School of Biomolecular Science and Engineering, Vidyasirimedhi Institute of Science and Technology (VISTEC), Wangchan Valley, Rayong, 21210, Thailand.
Department of Biotechnology, Faculty of Engineering and Industrial Technology, Silpakorn University, 6 Rajamankha Nai Road, Nakornpathom, 73000, Thailand.
Chembiochem. 2019 Dec 13;20(24):3020-3031. doi: 10.1002/cbic.201900413. Epub 2019 Sep 26.
HadA is a flavin-dependent monooxygenase that can catalyze the denitration and dehalogenation of a wide variety of toxicants such as pesticides. Although these enzymatic reactions are useful for bioremediation or biocatalysis, the application of HadA for these purposes is not yet possible because of its low thermostability. In this work we have engineered HadA to be more thermostable through the use of structural, in silico, and rational approaches. The X-ray structure of HadA was solved to obtain a reliable three-dimensional protein model for further prediction of thermostable variants. In silico analysis by using two bioinformatic tools-FireProt and Disulfide by Design-suggested 102 variants that we then further refined by applying rational criteria including the location of a particular residue and its nearby interactions, as well as other biophysical parameters to narrow down the list to six candidates. The G513Y variant was found to be an optimal engineered candidate because it has significantly improved stability relative to the wild-type enzyme and equivalent activity. G513Y has an activity half-life 72 (50 °C) and 160 times (45 °C) longer than that of the wild-type enzyme. Coupled together with thermostable reactions of reduced flavin and NADH-regenerating systems, the G513Y variant can be used to catalyze denitration of 4nitrophenol at 45 °C. Structure/sequence alignments of HadA and its homologues indicate that several flavin-dependent monooxygenases also contain amino acid residues homologous to the G513 of HadA, hence opening up the possibility of applying this engineering approach to improving their thermostabilities as well. Molecular dynamics (MD) simulations confirmed that the improved thermostability of the G513Y variant was due to aromatic hydrocarbon interactions between Y513 and N359, L347, G348, and F349.
HadA 是一种黄素依赖性单加氧酶，能够催化多种有毒物质（如农药）的脱硝和脱卤反应。尽管这些酶促反应在生物修复或生物催化方面很有用，但由于 HadA 的热稳定性较低，目前还无法将其应用于这些目的。在这项工作中，我们通过使用结构、计算机和合理的方法来设计 HadA 以提高其热稳定性。解析 HadA 的 X 射线结构，获得可靠的三维蛋白质模型，进一步预测耐热变体。通过使用两个生物信息学工具-FireProt 和 Disulfide by Design-进行的计算机分析表明，有 102 个变体，然后我们进一步应用合理的标准进行细化，包括特定残基的位置及其附近的相互作用，以及其他生物物理参数，以将列表缩小到六个候选者。G513Y 变体被发现是一个优化的工程候选者，因为它相对于野生型酶具有显著提高的稳定性和相当的活性。G513Y 的半衰期（50°C）是野生型酶的 72 倍，半衰期（45°C）是野生型酶的 160 倍。与黄素还原和 NADH 再生系统的热稳定反应结合，G513Y 变体可用于在 45°C 下催化 4-硝基苯酚的脱硝反应。HadA 及其同源物的结构/序列比对表明，几种黄素依赖性单加氧酶也含有与 HadA 的 G513 同源的氨基酸残基，因此有可能应用这种工程方法来提高它们的热稳定性。分子动力学（MD）模拟证实，G513Y 变体的热稳定性提高是由于 Y513 与 N359、L347、G348 和 F349 之间的芳烃相互作用所致。
