期刊
ACS CATALYSIS
卷 10, 期 23, 页码 13946-13956出版社
AMER CHEMICAL SOC
DOI: 10.1021/acscatal.0c03841
关键词
L-lysine hydroxylase; (2S,4R)-4-hydroxylysine; catalytic efficiency; binding pocket; molecular dynamics
资金
- National Natural Science Foundation of China [31771911]
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-Iysine to form the product. To overcome this bottleneck we modified the t-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 rnM(-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.
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