Jongkind Ewald P J, Domenech Jack, Govers Arthur, van den Broek Marcel, Daran Jean-Marc, Grogan Gideon, Paul Caroline E
Department of Biotechnology, Delft University of Technology, van der Maasweg 9, 2629 HZ Delft, The Netherlands.
York Structural Biology Laboratory, Department of Chemistry, University of York, Heslington, York YO10 5DD, United Kingdom.
ACS Catal. 2024 Dec 16;15(1):211-219. doi: 10.1021/acscatal.4c04935. eCollection 2025 Jan 3.
Reductive amination is one of the most synthetically direct routes to access chiral amines. Several Imine Reductases (IREDs) have been discovered to catalyze reductive amination (Reductive Aminases or RedAms), yet they are dependent on the expensive phosphorylated nicotinamide adenine dinucleotide cofactor NADPH and usually more active at basic pH. Here, we describe the discovery and synthetic potential of an IRED from (RedAm) that catalyzes reductive amination between a series of medium to large carbonyl and amine compounds with conversions of up to >99% and 99% enantiomeric excess at neutral pH. RedAm catalyzes the formation of a substituted γ-lactam and -methyl-1-phenylethanamine with stereochemistry opposite to that of fungal RedAms, giving the ()-enantiomer. This enzyme remarkably uses both NADPH and NADH cofactors with values of 15 and 247 μM and turnover numbers of 3.6 and 9.0 s, respectively, for the reductive amination of hexanal with allylamine. The crystal structure obtained provides insights into the flexibility to also accept NADH, with residues R35 and I69 diverging from that of other IREDs/RedAms in the otherwise conserved Rossmann fold. RedAm thus represents a subfamily of enzymes that enable synthetic applications using NADH-dependent reductive amination to access complementary chiral amine products.
还原胺化反应是获取手性胺类化合物最直接的合成途径之一。人们已经发现了几种亚胺还原酶（IREDs）可催化还原胺化反应（还原氨基酶或RedAms），但它们依赖于昂贵的磷酸化烟酰胺腺嘌呤二核苷酸辅因子NADPH，并且通常在碱性pH条件下活性更高。在此，我们描述了一种来自[具体来源未给出]的IRED（RedAm）的发现及其合成潜力，该酶可催化一系列中到大尺寸的羰基化合物与胺类化合物之间的还原胺化反应，在中性pH条件下转化率高达>99%，对映体过量值为99%。RedAm催化形成一种取代的γ-内酰胺和（）-甲基-1-苯基乙胺，其立体化学与真菌RedAms相反，生成（）-对映体。对于己醛与烯丙胺的还原胺化反应，该酶显著地同时使用NADPH和NADH作为辅因子，其Km值分别为15和247μM，周转数（kcat）分别为3.6和9.0 s⁻¹。所获得的晶体结构揭示了其能够接受NADH的灵活性，在其他方面保守的Rossmann折叠中，残基R35和I69与其他IREDs/RedAms不同。因此，RedAm代表了一类酶亚家族，它们能够利用依赖NADH的还原胺化反应进行合成应用，以获得互补的手性胺产物。
