8XIZ

Crystal structure of an epoxide hydrolase mutant A250IC/L344V from Aspergillus usamii E001 at 2.17 Angstroms resolution

Summary for 8XIZ

• Entry DOI: 10.2210/pdb8xiz/pdb
• Descriptor: Microsomal epoxide hyddrolase (2 entities in total)
• Functional Keywords: alpha and beta proteins, alpha/beta-hydrolases, styrene oxide, epoxide hydrolase, hydrolase
• Biological source: Aspergillus usamii
• Total number of polymer chains: 2
• Total formula weight: 97049.42

Authors

Hu, B.C.,Lu, Z.Y.,Tang, C.D.,Hu, D. (deposition date: 2023-12-20, release date: 2024-02-07, Last modification date: 2025-05-21)

Primary citation

Lu, Z.Y.,Liao, X.,Jing, W.W.,Liu, K.K.,Ren, Q.G.,He, Y.C.,Hu, D.
Rational mutagenesis of an epoxide hydrolase and its structural mechanism for the enantioselectivity improvement toward chiral ortho-fluorostyrene oxide.
Int.J.Biol.Macromol., 282:136864-136864, 2024

PubMed Abstract: 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, yielding (S)-oFSO (>99 % ee, 50 % yield) and (R)-oFPED (>99 % ee, 50 % yield) 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.

PubMed: 39476898
DOI: 10.1016/j.ijbiomac.2024.136864

Experimental method

X-RAY DIFFRACTION (2.175 Å)