Chiral non--amino acids are highly marketable compounds. However, present enzymatic synthesis predominantly targets the generation of - and amino acids, lacking biosynthetic routes for non-α-amino acids with amino substitutions at more remote positions due to specific polar interactions with substrate's carboxyl group. Substrate engineering via esterification is a crucial method for carboxyl group modification. Consequently, keto esters were selected as substrates, and asymmetric reductive amination of these esters with varying substitution positions was accomplished using amine dehydrogenase from AmDH The strategy of coupling substrate engineering with enzyme evolution was applied to enhance the compatibility between substrate and enzyme, thereby increasing the activity on and sters by 13, 8, 9, and 40 times, respectively. Molecular dynamics simulations and kinetic parameters analysis revealed that substrate engineering and enzyme evolution boost nonpolar interactions and improve substrate binding affinity within the enzyme's active site. Ultimately, diverse non--amino acids was synthesized employing exceptional mutants, yielding substantial conversion and enantioselectivity exceeding 99 %. The synthesis encompassed andamino acids, as well as amino acids with more distal substitution positions. Consequently, an eco-friendly, enzymatic-chemical cascade for the synthesis of non--amino acids with distinct substitution patterns was successfully established.

中文翻译：

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构建涉及工程胺脱氢酶的酶化学级联，用于通过底物工程和酶进化合成手性非α-氨基酸

手性非氨基酸是极具市场价值的化合物。然而，目前的酶促合成主要针对α-和氨基酸的生成，缺乏由于与底物羧基的特定极性相互作用而在更远位置进行氨基取代的非α-氨基酸的生物合成途径。通过酯化进行底物工程是羧基修饰的重要方法。因此，选择酮酯作为底物，并使用AmDH的胺脱氢酶完成这些具有不同取代位置的酯的不对称还原胺化。采用底物工程与酶进化耦合的策略来增强底物与酶之间的相容性，从而提高底物与酶之间的相容性。和 的活动分别为 13、8、9 和 40 次。分子动力学模拟和动力学参数分析表明，底物工程和酶进化增强了非极性相互作用，并提高了酶活性位点内的底物结合亲和力。最终，利用特殊的突变体合成了多种非氨基酸，产生了显着的转化率和超过 99% 的对映选择性。合成涵盖氨基酸以及具有更远端取代位置的氨基酸。因此，成功建立了一种生态友好的酶化学级联，用于合成具有不同取代模式的非氨基酸。