Structural studies of Baeyer-Villiger monooxygenases

Baeyer-Villiger单加氧酶（BMVOs）是黄素酶，催化环酮转化为内酯。这些酶提供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和氧作为共底物，产生NADP+和水作为副产物。这些特性使BVMOs成为工业生物催化剂的理想选择。

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.