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）是黄素酶，可促进环状酮转化为内酯。这些酶在作用于各种底物的同时提供精美的区域和对映体特异性。此外，它们仅将NADPH和氧气用作共蛋白酶，并产生NADP+和水与产品一样。这些特性使BVMOS理想用作工业生物催化剂。在过去的10年中，我们从事BVMO的结构功能研究。到目前为止，我们确定了原型BVMO的三维结构：环己酮Mooxygongenase（CHMO）。我们揭示了底物/产品结合的结构细节，并建模了Criegee过渡状态。这些研究允许假定详细的催化机制，并合理化了底物子集的区域和对映体特异性的结构基础。尽管我们的研究揭示了BVMO催化的结构性基础，但许多细节仍然未知。在接下来的五年中，我们计划检查以下三个方面：底物结合到迄今为止，我们仅检查了6元的循环环作为底物，目前尚不清楚较大的底物如何结合。对于这些底物，区域和对映体特异性的结构基础也尚待解决。因此，我们将研究与CHMO复合物中的多环基底物/产品的研究。同样，氧结合位点的位置尚不清楚。此外，氧气到达该部位的途径是猜测的问题。我们将使用计算方法以及结构功能研究来解决这些问题。构象柔韧性BVMO在催化周期中采用不同的构象。我们已经开始探究构象灵活性在催化中的相关性，并注意柔韧性对底物结合和催化效率产生了深远的影响。但是，这种影响的结构基础尚不清楚。我们将使用位置定向的诱变，动力学研究，结构生物学和生物物理方法来探索这一点。除了将环状酮转化为内酯外，BVMO还能够氧化杂原子，例如将硫化物的对映体选择性转化为相应的亚氧化物。但是，尚未检查这种催化活性的结构基础。因此，我们计划与包含底物/产物的各种杂种的CHMO进行CHMO的结构研究，以阐明BVMO的这种催化活性。结合在一起，这些研究将大大提高我们对BVMO介导的催化的结构基础的理解，并有助于为Baeyer-Villiger反应的生物催化剂的合理设计，这些反应可用于以绿色化学为中心的工业应用。