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<p>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 <i>Mycobacterium smegmatis</i> 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<sub>T163G</sub>, 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<sub>T163G</sub>. 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.</p> Graphical abstract <p></p>

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

  • 期刊论文

  • 发表日期：

    2026年4月20日

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  • Ming-Xin Sun,
  • Zi-Wen Xie,
  • Zheng-Heng Qian,
  • Jian-Zhong Xu

摘要

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 MsET163G, 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 MsET163G. 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