Department of Biotechnology and Enzyme Catalysis, Institute of Biochemistry, Greifswald University, Felix-Hausdorff-Str, 4, D-17487 Greifswald, Germany.
AMB Express. 2011 Jun 23;1(1):13. doi: 10.1186/2191-0855-1-13.
Three different Baeyer-Villiger monooxygenases (BVMOs) were reported to be involved in the camphor metabolism by Pseudomonas putida NCIMB 10007. During (+)-camphor degradation, 2,5-diketocamphane is formed serving as substrate for the 2,5-diketocamphane 1,2-monooxygenase. This enzyme is encoded on the CAM plasmid and depends on the cofactors FMN and NADH and hence belongs to the group of type II BVMOs. We have cloned and recombinantly expressed the oxygenating subunit of the 2,5-diketocamphane 1,2-monooxygenase (2,5-DKCMO) in E. coli followed by His-tag-based affinity purification. A range of compounds representing different BVMO substrate classes were then investigated, but only bicyclic ketones were converted by 2,5-DKCMO used as crude cell extract or after purification. Interestingly, also (-)-camphor was oxidized, but conversion was about 3-fold lower compared to (+)-camphor. Moreover, activity of purified 2,5-DKCMO was observed in the absence of an NADH-dehydrogenase subunit.
三种不同的 Baeyer-Villiger 单加氧酶（BVMO）被报道参与假单胞菌属 NCIMB 10007 中的樟脑代谢。在（+）-樟脑降解过程中，形成 2,5-二酮莰烷作为 2,5-二酮莰烷 1,2-单加氧酶的底物。该酶编码在 CAM 质粒上，依赖黄素单核苷酸（FMN）和烟酰胺腺嘌呤二核苷酸（NADH）等辅酶，因此属于 II 型 BVMO 酶。我们已在大肠杆菌中克隆和重组表达了 2,5-二酮莰烷 1,2-单加氧酶（2,5-DKCMO）的加氧酶亚基，并进行了基于 His 标签的亲和纯化。然后，我们研究了一系列代表不同 BVMO 底物类别的化合物，但只有二环酮被用作粗细胞提取物或纯化后的 2,5-DKCMO 转化。有趣的是，（-）-樟脑也被氧化，但转化率比（+）-樟脑低约 3 倍。此外，在没有 NADH 脱氢酶亚基的情况下，也观察到纯化的 2,5-DKCMO 的活性。
