Xing Jinming, Orderley Georgie, Bradshaw Allen Ruth T, Ahmad Nabieha N, Gourjault Camille, Akgul Ardil, Alhassan Sephia O, Ngernanek Nichapa, Salke Siddhika, Aleku Godwin A
Institute of Pharmaceutical Science, Franklin-Wilkins Building, King's College London, 150 Stamford Street, London SE1 9NH, United Kingdom.
JACS Au. 2025 Jun 27;5(7):3468-3482. doi: 10.1021/jacsau.5c00512. eCollection 2025 Jul 28.
Enzymatic reductive amination allows direct and stereoselective access to 1°, 2°, and 3° chiral amines under environmentally friendly reaction conditions. Enzyme discovery and engineering campaigns for this important transformation are crucial for industrial applications but currently rely on tedious and time-consuming screening of large libraries using expensive LC/LC-MS systems. Such engineering campaigns have also focused on optimizing a single aminase candidate per defined synthetic target. In this work, we have developed a versatile high-throughput (HTP) spectrophotometric/colorimetric assay for rapid and reliable quantitative monitoring of the aminase activity of recombinant aminase-expressing cells or unpurified cell extracts. The assay couples aminase product formation to an amine oxidase-HRP reporter system, yielding a colored dye/signal that can be monitored at 492/498 nm. We demonstrate its application to quantitatively monitor reductive amination reactions catalyzed by reductive aminases (RedAms), amine dehydrogenases (AmDHs), and amino acid dehydrogenases (AADHs). The assay enabled the HTP screening of 56 site saturation libraries in two RedAms, revealing 10 positions, including N113, D135, D188, Y196, W227, T234, Q257, A262, S270, and D297 in RedAm as important residues for RedAms' catalytic function/substrate specificity. Extending our screening to four substrate combinations enabled the identification of positions R53, T115, Y154, L189, M195, Y196, W227, and Q257 in RedAm (and equivalent positions: R35, T99, Y139, L174, M180, Y181, W211, and Q241 in RedAm) that yielded mutants with improved activity of up to 7-fold compared to the wild-type enzyme in at least three of the four transformations. We propose that these residues act as potential "universal" hotspots for engineering substrate specificity in these enzymes across diverse substrates. Our work lays an important foundation for mapping sequence-activity relationships in RedAms at the enzyme family level to pave the way for more predictable, faster, and cost-effective engineering of RedAms for applications.
酶促还原胺化反应能够在环境友好的反应条件下直接且立体选择性地合成伯、仲和叔手性胺。针对这一重要转化的酶发现和工程改造活动对于工业应用至关重要，但目前依赖于使用昂贵的液相色谱/液相色谱-质谱系统对大型文库进行繁琐且耗时的筛选。此类工程改造活动还专注于针对每个确定的合成目标优化单一的氨基酶候选物。在这项工作中，我们开发了一种通用的高通量（HTP）分光光度法/比色法，用于快速、可靠地定量监测表达重组氨基酶的细胞或未纯化的细胞提取物的氨基酶活性。该测定法将氨基酶产物的形成与胺氧化酶-辣根过氧化物酶（HRP）报告系统偶联，产生一种可在492/498 nm处监测的有色染料/信号。我们展示了其在定量监测由还原氨基酶（RedAms）、胺脱氢酶（AmDHs）和氨基酸脱氢酶（AADHs）催化的还原胺化反应中的应用。该测定法能够对两种RedAms中的56个位点饱和文库进行高通量筛选，揭示了RedAms中的10个位点，包括N113、D135、D188、Y196、W227、T234、Q257、A262、S270和D297，这些是RedAms催化功能/底物特异性的重要残基。将我们的筛选扩展到四种底物组合，使得能够鉴定出RedAms中的R53、T115、Y154、L189、M195、Y196、W227和Q257位点（以及RedAm中的等效位点：R35、T99、Y139、L174、M180、Y181、W211和Q241），这些位点产生的突变体在四种转化中的至少三种中，与野生型酶相比活性提高了7倍。我们提出这些残基作为潜在的“通用”热点，用于在这些酶中针对不同底物工程化底物特异性。我们的工作为在酶家族水平上绘制RedAms中的序列-活性关系奠定了重要基础，为更可预测、更快且更具成本效益的RedAms工程化应用铺平了道路。
