Abstract:
Baeyer-Villiger monooxygenases (BVMOs) are NAD(P)H-dependent flavoproteins that convert ketones to esters and lactones. While these enzymes offer an appealing alternative to traditional Baeyer-Villiger oxidations, these proteins tend to be either too unstable or exhibit too narrow of a substrate scope for implementation as industrial biocatalysts. Here, sequence similarity networks were used to search for novel BVMOs that are both stable and promiscuous. Our genome mining led to the identification of an enzyme from Chloroflexota bacterium (strain G233) dubbed ssnBVMO that exhibits i) the highest melting temperature of any naturally sourced BVMO (62.5 ºC), ii) a remarkable kinetic stability across a wide range of conditions, similar to those of PAMO and PockeMO, iii) optimal catalysis at 50 °C, and iv) a broad substrate scope that includes linear aliphatic, aromatic, and sterically bulky ketones. Subsequent quantitative assays using propiophenone demonstrated >95% conversion. Several fusions were also constructed that linked ssnBVMO to a thermostable phosphite dehydrogenase. These fusions can recycle NADPH and catalyze oxidations with sub-stoichiometric quantities of this expensive cofactor. Characterization of these fusions permitted identification of PTDH-L1-ssnBVMO as the most promising protein that could have utility as a seed sequence for enzyme engineering campaigns aiming to develop biocatalysts for Baeyer-Villiger oxidations.
摘要:
Baeyer-Villiger单加氧酶(BVMO)是NAD(P)H依赖性黄素蛋白,可将酮转化为酯和内酯。虽然这些酶为传统的Baeyer-Villiger氧化提供了一个有吸引力的替代品,这些蛋白质倾向于要么太不稳定,要么表现出作为工业生物催化剂的底物范围太窄。这里,序列相似性网络用于搜索既稳定又混杂的新型BVMO。我们的基因组挖掘导致鉴定了来自Chloroflexota细菌(菌株G233)的一种酶,称为ssnBVMO,该酶表现出i)任何天然来源的BVMO的最高解链温度(62.5ºC),ii)在广泛的条件下具有显著的动力学稳定性,类似于PAMO和PockeMO,iii)在50°C下的最佳催化,和iv)广泛的底物范围,包括线性脂肪族,芳香,和空间庞大的酮。随后的使用苯丙酮的定量测定证明了>95%的转化率。还构建了几种融合体,将ssnBVMO连接到热稳定的亚磷酸盐脱氢酶。这些融合物可以再循环NADPH并且用亚化学计量量的这种昂贵的辅因子催化氧化。这些融合体的表征允许将PTDH-L1-ssnBVMO鉴定为最有前途的蛋白质,该蛋白质可用作旨在开发Baeyer-Villiger氧化生物催化剂的酶工程活动的种子序列。