Ulvans, complex polysaccharides found in the ulvales (green seaweed) cell wall, contain predominantly 3-sulfated rhamnose (Rha3S) linked to either d-glucuronic acid, l-iduronic acid or d-xylose.
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 substratebinding 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.
BackgroundBiosynthesis of steroidal drugs is of great benefit in pharmaceutical manufacturing as the process involves efficient enzymatic catalysis at ambient temperature and atmospheric pressure compared to chemical synthesis. 3-ketosteroid-∆1-dehydrogenase from Arthrobacter simplex (KsdD3) catalyzes 1,2-desaturation of steroidal substrates with FAD as a cofactor.ResultsRecombinant KsdD3 exhibited organic solvent tolerance. W117, F296, W299, et al., which were located in substrate-binding cavity, were predicted to form hydrophobic interaction with the substrate. Structure-based site-directed saturation mutagenesis of KsdD3 was performed with W299 mutants, which resulted in improved catalytic activities toward various steroidal substrates. W299A showed the highest increase in catalytic efficiency (kcat/Km) compared with the wild-type enzyme. Homology modelling revealed that the mutants enlarged the active site cavity and relieved the steric interference facilitating recognition of C17 hydroxyl/carbonyl steroidal substrates. Steered molecular dynamics simulations revealed that W299A/G decreased the potential energy barrier of association of substrates and dissociation of the corresponding products. The biotransformation of AD with enzymatic catalysis and resting cells harbouring KsdD3 WT/mutants revealed that W299A catalyzed the maximum ADD yields of 71 and 95% by enzymatic catalysis and resting cell conversion respectively, compared with the wild type (38 and 75%, respectively).ConclusionsThe successful rational design of functional KsdD3 greatly advanced our understanding of KsdD family enzymes. Structure-based site-directed saturation mutagenesis and biochemical data were used to design KsdD3 mutants with a higher catalytic activity and broader selectivity.
Electronic supplementary materialThe online version of this article (10.1186/s12934-018-0981-0) contains supplementary material, which is available to authorized users.
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