Baeyer-Villiger单加氧酶非保守Hinge影响酶的催化活性和立体选择性  

Nonconserved hinge in Baeyer-Villiger monooxygenase affects catalytic activity and stereoselectivity

作　　者：梁秋玲[1,2] 吴胜[1] 

机构地区：[1]中国科学院微生物研究所微生物资源前期开发国家重点实验室,北京100101 [2]中国科学院大学,北京100049

出　　处：《生物工程学报》2015年第3期361-374,共14页Chinese Journal of Biotechnology

基　　金：国家自然科学基金(No.31070718);中国科学院微生物所基础前沿研究项目(No.KSCX2-EW-J-6)资助~~

摘　　要：Baeyer-Villiger单加氧酶是一种重要的生物催化剂,可用于合成一系列有价值的酯和内酯化合物。通过序列比对和晶体结构分析推测连接NADPH结构域和FAD结构域的一段非保守Hinge可能在酶对底物识别和催化氧化过程中扮演着重要角色。在以环己酮单加氧酶为模型的研究中发现,对该Hinge结构进行同源序列替换得到的突变体几乎完全丧失了催化活性,证明了其整体水平的重要性。丙氨酸扫描突变揭示其中一些位点对酶的功能有显著影响:K153位点的改变使酶的活性下降,立体选择性却更优化;L143位点的改变对酶的活性影响较小,却降低了立体选择性;L144位点的改变则同时大幅度削弱酶的活性和立体选择性。将同样的方法运用在苯丙酮单加氧酶中,我们得到了相似的结论,证明这些位点的重要功能在Baeyer-Villiger单加氧酶家族中有一定的普遍性。这一研究增进了对Baeyer-Villiger单加氧酶的结构与功能关系的认识,有助于底物结合口袋的精确描述和Baeyer-Villiger单加氧酶催化图景的进一步细化,对未来相关的理性设计和定向改造研究提供了借鉴。Baeyer-Villiger monooxygenases（BVMOs） are important biocatalysts to synthesize a series of valuable esters and lactones. Based on protein sequence alignment and crystal structure analysis, a nonconserved hinge which linked NADPH domain and FAD domain was speculated to play an important role in substrate recognition and catalytic oxidation process. Cyclohexanone monooxygenase（CHMO） was selected as a model. Mutants obtained by homologous replacement of the whole hinge almost completely lost its original catalytic activity, demonstrating that the overall hinge structure was of great importance. Some significant sites were identified to greatly affect the catalytic activity and stereoselectivity by alanine scanning mutagenesis, accompanied by enzyme activity assessments and chiral kinetic resolutions. Altering K153 decreased the activity of the enzyme but enhanced the stereoselectivity. Changing L143 site reduced stereoselectivity but had little effect on enzyme activity. Mutation at L144 site dramatically weakened both activity and stereoselectivity. Subsequently, these corresponding sites in phenylacetone monooxygenase were also illustrated to follow a similar rule, revealing a universal importance of these sites in the BVMO family. These results expanded our understanding of the structure-activity relationship of these enzymes and provided more proofs for future directed evolution of BVMOs.

关 键 词：Baeyer-Villiger单加氧酶 环己酮单加氧酶 同源替换 丙氨酸扫描突变 

分 类 号：Q814[生物学—生物工程]