Rotticci D, Rotticci-Mulder J C, Denman S, Norin T, Hult K
Department of Chemistry, Organic Chemistry, Royal Institute of Technology, 10044 Stockholm, Sweden.
Chembiochem. 2001 Oct 1;2(10):766-70. doi: 10.1002/1439-7633(20011001)2:10<766::AID-CBIC766>3.0.CO;2-K.
A model based on two different binding modes for alcohol enantiomers in the active site of a lipase allowed rational redesign of its enantioselectivity. 1-Halo-2-octanols were poorly resolved by Candida antarctica lipase B. Interactions between the substrates and the lipase were investigated with molecular modeling. Unfavorable interactions were found between the halogen moiety of the fast-reacting S enantiomer and a region situated at the bottom of the active site (stereoselectivity pocket). The lipase was virtually mutated in this region and energy contour maps of some variants displayed better interactions for the target substrates. Four selected variants of the lipase were produced and kinetic resolution experiments were undertaken with these mutants. Single point mutations gave rise to one variant with doubled enantioselectivity as well as one variant with annihilated enantioselectivity towards the target halohydrins. An increased volume of the stereoselectivity pocket caused a decrease in enantioselectivity, while changes in electrostatic potential increased enantioselectivity. The enantioselectivity of these new lipase variants towards other types of alcohols was also investigated. The changes in enantioselectivity caused by the mutations were well in agreement with the proposed model concerning the chiral recognition of alcohol enantiomers by this lipase.
基于脂肪酶活性位点中醇对映体的两种不同结合模式建立的模型，实现了其对映选择性的合理重新设计。南极假丝酵母脂肪酶B对1-卤代-2-辛醇的拆分效果不佳。采用分子模拟研究了底物与脂肪酶之间的相互作用。发现反应较快的S对映体的卤素部分与活性位点底部（立体选择性口袋）的一个区域之间存在不利相互作用。对该区域的脂肪酶进行虚拟突变，一些变体的能量等高线图显示出与目标底物有更好的相互作用。制备了脂肪酶的四个选定变体，并对这些突变体进行了动力学拆分实验。单点突变产生了一个对映选择性加倍的变体，以及一个对目标卤代醇对映选择性消失的变体。立体选择性口袋体积的增加导致对映选择性降低，而静电势的变化则提高了对映选择性。还研究了这些新的脂肪酶变体对其他类型醇的对映选择性。突变引起的对映选择性变化与所提出的关于该脂肪酶对醇对映体的手性识别模型非常吻合。
