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Tytuł:
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Molecular cloning, expression, and functional analysis of copper amine oxidase gene from mulberry (Morus alba L.).
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Autorzy:
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Wang D; School of Marine and Bioengineering, Yancheng Institute of Technology, Yancheng, 224051, China.
Zhao L; School of Pharmacy, Jiangsu University, Zhenjiang, Jiangsu, 212013, China.
Wan J; School of Pharmacy, Jiangsu University, Zhenjiang, Jiangsu, 212013, China.
Liu J; School of Pharmacy, Jiangsu University, Zhenjiang, Jiangsu, 212013, China.
Wei Y; School of Pharmacy, Jiangsu University, Zhenjiang, Jiangsu, 212013, China.
Ouyang Z; School of Pharmacy, Jiangsu University, Zhenjiang, Jiangsu, 212013, China. Electronic address: .
Yu X; School of Marine and Bioengineering, Yancheng Institute of Technology, Yancheng, 224051, China.
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Źródło:
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Protein expression and purification [Protein Expr Purif] 2023 Jan; Vol. 201, pp. 106166. Date of Electronic Publication: 2022 Sep 27.
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Typ publikacji:
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Journal Article; Research Support, Non-U.S. Gov't
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Język:
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English
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Imprint Name(s):
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Publication: Orlando, FL : Academic Press
Original Publication: San Diego : Academic Press, c1990-
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MeSH Terms:
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Amine Oxidase (Copper-Containing)*/genetics
Morus*/chemistry
1-Deoxynojirimycin/metabolism ; Cadaverine/metabolism ; Cloning, Molecular ; Copper/metabolism ; Plant Leaves/metabolism
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Contributed Indexing:
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Keywords: 1-Deoxynojirimycin; Copper amine oxidase; Functional analysis; Gene cloning; Mulberry
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Substance Nomenclature:
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19130-96-2 (1-Deoxynojirimycin)
789U1901C5 (Copper)
EC 1.4.3.21 (Amine Oxidase (Copper-Containing))
L90BEN6OLL (Cadaverine)
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Entry Date(s):
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Date Created: 20220929 Date Completed: 20221017 Latest Revision: 20221027
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Update Code:
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20240105
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DOI:
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10.1016/j.pep.2022.106166
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PMID:
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36174814
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In this study, we investigated a key enzyme encoded by the gene copper amine oxidase (MaCAO), which is involved in the biosynthetic pathway of 1-deoxynojirimycin (DNJ) 1 , an active ingredient in mulberry leaves. The 1680 bp long MaCAO was successfully cloned (GenBank accession no: MH205733). Subsequently, MaCAO was heterologously expressed using a recombinant plasmid, pET-22b (+)/MaCAO in Escherichia coli BL21 (DE3). A protein with a molecular mass of 62.9 kDa was obtained, whose function was validated through enzymatic reaction. Bioinformatics analysis identified that MaCAO contained the same conserved domain as that of copper amine oxidases ("NYDY"). Furthermore, the tertiary structure of the predicted protein using homology modeling revealed 46% similarity with that of copper amine oxidase (Protein Data Bank ID: 1W2Z). Gas chromatography-mass spectrometry analysis of the enzymatic reaction revealed that MaCAO could catalyze 1,5-pentanediamine to produce 5-aminopentanal. Additionally, levels of mulberry leaf DNJ content were significantly positively correlated with expression levels of MaCAO (P < 0.001). Our results conclude that MaCAO is the key enzyme involved in the biosynthetic pathway of DNJ. The function of MaCAO is validated, providing a foundation for the further analysis of biosynthetic pathways of DNJ in mulberry leaves using tools of synthetic biology.
Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
(Copyright © 2022 Elsevier Inc. All rights reserved.)