School of Chemistry, Main Building, Cardiff University, Cardiff, CF10 3AT, UK.
Department de Química Física i Analítica, Universitat Jaume I, Castellón, 12071, Spain.
Org Biomol Chem. 2021 Dec 8;19(47):10424-10431. doi: 10.1039/d1ob01947c.
Here, we combine the use of host screening, protein crystallography and QM/MM molecular dynamics simulations to investigate how the protein structure affects iminium catalysis by biotinylated secondary amines in a model 1,4 conjugate addition reaction. Monomeric streptavidin (M-Sav) lacks a quaternary structure and the solvent-exposed reaction site resulted in poor product conversion in the model reaction with low enantio- and regioselectivities. These parameters were much improved when the tetrameric host T-Sav was used; indeed, residues at the symmetrical subunit interface were proven to be critical for catalysis through a mutagenesis study. The use of QM/MM simulations and the asymmetric dimeric variant D-Sav revealed that both Lys121 residues which are located in the hosting and neighboring subunits play a critical role in controlling the stereoselectivity and reactivity. Lastly, the D-Sav template, though providing a lower conversion than that of the symmetric tetrameric counterpart, is likely a better starting point for future protein engineering because each surrounding residue within the asymmetric scaffold can be refined for secondary amine catalysis.
在这里，我们结合使用宿主筛选、蛋白质晶体学和 QM/MM 分子动力学模拟，研究蛋白质结构如何影响生物素化仲胺在模型 1,4 共轭加成反应中的亚胺催化作用。单体链霉亲和素（M-Sav）缺乏四级结构，溶剂暴露的反应位点导致模型反应中产物转化率低，对映选择性和区域选择性差。当使用四聚体宿主 T-Sav 时，这些参数得到了很大的改善；事实上，通过突变研究证明，对称亚基界面上的残基对于催化至关重要。使用 QM/MM 模拟和不对称二聚体变体 D-Sav 表明，位于宿主和相邻亚基中的两个 Lys121 残基在控制立体选择性和反应性方面起着关键作用。最后，尽管 D-Sav 模板的转化率低于对称四聚体模板，但它可能是未来蛋白质工程的更好起点，因为不对称支架内的每个周围残基都可以针对仲胺催化进行优化。
