由于一系列挑战,分子建模在金属酶设计中的应用仍然很少。最重要的是,金属介导的结合的模拟和催化主管几何形状的识别需要大量的构象探索和精细电子性质的模拟。这里,我们展示了如何将新工具纳入多尺度战略,即底物扩散勘探,允许更进一步。作为展示,基于Rh2的人工环丙烷酶(GSH,HFF和RFY)由刘易斯和同事(NAT。Commun.,2015、6、7789和NAT。Chem.,2018,10,318-324)已经合理化。自由辅因子介导过程的DFT计算将卡宾插入和环丙烷形成确定为关键事件,后者是对映确定步骤,它显示多达8个竞争方向,很容易被蛋白质环境改变。将反应的关键中间体对接到蛋白质支架中,显示一些突变的残基与辅因子和/或共底物具有直接相互作用。这些相互作用采取Rh在GSH和HFF中的直接配位以及在RFY中与卡宾部分的强疏水斑块的形式。后分子动力学维持辅因子诱导蛋白质的整体重排。最后,对基质扩散的大规模探索,基于GPathFinder方法,将此事件定义为GSH和RFY中对映选择性的起源。对于HFF,精细的分子对接表明,它可能与扩散时的局部相互作用有关。这项工作表明,如何对金属酶的催化谱进行远距离突变建模可能是不可避免的,并且应应用模拟底物扩散的软件。
Molecular modelling applications in metalloenzyme design are still scarce due to a series of challenges. On top of that, the simulations of metal-mediated binding and the identification of catalytic competent geometries require both large conformational exploration and simulation of fine electronic properties. Here, we demonstrate how the incorporation of new tools in multiscale strategies, namely substrate diffusion exploration, allows taking a step further. As a showcase, the enantioselective profiles of the most outstanding variants of an artificial Rh2-based cyclopropanase (GSH, HFF and RFY) developed by Lewis and co-workers (Nat. Commun., 2015, 6, 7789 and Nat. Chem., 2018, 10, 318-324) have been rationalized. DFT calculations on the free-cofactor-mediated process identify the carbene insertion and the cyclopropanoid formation as crucial events, the latter being the enantiodetermining step, which displays up to 8 competitive orientations easily altered by the protein environment. The key intermediates of the reaction were docked into the protein scaffold showing that some mutated residues have direct interaction with the cofactor and/or the co-substrate. These interactions take the form of a direct coordination of Rh in GSH and HFF and a strong hydrophobic patch with the carbene moiety in RFY. Posterior molecular dynamics sustain that the cofactor induces global re-arrangements of the protein. Finally, massive exploration of substrate diffusion, based on the GPathFinder approach, defines this event as the origin of the enantioselectivity in GSH and RFY. For HFF, fine molecular dockings suggest that it is likely related to local interactions upon diffusion. This work shows how modelling of long-range mutations on the catalytic profiles of metalloenzymes may be unavoidable and software simulating substrate diffusion should be applied.