Rational design of a cyclohexanone dehydrogenase for enhanced a,ß-desaturation and substrate specificity
Rational design of a cyclohexanone dehydrogenase for enhanced a,ß-desaturation and substrate specificity
复制标题
合理设计环己酮脱氢酶以增强α，β-去饱和度和底物特异性
DOI：
10.1039/d3sc04009g
复制
发表时间：
2024
期刊：
影响因子：
8.4
通讯作者：
Singh W
中科院分区：
文献类型：
--
作者：
Singh W
环状羰基化合物存在于许多类固醇和生物活性分子的核心中，利用绿色化学对其进行选择性α，β去饱和化是非常可取的。为了实现这一任务，我们首次描述并解决了环己酮脱氢酶类酶的从头结构。通过对脱氮耐热杆菌的环己酮脱氢酶与环酮、内酯和内酰胺类化合物的比对，考察了底物专一性的广度。为了研究底物结合，生成了催化变异体Y195F，并用于获得与天然底物环己酮的结晶学络合物。这揭示了底物-活性位点的相互作用，以及辅因子黄素腺嘌呤二核苷酸的接近，并使我们能够提出关键氨基酸的机械功能。然后，我们使用分子动力学模拟来指导设计，以增加环己酮脱氢酶的功能。由此产生的W113A变异体总体上提高了酶活性和底物范围，即接受更大的羰基化合物二氢香豆素。对W113A变异体的结构分析揭示了一个更广泛、更开放的活性部位，这有助于解释修饰底物的特异性。这项工作为未来通过合理的酶工程合成重要生物活性分子的定制区域选择性α，β去饱和化铺平了道路。
The selective α,β-desaturation of cyclic carbonyl compounds, which are found in the core of many steroid and bioactive molecules, using green chemistry is highly desirable. To achieve this task, we have for the first time described and solved the de novo structure of a member of the cyclohexanone dehydrogenase class of enzymes. The breadth of substrate specificity was investigated by assaying the cyclohexanone dehydrogenase, from Alicycliphilus denitrificans, against several cyclic ketones, lactones and lactams. To investigate substrate binding, a catalytic variant, Y195F, was generated and used to obtain a crystallographic complex with the natural substrate, cyclohexanone. This revealed substrate–active site interactions, as well as the proximity of the cofactor, flavin adenine dinucleotide, and enabled us to propose a mechanistic function to key amino acids. We then used molecular dynamic simulations to guide design to add functionality to the cyclohexanone dehydrogenase enzyme. The resulting W113A variant had overall improved enzyme activity and substrate scope, i.e., accepting the bulkier carbonyl compound, dihydrocoumarin. Structural analysis of the W113A variant revealed a broader, more open active site, which helped explain the modified substrate specificity. This work paves the way for future bespoke regioselective α,β-desaturation in the synthesis of important bioactive molecules via rational enzyme engineering.