Tetrahydrofuran-3-one (3TF) was regarded as a difficult-to-reduce ketone for carbonyl reductases due to its high stereosymmetry, result of which, the biosynthesis of chiral 3-hydroxytetrahydrofuran (3HTF), a key precursor of pharmaceuticals for treatment of HIV or diabetes, has been limited. Present study mined a robust carbonyl reductase CmCR from Candida metapsilosis with high activity towards 3TF using isopropanol as co-substrate (500 mM, 22 g/L/h space-time yield, 67% ee). Based on the orientation of substrate's etheryl oxygen in the substrate binding pocket, CmCR was rationally designed and two mutants MuR and MuS were screened out to completely reduce 3TF into (R)-HTF and (S)-HTF respectively with the highest 3TF loading and productivity (200 mM, 4.4 g/L/h space-time yield, 99% ee for MuR while 500 mM, 11 g/L/h space-time yield, 99% ee for MuS). The structural mechanism for the enhanced stereoselectivity was revealed that the mutagenesis changed the electrostatic potential surrounding the substrate entrance and promoted 3TF to approach, bind and form prereaction state with MuR or MuS in one certain direction, by which 3TF was converted into 3HTF with excellent optical purity. The success in this study provides a viable approach for rational design of carbonyl reductases with high enantioselectivity towards target substrates of high symmetry.

四氢呋喃-3-酮（3TF）因其高度立体对称性而被认为是羰基还原酶难以还原的酮，因此，生物合成手性3-羟基四氢呋喃（3HTF）是治疗糖尿病药物的关键前体。 HIV或糖尿病，已受到限制。目前的研究使用异丙醇作为共底物（500 mM，22 g/L/h 时空产率，67% ee），从念珠菌化生中挖掘出一种对 3TF 具有高活性的强大羰基还原酶 CmCR 。根据底物结合口袋中底物醚氧的取向，合理设计CmCR，筛选出两个突变体MuR和MuS，将3TF完全还原为( R )-HTF和( S))-HTF 分别具有最高的 3TF 负载量和生产率（ MuR 为200 mM，4.4 g/L/h 时空产率，99% ee，而500 mM，11 g/ L /h 时空产率，99% ee ）穆斯）。揭示了立体选择性增强的结构机制：诱变改变了底物入口周围的静电势，促进3TF与MuR或MuS在某一方向接近、结合并形成预反应状态，从而将3TF转化为具有优异光学性能的3HTF。纯度。这项研究的成功为合理设计对高对称性目标底物具有高对映选择性的羰基还原酶提供了一种可行的方法。