Molecular cloning and characterization of copper amine oxidase from Huperzia serrata.
A cDNA encoding a novel copper amine oxidase (CAO) was cloned and sequenced from the Chinese club moss Huperzia serrata (Huperziaceae), which produces the Lycopodium alkaloid huperzine A. A 2043-bp open reading frame encoded an Mr 76,854 protein with 681 amino acids. The deduced amino acid sequence shared 44-56% identity with the known CAOs of plant origin, and contained the active site consensus sequence of Asn-Tyr-Asp/Glu. The phylogenetic tree analysis revealed that HsCAO from the primitive vascular plant H. serrata is closely related to Physcomitrella patens subsp CAO. The recombinant enzyme, heterologously expressed in Escherichia coli, catalyzed the oxidative deamination of aliphatic and aromatic amines. Among them, the enzyme accepted cadaverine as the best substrate to catalyze the oxidative deamination to Δ(1)-piperideine, which is the precursor of the Lycopodium alkaloids. Furthermore, a homology modeling and site-directed mutagenesis studies predicted the active site architecture, which suggested the crucial active site residues for the observed substrate preference. This is the first report of the cloning and characterization of a CAO enzyme from the primitive Lycopodium plant.
Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo,Tokyo 113-0033, Japan.
Jieyin
J
Sun
Hiroyuki
H
Morita
Guoshen
G
Chen
Hiroshi
H
Noguchi
Ikuro
I
Abe
D000447:Aldehydes / Q000737:chemistry / Q000378:metabolism; D006631:Amine Oxidase (Copper-Containing) / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000588:Amines / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D055162:Biocatalysis; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D032501:Huperzia / Q000201:enzymology* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010084:Oxidation-Reduction; D010802:Phylogeny; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D000447:Aldehydes; D000588:Amines; D011994:Recombinant Proteins; D006631:Amine Oxidase (Copper-Containing)