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Functional characterization and key residues engineering of a regiopromiscuity O-methyltransferase involved in benzylisoquinoline alkaloid biosynthesis in Nelumbo nucifera.

ABSTRACT: Lotus (Nelumbo nucifera), an ancient aquatic plant, possesses a unique pharmacological activity that is primarily contributed by benzylisoquinoline alkaloids (BIAs). However, only few genes and enzymes involved in BIA biosynthesis in N. nucifera have been isolated and characterized. In the present study we identified the regiopromiscuity of an O-methyltransferase, designated NnOMT6, isolated from N. nucifera; NnOMT6 was found to catalyze the methylation of monobenzylisoquinoline 6-O/7-O, aporphine skeleton 6-O, phenylpropanoid 3-O, and protoberberine 2-O. We further probed the key residues affecting NnOMT6 activity via molecular docking and molecular dynamics simulation. Verification using site-directed mutagenesis revealed that residues D316, N130, L135, N176A, D269, and E328 were critical for BIA O-methyltransferase activities; furthermore, N323A, a mutant of NnOMT6, demonstrated a substantial increase in catalytic efficiency for BIAs and a broader acceptor scope compared with wild-type NnOMT6. To the best of our knowledge, this is the first study to report the O-methyltransferase activity of an aporphine skeleton without benzyl moiety substitutions in N. nucifera. The study findings provide biocatalysts for the semisynthesis of related medical compounds and give insights into protein engineering to strengthen O-methyltransferase activity in plants.

SUBMITTER: Yu Y

PROVIDER: S-EPMC9923211 | biostudies-literature | 2023 Feb

REPOSITORIES: biostudies-literature

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Functional characterization and key residues engineering of a regiopromiscuity O-methyltransferase involved in benzylisoquinoline alkaloid biosynthesis in Nelumbo nucifera.

Yu Yuetong Y Liu Yan Y Dong Gangqiang G Jiang JinZhu J Leng Liang L Liu XianJu X Zhang Jun J Liu An A Chen Sha S

Horticulture research 20221209 2

Lotus (Nelumbo nucifera), an ancient aquatic plant, possesses a unique pharmacological activity that is primarily contributed by benzylisoquinoline alkaloids (BIAs). However, only few genes and enzymes involved in BIA biosynthesis in N. nucifera have been isolated and characterized. In the present study we identified the regiopromiscuity of an O-methyltransferase, designated NnOMT6, isolated from N. nucifera; NnOMT6 was found to catalyze the methylation of monobenzyli ...[more]

PMID: 36789257

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Project description:The predominant alkaloids in lotus leaves are aporphine alkaloids. These are the most important active components and have many pharmacological properties, but little is known about their biosynthesis. We used digital gene expression (DGE) technology to identify differentially-expressed genes (DEGs) between two lotus cultivars with different alkaloid contents at four leaf development stages. We also predicted potential genes involved in aporphine alkaloid biosynthesis by weighted gene co-expression network analysis (WGCNA). Approximately 335 billion nucleotides were generated; and 94% of which were aligned against the reference genome. Of 22 thousand expressed genes, 19,000 were differentially expressed between the two cultivars at the four stages. Gene Ontology (GO) enrichment analysis revealed that catalytic activity and oxidoreductase activity were enriched significantly in most pairwise comparisons. In Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, dozens of DEGs were assigned to the categories of biosynthesis of secondary metabolites, isoquinoline alkaloid biosynthesis, and flavonoid biosynthesis. The genes encoding norcoclaurine synthase (NCS), norcoclaurine 6-O-methyltransferase (6OMT), coclaurine N-methyltransferase (CNMT), N-methylcoclaurine 3'-hydroxylase (NMCH), and 3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase (4'OMT) in the common pathways of benzylisoquinoline alkaloid biosynthesis and the ones encoding corytuberine synthase (CTS) in aporphine alkaloid biosynthetic pathway, which have been characterized in other plants, were identified in lotus. These genes had positive effects on alkaloid content, albeit with phenotypic lag. The WGCNA of DEGs revealed that one network module was associated with the dynamic change of alkaloid content. Eleven genes encoding proteins with methyltransferase, oxidoreductase and CYP450 activities were identified. These were surmised to be genes involved in aporphine alkaloid biosynthesis. This transcriptomic database provides new directions for future studies on clarifying the aporphine alkaloid pathway.

Dataset Information

Functional characterization and key residues engineering of a regiopromiscuity O-methyltransferase involved in benzylisoquinoline alkaloid biosynthesis in Nelumbo nucifera.

Publications

Functional characterization and key residues engineering of a regiopromiscuity <i>O</i>-methyltransferase involved in benzylisoquinoline alkaloid biosynthesis in <i>Nelumbo nucifera</i>.

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