Sustainable Process Technologies, Faculty of Engineering, University of Nottingham, Nottingham, UK.
Faraday Discuss. 2022 May 18;234(0):315-335. doi: 10.1039/d1fd00070e.
Artificial metalloenzymes (ArMs) confer non-biological reactivities to biomolecules, whilst taking advantage of the biomolecular architecture in terms of their selectivity and renewable origin. In particular, the design of ArMs by the supramolecular anchoring of metal catalysts to protein hosts provides flexible and easy to optimise systems. The use of cofactor dependent enzymes as hosts gives the advantage of both a (hydrophobic) binding site for the substrate and a cofactor pocket to accommodate the catalyst. Here, we present a computationally driven design approach of ArMs for the transfer hydrogenation reaction of cyclic imines, starting from the NADP-dependent alcohol dehydrogenase from (TbADH). We tested and developed a molecular docking workflow to define and optimize iridium catalysts with high affinity for the cofactor binding site of TbADH. The workflow uses high throughput docking of compound libraries to identify key structural motifs for high affinity, followed by higher accuracy docking methods on smaller, focused ligand and catalyst libraries. Iridium sulfonamide catalysts were selected and synthesised, containing either a triol, a furane, or a carboxylic acid to provide the interaction with the cofactor binding pocket. IC values of the resulting complexes during TbADH-catalysed alcohol oxidation were determined by competition experiments and were between 4.410 mM and 0.052 mM, demonstrating the affinity of the iridium complexes for either the substrate or the cofactor binding pocket of TbADH. The catalytic activity of the free iridium complexes in solution showed a maximal turnover number (TON) of 90 for the reduction of salsolidine by the triol-functionalised iridium catalyst, whilst in the presence of TbADH, only the iridium catalyst with the triol anchoring functionality showed activity for the same reaction (TON of 36 after 24 h). The observation that the artificial metalloenzymes developed here lacked stereoselectivity demonstrates the need for the further investigation and optimisation of the ArM. Our results serve as a starting point for the design of robust artificial metalloenzymes, exploiting supramolecular anchoring to natural NAD(P)H binding pockets.
人工金属酶（ArMs）使生物分子具有非生物反应性，同时利用生物分子的结构在选择性和可再生性方面的优势。特别是，通过将金属催化剂通过超分子锚定到蛋白质宿主上来设计 ArMs，可以提供灵活且易于优化的系统。使用辅因子依赖性酶作为宿主具有两个优点：一个是（疏水性）底物结合位点，另一个是容纳催化剂的辅因子口袋。在这里，我们从依赖 NADP 的醇脱氢酶（TbADH）出发，提出了一种用于环状亚胺转移氢化反应的 ArM 的计算驱动设计方法。我们测试并开发了一种分子对接工作流程，以定义和优化与 TbADH 辅因子结合位点具有高亲和力的铱催化剂。该工作流程使用高通量对接化合物库来识别高亲和力的关键结构基序，然后在较小的、重点的配体和催化剂库上使用更高精度的对接方法。选择并合成了铱磺酰胺催化剂，其中包含三醇、呋喃或羧酸，以提供与辅因子结合口袋的相互作用。通过竞争实验确定了在 TbADH 催化的醇氧化过程中生成的配合物的 IC 值在 4.410 mM 至 0.052 mM 之间，这表明铱配合物对 TbADH 的底物或辅因子结合口袋的亲和力。在游离铱配合物在溶液中的催化活性中，三醇功能化的铱催化剂将 salsolidine 还原的最大周转数（TON）显示为 90，而在 TbADH 存在下，只有具有三醇锚定功能的铱催化剂对相同反应表现出活性（24 小时后 TON 为 36）。这里开发的人工金属酶缺乏立体选择性的观察结果表明需要进一步研究和优化 ArM。我们的结果为利用超分子锚定天然 NAD(P)H 结合口袋设计稳健的人工金属酶提供了起点。
