| 1. |
- Bu, Junling, et al.
(författare)
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Catalytic promiscuity of O-methyltransferases from Corydalis yanhusuo leading to the structural diversity of benzylisoquinoline alkaloids
- 2022
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Ingår i: Horticulture Research. - : Oxford University Press (OUP). - 2662-6810 .- 2052-7276. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- O-methyltransferases play essential roles in producing structural diversity and improving the biological properties of benzylisoquinoline alkaloids (BIAs) in plants. In this study, Corydalis yanhusuo, a plant used in traditional Chinese medicine due to the analgesic effects of its BIA-active compounds, was employed to analyze the catalytic characteristics of O-methyltransferases in the formation of BIA diversity. Seven genes encoding O-methyltransferases were cloned, and functionally characterized using seven potential BIA substrates. Specifically, an O-methyltransferase (CyOMT2) with highly efficient catalytic activity of both 4′- and 6-O-methylations of 1-BIAs was found. CyOMT6 was found to perform two sequential methylations at both 9- and 2-positions of the essential intermediate of tetrahydroprotoberberines, (S)-scoulerine. Two O-methyltransferases (CyOMT5 and CyOMT7) with wide substrate promiscuity were found, with the 2-position of tetrahydroprotoberberines as the preferential catalytic site for CyOMT5 (named scoulerine 2-O-methyltransferase) and the 6-position of 1-BIAs as the preferential site for CyOMT7. In addition, results of integrated phylogenetic molecular docking analysis and site-directed mutation suggested that residues at sites 172, 306, 313, and 314 in CyOMT5 are important for enzyme promiscuity related to O-methylations at the 6- and 7-positions of isoquinoline. Cys at site 253 in CyOMT2 was proved to promote the methylation activity of the 6-position and to expand substrate scopes. This work provides insight into O-methyltransferases in producing BIA diversity in C. yanhusuo and genetic elements for producing BIAs by metabolic engineering and synthetic biology.
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| 2. |
- Liu, Xiuyu, et al.
(författare)
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Characterization of CYP82 genes involved in the biosynthesis of structurally diverse benzylisoquinoline alkaloids in Corydalis yanhusuo
- 2024
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Ingår i: Plant Molecular Biology. - 0167-4412 .- 1573-5028. ; 114:2
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Tidskriftsartikel (refereegranskat)abstract
- Benzylisoquinoline alkaloids (BIAs) represent a significant class of secondary metabolites with crucial roles in plant physiology and substantial potential for clinical applications. CYP82 genes are involved in the formation and modification of various BIA skeletons, contributing to the structural diversity of compounds. In this study, Corydalis yanhusuo, a traditional Chinese medicine rich in BIAs, was investigated to identify the catalytic function of CYP82s during BIA formation. Specifically, 20 CyCYP82-encoding genes were cloned, and their functions were identified in vitro. Ten of these CyCYP82s were observed to catalyze hydroxylation, leading to the formation of protopine and benzophenanthridine scaffolds. Furthermore, the correlation between BIA accumulation and the expression of CyCYP82s in different tissues of C. yanhusuo was assessed their. The identification and characterization of CyCYP82s provide novel genetic elements that can advance the synthetic biology of BIA compounds such as protopine and benzophenanthridine, and offer insights into the biosynthesis of BIAs with diverse structures in C. yanhusuo.
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| 3. |
- Liu, Xiuyu, et al.
(författare)
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Functional characterization of (S)–N-methylcoclaurine 3′-hydroxylase (NMCH) involved in the biosynthesis of benzylisoquinoline alkaloids in Corydalis yanhusuo
- 2021
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Ingår i: Plant Physiology and Biochemistry. - : Elsevier BV. - 0981-9428. ; 168, s. 507-515
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Tidskriftsartikel (refereegranskat)abstract
- Benzylisoquinoline alkaloids (BIAs) are compounds naturally found in plants and can have significant value in clinical settings. Metabolic engineering and synthetic biology are both promising approaches for the heterologous acquisition of benzylisoquinoline alkaloids. (S)–N-methylcoclaurine 3′-hydroxylase (NMCH), a member of the CYP80 family of CYP450, is the penultimate catalytic enzyme that forms the central branch-point intermediate (S)-reticuline and plays a key role in the biosynthesis of BIAs. In this study, an NMCH gene was cloned from Corydalis yanhusuo, while in vitro reactions demonstrated that CyNMCH can catalyze (S)–N-methylcoclaurine to produce (S)-3′-hydroxy-N-methylcoclaurine. The Km and Kcat of CyNMCH were estimated and compared with those identified in Eschscholzia californica and Coptis japonica. This newly discovered CyNMCH will provide alternative genetic resources for the synthetic biological production of benzylisoquinoline alkaloids and provides a foundation to help analyze the biosynthetic pathway of BIAs biosynthesis in C. yanhusuo.
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| 4. |
- Liu, Xiuyu, et al.
(författare)
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Structure-function analysis of CYP719As involved in methylenedioxy bridge-formation in the biosynthesis of benzylisoquinoline alkaloids and its de novo production
- 2023
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Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 22:1
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Tidskriftsartikel (refereegranskat)abstract
- Benzylisoquinoline alkaloids (BIAs) are a type of secondary metabolite with clinical application value. (S)-stylopine is a special BIA which contains methylenedioxy bridge structures. CYP719As could catalyze the methylenedioxy bridge-formation on the A or D rings of protoberberine alkaloids, while displaying significant substrate regiospecificity. To explore the substrate preference of CYP719As, we cloned and identified five CyCYP719A candidates from Corydalis yanhusuo. Two CyCYP719As (CyCYP719A39 and CyCYP719A42) with high catalytic efficiency for the methylenedioxy bridge-formation on the D or A rings were characterized, respectively. The residues (Leu 294 for CyCYP719A42 and Asp 289 for CyCYP719A39) were identified as the key to controlling the regioselectivity of CYP719As affecting the methylenedioxy bridge-formation on the A or D rings by homology modeling and mutation analysis. Furthermore, for de novo production of BIAs, CyCYP719A39, CyCYP719A42, and their mutants were introduced into the (S)-scoulerine-producing yeast to produce 32 mg/L (S)-stylopine. These results lay a foundation for understanding the structure-function relationship of CYP719A-mediated methylenedioxy bridge-formation and provide yeast strains for the BIAs production by synthetic biology.
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