Reshaping the Binding Pocket of Lysine Hydroxylase for Enhanced Activity

The versatile synthetic intermediate (2S,4R)-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 (2S,4R)-4-hydroxylysine.