Structural studies of Baeyer-Villiger monooxygenases

Baeyer-Villiger 单加氧酶的结构研究

• 批准号: RGPIN-2020-04270
• 负责人: Berghuis, Albert
• 金额: $ 3.06万
• 依托单位: McGill University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=744063
• 关键词: Structural; studies; Baeyer; Villiger; monooxygenases

The Baeyer-Villiger monooxygenases (BMVOs) are flavin enzymes that catalyze the conversion of cyclic ketones into lactones. These enzymes offer exquisite regio- and enantio-specificity, while acting on a wide range of substrates. Furthermore, they only use NADPH and oxygen as co-substrates, and produce NADP+ and water as by products. These properties make BVMOs ideal for use as industrial biocatalysts. For the past 10+ years, we have pursued structure-function studies of BVMOs. Thus far, we determined the three-dimensional structure for the archetypal BVMO: cyclohexanone mooxygenase (CHMO). We revealed the structural details of substrate/product binding, and modeled the Criegee transition state. These studies allowed for a detailed mechanism of catalysis to be postulated, and rationalize the structural basis for regio- and enantio-specificity for a subset of substrates. While our research has revealed much about the structural basis for BVMO catalysis, many details are still unknown. In the coming five years, we plan to examine the following three aspects: Substrate binding We have thus far only examined 6-membered cyclic rings as substrates, and it is unclear how larger substrates are bound. For these substrates, the structural basis for regio- and enantio-specificity also remains to be resolved. We will therefore pursue studies of polycyclic substrates/products in complex with CHMO. Also, the location of the oxygen binding site is not known. Furthermore, the route by which oxygen reaches this site is a matter of speculation. We will address these questions using computational approaches together with structure-function studies. Conformational flexibility BVMOs adopt different conformations during the catalytic cycle. We have begun to probe the relevance of conformational flexibility for catalysis, and note that flexibility has a profound impact on substrate binding and catalytic efficiency. However, the structural basis for this impact is unknown. We will use site-directed mutagenesis, kinetic studies, structural biology and biophysical approaches to explore this. Catalytic activity In addition to converting cyclic ketones into lactones, BVMOs are able to oxygenate heteroatoms, such as the enantio-selective conversion of sulfides to their corresponding sulfoxides. However, the structural basis for this catalytic activity has not been examined. Thus, we plan to perform structural studies of CHMO in complex with various heteroatom containing substrates/products, to shed light on this catalytic activity of BVMOs. Combined, these studies will significantly advance our understanding for the structural basis of BVMO mediated catalysis and aid in the rational design of biocatalyst for Baeyer-Villiger reactions that can be used in green-chemistry focused industrial applications.

Baeyer-Villiger单加氧酶(BMVO)是一种黄素酶，催化环酮转化为内酯。这些酶提供了精致的区域和对映体特异性，同时作用于广泛的底物。此外，它们只使用NADPH和氧气作为共基质，并产生NADP+和水作为副产物。这些特性使BVMO成为工业生物催化剂的理想选择。在过去的10多年里，我们一直在进行BVMO的结构和功能研究。到目前为止，我们确定了BVMO的原型：环己酮加氧酶(CHMO)的三维结构。我们揭示了底物/产物结合的结构细节，并模拟了Criegee过渡态。这些研究可以推测催化的详细机制，并使部分底物的区域和对映体专一性的结构基础合理化。虽然我们的研究已经揭示了BVMO催化的结构基础，但许多细节仍然未知。在接下来的五年里，我们计划研究以下三个方面：底物结合我们到目前为止只研究了六元环作为底物，还不清楚更大的底物是如何结合的。对于这些底物，区域和对映体特异性的结构基础还有待解决。因此，我们将继续研究与CHMO形成络合物的多环底物/产物。此外，氧结合部位的位置也是未知的。此外，氧气到达这个地点的路线也是一个猜测。我们将使用计算方法和结构-功能研究来解决这些问题。构象柔性BVMO在催化循环中采用不同的构象。我们已经开始探索构象柔性与催化的相关性，并注意到柔性对底物结合和催化效率有深远的影响。然而，这种影响的结构基础尚不清楚。我们将使用定点突变、动力学研究、结构生物学和生物物理方法来探索这一点。催化活性除了将环酮转化为内酯外，BVMO还能够氧化杂原子，例如将硫化物对映体选择性转化为相应的亚硫醚。然而，这种催化活性的结构基础还没有被研究。因此，我们计划对CHMO与各种含杂原子底物/产物的络合物进行结构研究，以阐明BVMO的这种催化活性。总而言之，这些研究将极大地促进我们对BVMO介导的催化的结构基础的理解，并有助于合理设计可用于绿色化学重点工业应用的Baeyer-Villiger反应的生物催化剂。