Reshaping the Binding Pocket of Lysine Hydroxylase for Enhanced Activity

The versatile synthetic intermediate (2 S,4 R )-4-hydroxylysine can be produced using l -lysine hydroxylase. However, the wild-type enzyme cannot effectively catalyze the C4 hydroxylation of l -lysine to form the product. To overcome this bottleneck, we modified the l -lysine hydroxylase from Niastella koreensis (NkLH4), using the semirational combinatorial active-site saturation test (CAST). We obtained a highly active mutant MT3 (Q161N/T162A/F178Y/E260D) with a 24.97-fold increase of k cat / K m, compared with the wild-type enzyme (791.33 mM –1 s –1 vs 31.69 mM –1 s –1 ). Further analysis of the structure–activity relationship via molecular dynamics (MD) simulations suggested that MT3 had a more flexible conformation, as well as an enlarged substrate-binding pocket with decreased steric hindrance and increased binding energy in substrate recognition. Our study provides a highly active NkLH4 mutant for potential commercial use in the production of enantiomerically pure (2 S,4 R )-4-hydroxylysine.