Reshaping the Binding Pocket of Lysine Hydroxylase for Enhanced Activity
Fenghua Wang, Menglu Zhu, Zhan Song, Chao Li, Yuying Wang, Zhangliang Zhu, Dengyue Sun, Fuping Lu, Hui‐Min Qin
Abstract
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 kcat/Km, 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.