摘要
Objective Benzylisoquinoline alkaloids(BIAs)are valuable plant metabolites whose structural diversity largely depends on O-/N-methyltransferases(OMTs/NMTs).Although the CYP450-mediated backbone formation of BIAs was previously elucidated in Menispermum dauricum DC.,the specific OMTs/NMTs responsible for their functional methylation remain unknown.This study aims to systematically identify and characterize these methyltransferases to define their roles in BIA biosynthesis.Methods Combining genomic and biochemical approaches,a genome-wide identification of methyltransferase(MT)genes was conducted using the published M.dauricum genome.The candidate MTs were functionally evaluated through in vitro enzymatic assays employing diverse BIA substrates to determine their methylation specificities.The substrate-binding modes of MdOMT1 and MdOMT11 were predicted and compared by protein modeling and molecular docking.Results Functional characterization of the 75 methyltransferases identified in the M.dauricum genome revealed four key enzymes(three OMTs and one NMT)that contribute to the diversification of BIA scaffolds through their distinct substrate specificities and positional preferences.Specifically,MdOMT1 preferentially catalyzed O-methylation at C7 position of 1-benzylisoquinolines(1-BIAs)and C2 position of tetrahydroprotoberberines.In contrast,MdOMT11 exhibited superior affinity and a strong preference for O-methylation at the C9 position of(S)-scoulerine.Meanwhile,MdNMT3 demonstrated effective N-methylation activity toward both 1-BIAs and tetrahydroprotoberberines.Conclusion This research elucidates the functional landscape of OMTs/NMTs in M.dauricum,revealing their crucial roles in BIA structural diversification.The newly identified enzymes provide valuable biocatalytic tools for synthetic biology approaches aimed at the sustainable and optimized production of high-value BIAs.
基金
supported by the National Natural Science Foundation of China(Nos.82274037,32460117)
Beijing Life Science Academy(BLSA,No.2024500CD0120)
YNDG202402ZY02,YNDG202302ZY02
CNTC-110202201016(JY-16)
YNTC-2023530000241009
the Fundamental Research Funds for the central public welfare research institutes(Nos.ZZ16-YQ-047,ZZ18-ND-10-11).
