacteria with significantly different preferences for pH, temperature, metal ion, and organic solvent tolerance despite high sequence similarity. The crystal structures of these two esterases, including wild type and mutants, were determined to high resolutions ranging from 1.18 Å to 2.24 Å. Both CrmE10 and AlinE4 were composed of five β-strands and nine α-helices, which formed one compact N-terminal α/β globular domain and one extended C-terminal domain. The aspartic residues (D178 in CrmE10/D162 in AlinE4) destabilized the conformations of the catalytic triad (Ser-Asp-His) in both esterases, and the metal ion Cd2+ might reduce enzymatic activity by blocking proton transfer or substrate binding. CrmE10 and AlinE4 showed distinctly different electrostatic surface potentials, despite the similar atomic architectures and a similar swap catalytic mechanism. When five negatively charged residues (Asp or Glu) were mutated to residue Lys, CrmE10 obtained elevated alkaline adaptability and significantly increased the enzymatic activity from 0 to 20% at pH 10.5. Also, CrmE10 mutants exhibited dramatic change for enzymatic properties when compared with the wide-type enzyme. These findings offer a perspective for understanding the catalytic mechanism of different esterases and might facilitate the industrial biocatalytic applications.

中文翻译：

---

结构引导的蛋白质工程增加了 SGNH 家族酯酶的酶活性。

酯酶和脂肪酶分别水解短链酯和长链甘油三酯，因此在制药和食品工业中酯键的合成和分解中发挥着重要作用。许多 SGNH 家族酯酶在序列上具有高度相似性。然而，它们对相同的底物具有不同的酶活性。由于缺乏结构信息，这些酯酶的详细催化机制仍然很少研究。在这项研究中，我们从海洋细菌中鉴定出两种 SGNH 家族酯酶 CrmE10 和 AlinE4，尽管序列相似性很高，但它们对 pH、温度、金属离子和有机溶剂耐受性的偏好明显不同。这两种酯酶的晶体结构，包括野生型和突变体，被确定为从 1.18 到 2 的高分辨率。24 埃。CrmE10和AlinE4均由5条β链和9条α螺旋组成，形成一个紧凑的N端α/β球状结构域和一个延伸的C端结构域。天冬氨酸残基（CrmE10 中的 D178/AlinE4 中的 D162）破坏了两种酯酶中催化三联体（Ser-Asp-His）的构象，金属离子 Cd2+ 可能通过阻断质子转移或底物结合来降低酶活性。CrmE10 和 AlinE4 表现出明显不同的静电表面电位，尽管具有相似的原子结构和相似的交换催化机制。当五个带负电荷的残基（Asp 或 Glu）突变为残基 Lys 时，CrmE10 获得了更高的碱性适应性，并在 pH 10.5 下将酶活性从 0% 显着提高到 20%。还，与宽型酶相比，CrmE10 突变体的酶特性发生了巨大变化。这些发现为理解不同酯酶的催化机制提供了一个视角，并可能促进工业生物催化应用。