A novel ketone reductase KaKR was identified from Kaistia algarum through genome mining with high activity and enantioselectivity toward 3-quinuclidione. Sequence alignment and conserved motifs analysis revealed that KaKR was classified into classical short-chain dehydrogenase/reductase family. KaKR displayed the highest activity at pH 7.0 and 50°C. Substrate spectrum analysis revealed that KaKR exhibited high substrate specificity toward N-heterocyclic ketones. The k_cat/K_M toward 3-quinuclidione and NADH were 82.8 and 1697 s^–1⋅mM^–1. KaKR is a robust biocatalyst and could tolerate high concentrations of 3-quinuclidione. Without assistance of external cofactor, as high as 5.0 M 3-quinuclidione could be completely and enantioselectivity reduced into (R)-quinuclidinol with ee of >99.9%, S/C of 42.6 g⋅g^–1 and space-time yield of 1027 g⋅L^–1⋅d^–1. Alanine scanning was performed to elucidate the molecular mechanism of the high substrate specificity and enantioselectivity. Two critical residues, V94 and E198, were identified with important roles in determining the catalytic efficiency and enantioselectivity. This study provides a novel and robust ketone reductase, and establishes an efficient and economic bioprocess for the synthesis of (R)-3-quinuclidinol without addition of external cofactor.

通过基因组挖掘从Kaistia algarum中鉴定出一种新的酮还原酶Ka KR，该酶对 3-奎宁二酮具有高活性和对映选择性。序列比对和保守基序分析表明，Ka KR 属于经典的短链脱氢酶/还原酶家族。Ka KR 在 pH 7.0 和 50°C 下表现出最高的活性。底物光谱分析表明，Ka KR 对N-杂环酮表现出高底物特异性。3-奎宁二酮和 NADH的k _cat / K _M分别为 82.8 和 1697 s ^–1 ⋅mM ^–1。K aKR 是一种强大的生物催化剂，可以耐受高浓度的 3-奎宁二酮。在没有外部辅助因子的帮助下，高达 5.0 M 的 3-quinuclidione 可以被完全和对映选择性还原为 ( R )-quinuclidinol，ee > 99.9%，S/C 为 42.6 g⋅g ^–1，时空产率为 1027 g⋅L ^–1 ⋅d ^–1。进行丙氨酸扫描以阐明高底物特异性和对映选择性的分子机制。确定了两个关键残基 V94 和 E198，它们在确定催化效率和对映选择性方面具有重要作用。该研究提供了一种新型且稳健的酮还原酶，并建立了一种高效且经济的生物工艺来合成（R )-3-奎宁醇，无需添加外部辅助因子。