Abstract Halohydrin dehalogenases (HHDHs) represent as important biocatalysts for the production of valuable chiral epoxides and β-substituted alcohols. The HHDH from A. radiobacter (HheC) showed great potential for biosynthesis of (S)-epichlorohydrin ((S)-ECH). However, two bottlenecks including self-degradation and enzyme-mediated racemization of product in aqueous solution restricted its wide-spread applications. To solve the problems, the biocatalytic process was performed in aqueous-organic two-phase system and structure-based engineered of HheC was performed. A newly found zigzag tunnel connecting the active site region and outside space, as well as a depressed cave structure in formation of an extra three-pot herringbone channel were demonstrated to play a pivotal role affecting the activity and enantioselectivity of the enzyme. By mutation of the involved amino acid residues, two variants E255 T and E255 G showed significantly improved activity, with specific activity increased from 59.9 U/mg to 101.6 U/mg and 77.5 U/mg, respectively, while one variant Y93 L exhibited high enantioselectivity, with ee value of (S)-ECH maintained at >99 %. In addition, the synergic variant Y93 L/E255 T had both improved catalytic efficiency and enantioselectivity. The results not only facilitate the applicability of HheC for efficient production of (S)-ECH, but also give new insights into the structural-functional relationship of the enzyme.

中文翻译：

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卤代醇脱卤酶的合理设计在水-有机两相体系中高效生产高活性和对映选择性的手性环氧氯丙烷

摘要 卤代醇脱卤酶（HHDHs）是生产有价值的手性环氧化物和β-取代醇的重要生物催化剂。来自放射杆菌 (HheC) 的 HHDH 显示出 (S)-表氯醇 ((S)-ECH) 生物合成的巨大潜力。然而，包括自降解和酶介导的产物在水溶液中的外消旋化在内的两个瓶颈限制了其广泛应用。为了解决这些问题，在水-有机两相体系中进行了生物催化过程，并进行了基于结构的HheC工程。新发现的连接活性部位区域和外部空间的曲折隧道以及形成额外的三锅人字形通道的凹陷洞穴结构被证明在影响酶的活性和对映选择性方面起着关键作用。通过对相关氨基酸残基的突变，E255 T和E255 G两种变体的活性显着提高，比活性分别从59.9 U/mg增加到101.6 U/mg和77.5 U/mg，而一种变体Y93 L表现出较高的活性。对映选择性，(S)-ECH 的 ee 值保持在 >99%。此外，协同变体 Y93 L/E255 T 具有更高的催化效率和对映选择性。结果不仅促进了 HheC 在 (S)-ECH 的高效生产中的适用性，而且为酶的结构 - 功能关系提供了新的见解。而一种变体 Y93 L 表现出高对映选择性，(S)-ECH 的 ee 值保持在 >99%。此外，协同变体 Y93 L/E255 T 具有更高的催化效率和对映选择性。结果不仅促进了 HheC 在 (S)-ECH 的高效生产中的适用性，而且为酶的结构 - 功能关系提供了新的见解。而一种变体 Y93 L 表现出高对映选择性，(S)-ECH 的 ee 值保持在 >99%。此外，协同变体 Y93 L/E255 T 具有更高的催化效率和对映选择性。结果不仅促进了 HheC 在 (S)-ECH 的高效生产中的适用性，而且还为酶的结构 - 功能关系提供了新的见解。