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Publication Abstract from PubMed

Chiral (S)-o-fluorostyrene oxide (oFSO) and vicinal diol (R)-o-fluorophenylethane-1,2-diol (oFPED) are important intermediates for synthesizing treatments for neuropathic diseases. This study aimed to engineer Aspergillus usamii epoxide hydrolase (AuEH2) through a rational mutagenesis strategy to customize high enantioselectivity mutant for rac-oFSO. Out of 181 single-site mutants screened, six showed elevated enantiomeric ratio (E value) ranging from 32 to 108 according to E value and activity mutability landscapes. By combinatorial mutagenesis of A250I with other five single-site mutants, we constructed five double-site mutants, with the best-performing mutant, D5 (A250I/L344V), achieving an E value of 180. This mutant enabled the efficient kinetic resolution of 400 mM rac-oFSO in pure water system using E. coli/Aueh2(A250I/L344V), yielding (S)-oFSO (>99 % ee(s), 50 % yield(s)) and (R)-oFPED (>99 % ee(p), 50 % yield(p)) with space-time yields (STYs) of 331.5 and 376.1 g/L/d, respectively. Combining crystal structure resolution with theoretical computations clarified the enantioselectivity mechanism of D5, demonstrating that A250I reduced the funnel-shaped substrate binding pocket (SBP) while L344V extended its bottom, enhancing specific recognition of (R)-oFSO and inhibiting (S)-oFSO hydrolysis. These findings provide valuable insights for designing highly enantioselective enzyme mutants, advancing the field of asymmetric synthesis of chiral compounds using green biocatalytic processes.

Rational mutagenesis of an epoxide hydrolase and its structural mechanism for the enantioselectivity improvement toward chiral ortho-fluorostyrene oxide.,Lu ZY, Liao X, Jing WW, Liu KK, Ren QG, He YC, Hu D Int J Biol Macromol. 2024 Dec;282(Pt 3):136864. doi: , 10.1016/j.ijbiomac.2024.136864. Epub 2024 Oct 29. PMID:39476898^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.