Departament de Química, Universitat Autònoma de Barcelona , Edifici C.n., 08193 Cerdanyola del Vallés, Barcelona, Spain.
J Am Chem Soc. 2014 Nov 5;136(44):15676-83. doi: 10.1021/ja508258t. Epub 2014 Oct 28.
An artificial imine reductase results upon incorporation of a biotinylated CpIr moiety (Cp = C5Me5(-)) within homotetrameric streptavidin (Sav) (referred to as CpIr(Biot-p-L)Cl] ⊂ Sav). Mutation of S112 reveals a marked effect of the Ir/streptavidin ratio on both the saturation kinetics as well as the enantioselectivity for the production of salsolidine. For [CpIr(Biot-p-L)Cl] ⊂ S112A Sav, both the reaction rate and the selectivity (up to 96% ee (R)-salsolidine, kcat 14-4 min(-1) vs [Ir], KM 65-370 mM) decrease upon fully saturating all biotin binding sites (the ee varying between 96% ee and 45% ee R). In contrast, for [Cp*Ir(Biot-p-L)Cl] ⊂ S112K Sav, both the rate and the selectivity remain nearly constant upon varying the Ir/streptavidin ratio [up to 78% ee (S)-salsolidine, kcat 2.6 min(-1), KM 95 mM]. X-ray analysis complemented with docking studies highlight a marked preference of the S112A and S112K Sav mutants for the SIr and RIr enantiomeric forms of the cofactor, respectively. Combining both docking and saturation kinetic studies led to the formulation of an enantioselection mechanism relying on an "induced lock-and-key" hypothesis: the host protein dictates the configuration of the biotinylated Ir-cofactor which, in turn, by and large determines the enantioselectivity of the imine reductase.
在同三聚体链霉亲和素（Sav）（称为 CpIr（Biot-p-L）Cl]⊂Sav）中加入生物素化 CpIr 部分，得到人工亚胺还原酶。突变 S112 揭示了 Ir/链霉亲和素比值对产生沙尔索丁的饱和动力学以及对映选择性都有显著影响。对于[CpIr（Biot-p-L）Cl]⊂S112A Sav，当完全饱和所有生物素结合位点时，反应速率和选择性（最高 96%ee（R）-沙尔索丁，kcat 14-4 min-1 与[Ir]相比，KM 65-370 mM）都会降低（ee 在 96%ee 和 45%ee R 之间变化）。相比之下，对于[CpIr（Biot-p-L）Cl]⊂S112K Sav，当改变 Ir/链霉亲和素比值时，速率和选择性几乎保持不变[最高 78%ee（S）-沙尔索丁，kcat 2.6 min-1，KM 95 mM]。X 射线分析与对接研究相结合，突出了 S112A 和 S112K Sav 突变体对辅因子的 SIr 和 RIr 对映体形式的明显偏好。将对接和饱和动力学研究相结合，提出了一种对映选择性机制，该机制依赖于“诱导锁钥”假说：宿主蛋白决定了生物素化 Ir 辅因子的构象，而辅因子的构象在很大程度上决定了亚胺还原酶的对映选择性。
