State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China.
Department of Engineering Science, University of Oxford, Oxford, United Kingdom.
Appl Environ Microbiol. 2024 Jun 18;90(6):e0143623. doi: 10.1128/aem.01436-23. Epub 2024 May 6.
Rieske non-heme dioxygenase family enzymes play an important role in the aerobic biodegradation of nitroaromatic pollutants, but no active dioxygenases are available in nature for initial reactions in the degradation of many refractory pollutants like 2,4-dichloronitrobenzene (24DCNB). Here, we report the engineering of hotspots in 2,3-dichloronitrobenzene dioxygenase from sp. strain JS3051, achieved through molecular dynamic simulation analysis and site-directed mutagenesis, with the aim of enhancing its catalytic activity toward 24DCNB. The computationally predicted activity scores were largely consistent with the detected activities in wet experiments. Among them, the two most beneficial mutations (E204M and M248I) were obtained, and the combined mutant reached up to a 62-fold increase in activity toward 24DCNB, generating a single product, 3,5-dichlorocatechol, which is a naturally occurring compound. analysis confirmed that residue 204 affected the substrate preference for -substituted nitroarenes, while residue 248 may influence substrate preference by interaction with residue 295. Overall, this study provides a framework for manipulating nitroarene dioxygenases using computational methods to address various nitroarene contamination problems.IMPORTANCEAs a result of human activities, various nitroaromatic pollutants continue to enter the biosphere with poor degradability, and dioxygenation is an important kickoff step to remove toxic nitro-groups and convert them into degradable products. The biodegradation of many nitroarenes has been reported over the decades; however, many others still lack corresponding enzymes to initiate their degradation. Although rieske non-heme dioxygenase family enzymes play extraordinarily important roles in the aerobic biodegradation of various nitroaromatic pollutants, prediction of their substrate specificity is difficult. This work greatly improved the catalytic activity of dioxygenase against 2,4-dichloronitrobenzene by computer-aided semi-rational design, paving a new way for the evolution strategy of nitroarene dioxygenase. This study highlights the potential for using enzyme structure-function information with computational pre-screening methods to rapidly tailor the catalytic functions of enzymes toward poorly biodegradable contaminants.
Rieske 非血红素双氧酶家族酶在芳香族污染物的需氧生物降解中起着重要作用，但自然界中没有活性双氧酶可用于许多难降解污染物（如 2,4-二氯硝基苯（24DCNB））初始降解反应。在这里，我们报道了通过分子动力学模拟分析和定点突变工程改造 sp. JS3051 菌株 2,3-二氯硝基苯双氧酶的热点，旨在提高其对 24DCNB 的催化活性。通过计算预测的活性评分与湿实验中检测到的活性有很大的一致性。其中，获得了两个最有益的突变（E204M 和 M248I），而组合突变体对 24DCNB 的活性最高可达 62 倍，生成一种天然存在的化合物 3,5-二氯邻苯二酚。 分析证实，残基 204 影响 -取代硝基芳烃的底物偏好，而残基 248 可能通过与残基 295 的相互作用影响底物偏好。总的来说，这项研究为使用计算方法操纵硝基芳烃双氧酶提供了一个框架，以解决各种硝基芳烃污染问题。
重要性
由于人类活动，各种硝基芳烃污染物不断进入生物圈，降解性差，而加氧作用是去除有毒硝基基团并将其转化为可降解产物的重要起始步骤。几十年来，已经报道了许多硝基芳烃的生物降解；然而，还有许多其他的硝基芳烃仍然缺乏相应的酶来启动它们的降解。尽管 Rieske 非血红素双氧酶家族酶在各种硝基芳烃污染物的需氧生物降解中起着非常重要的作用，但预测其底物特异性是困难的。这项工作通过计算机辅助半理性设计大大提高了双氧酶对 2,4-二氯硝基苯的催化活性，为硝基芳烃双氧酶的进化策略开辟了新途径。这项研究强调了利用酶结构-功能信息与计算预筛选方法相结合，快速调整酶对难降解污染物的催化功能的潜力。
