title: Molecular cloning, expression, and functional analysis of the copper amine oxidase gene in the endophytic fungus Shiraia sp. Slf14 from Huperzia serrata.
abstract: Huperzine A (HupA) is a drug used for the treatment of Alzheimer's disease. However, the biosynthesis of this medicinally important compound is not well understood. The HupA biosynthetic pathway is thought to be initiated by the decarboxylation of lysine to form cadaverine, which is then converted to 5-aminopentanal by copper amine oxidase (CAO). In this study, we cloned and expressed an SsCAO gene from a HupA-producing endophytic fungus, Shiraia sp. Slf14. Analysis of the deduced protein amino acid sequence showed that it contained the Asp catalytic base, conserved motif Asn-Tyr-Asp/Glu, and three copper-binding histidines. The cDNA of SsCAO was amplified and expressed in Escherichia coli BL21(DE3), from which a 76 kDa protein was obtained. The activity of this enzyme was tested, which provided more information about the SsCAO gene in the endophytic fungus. Gas Chromatograph-Mass Spectrometry (GC-MS) revealed that this SsCAO could accept cadaverine as a substrate to produce 5-aminopentanal, the precursor of HupA. Phylogenetic tree analysis indicated that the SsCAO from Shiraia sp. Slf14 was closely related to Stemphylium lycopersici CAO. This is the first report on the cloning and expression of a CAO gene from HupA-producing endophytic fungi. Functional characterization of this enzyme provides new insights into the biosynthesis of the HupA an anti-Alzheimer's drug.
authors.affiliation: Jiangxi Key Laboratory of Functional Organic Molecules, Jiangxi Science and Technology Normal University, Nanchang, Jiangxi, 330013, China; Key Laboratory of Protection and Utilization of Subtropic Plant Resources of Jiangxi Province, College of Life Science, Jiangxi Normal University, Nanchang, Jiangxi, 330022, China.
authors.forename: Huilin
authors.initials: H
authors.lastname: Yang
authors.affiliation: Key Laboratory of Protection and Utilization of Subtropic Plant Resources of Jiangxi Province, College of Life Science, Jiangxi Normal University, Nanchang, Jiangxi, 330022, China.
authors.forename: Silu
authors.initials: S
authors.lastname: Peng
authors.affiliation: Key Laboratory of Protection and Utilization of Subtropic Plant Resources of Jiangxi Province, College of Life Science, Jiangxi Normal University, Nanchang, Jiangxi, 330022, China.
authors.forename: Zhibin
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Protection and Utilization of Subtropic Plant Resources of Jiangxi Province, College of Life Science, Jiangxi Normal University, Nanchang, Jiangxi, 330022, China.
authors.forename: Riming
authors.initials: R
authors.lastname: Yan
authors.affiliation: Jiangxi Key Laboratory of Functional Organic Molecules, Jiangxi Science and Technology Normal University, Nanchang, Jiangxi, 330013, China.
authors.forename: Ya
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Department of Biological Engineering, Utah State University, 4105 Old Main Hill, Logan, UT, 84322-4105, USA.
authors.forename: Jixun
authors.initials: J
authors.lastname: Zhan
authors.affiliation: Jiangxi Key Laboratory of Functional Organic Molecules, Jiangxi Science and Technology Normal University, Nanchang, Jiangxi, 330013, China; Key Laboratory of Protection and Utilization of Subtropic Plant Resources of Jiangxi Province, College of Life Science, Jiangxi Normal University, Nanchang, Jiangxi, 330022, China. Electronic address: zhudu12@163.com.
authors.forename: Du
authors.initials: D
authors.lastname: Zhu
mesh_terms: D000544:Alzheimer Disease / Q000188:drug therapy; D006631:Amine Oxidase (Copper-Containing) / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000627:therapeutic use; D001203:Ascomycota / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli; D005656:Fungal Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000627:therapeutic use; D006801:Humans; D032501:Huperzia / Q000382:microbiology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000627:therapeutic use
chemical_list: D005656:Fungal Proteins; D011994:Recombinant Proteins; D006631:Amine Oxidase (Copper-Containing)
keywords: Cloning and expression; Copper amine oxidase; Endophytic fungus; Enzyme activity; Huperzine A