Resolving the L-abrine residual bottleneck: enzyme engineering facilitates its dimethylation into L-hypaphorine

Abstract

L-Hypaphorine (L-HYP) is a natural indole alkaloid with significant medicinal value. It can be biosynthesized via the triple methylation of the nitrogen terminus of L-Tryptophan (L-Trp), which can be catalyzed by an engineered double mutant MSE derived from the Mycobacterium smegmatis methyltransferase EgtD. In this methylation reaction, the intermediate product L-Abrine often accumulates in large quantities, limiting the yield of the final product L-HYP. To address this bottleneck, this study employed a semi-rational design strategy to modify the substrate-binding pocket of MSE. Key residues were subjected to site-directed mutagenesis, resulting in the mutant KSA (P34K/T213S/S284A). Compared with the previously screened monomethylation mutant MsE_T163G, KSA exhibited significantly enhanced catalytic activity toward the substrate L-Abrine at the purified enzyme level, with a relative activity reaching 1.83 times that of MsE_T163G. Under whole-cell catalysis conditions, its conversion rate increased from 29.16% to 56.68%. Further molecular dynamics simulations revealed the optimized mechanism of KSA in substrate binding and the catalytic microenvironment, confirming its efficacy in promoting the conversion of L-Abrine to L-HYP. This provides a critical foundation for the subsequent realization of a complete cascade reaction.

Graphical abstract

Keywords

SAM-dependent methyltransferase / L-Abrine / Biocatalysis / Semi-rational engineering / Molecular dynamics simulations

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Funding

Innovative Research Group Project of the National Natural Science Foundation of China(32271534)

RIGHTS & PERMISSIONS

Jiangnan University