Apple fruit copper amine oxidase isoforms: peroxisomal MdAO1 prefers diamines as substrates, whereas extracellular MdAO2 exclusively utilizes monoamines.
4-Aminobutyrate (GABA) accumulates in apple fruit during controlled atmosphere storage. A potential source of GABA is the polyamine putrescine, which can be oxidized via copper-containing amine oxidase (CuAO), resulting in the production 4-aminobutanal/Δ(1)-pyrroline, with the consumption of O2 and release of H2O2 and ammonia. Five putative CuAO genes (MdAO genes) were cloned from apple (Malus domestica Borkh. cv. Empire) fruit, and the deduced amino acid sequences found to contain the active sites typically conserved in CuAOs. Genes encoding two of these enzymes, MdAO1 and MdAO2, were highly expressed in apple fruit and selected for further analysis. Amino acid sequence analysis predicted the presence of a C-terminal peroxisomal targeting signal 1 tripeptide in MdAO1 and an N-terminal signal peptide and N-glycosylation site in MdAO2. Transient expression of green fluorescent fusion proteins in Arabidopsis protoplasts or onion epidermal cells revealed a peroxisomal localization for MdAO1 and an extracellular localization for MdAO2. The enzymatic activities of purified recombinant MdAO1 and MdAO2 were measured continuously as H2O2 production using a coupled reaction. MdAO1 did not use monoamines or polyamines and displayed high catalytic efficiency for 1,3-diaminopropane, putrescine and cadaverine, whereas MdAO2 exclusively utilized aliphatic and aromatic monoamines, including 2-phenylethylamine and tyramine. Together, these results indicate that MdAO1 may contribute to GABA production via putrescine oxidation in the peroxisome of apple fruit under controlled atmosphere conditions. MdAO2 seems to be involved in deamination of 2-phenylethylamine, which is a step in the biosynthesis of 2-phenylethanol, a contributor to fruit flavor and flower fragrance.
Department of Plant Agriculture, University of Guelph, 50 Stone Road E, Guelph, Canada N1G 2W1.
Adel
A
Zarei
Department of Plant Agriculture, University of Guelph, 50 Stone Road E, Guelph, Canada N1G 2W1.
Christopher P
CP
Trobacher
Department of Plant Agriculture, University of Guelph, 50 Stone Road E, Guelph, Canada N1G 2W1.
Alison R
AR
Cooke
School of Environmental Sciences, University of Guelph, 50 Stone Road E, Guelph, Canada N1G 2W1.
Ashley J
AJ
Meyers
School of Environmental Sciences, University of Guelph, 50 Stone Road E, Guelph, Canada N1G 2W1.
J Christopher
JC
Hall
Department of Plant Agriculture, University of Guelph, 50 Stone Road E, Guelph, Canada N1G 2W1 bshelp@uoguelph.ca.
Barry J
BJ
Shelp
D006631:Amine Oxidase (Copper-Containing) / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000166:cytology / Q000201:enzymology / Q000235:genetics; D015306:Biogenic Monoamines / Q000378:metabolism*; D053898:Biosynthetic Pathways; D003959:Diamines / Q000378:metabolism*; D005110:Extracellular Space / Q000201:enzymology; D005638:Fruit / Q000166:cytology / Q000201:enzymology* / Q000235:genetics; D018506:Gene Expression Regulation, Plant; D007527:Isoenzymes; D027845:Malus / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D019697:Onions / Q000166:cytology / Q000201:enzymology / Q000235:genetics; D009928:Organ Specificity; D010084:Oxidation-Reduction; D020675:Peroxisomes / Q000201:enzymology; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D011073:Polyamines / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
D015306:Biogenic Monoamines; D003959:Diamines; D007527:Isoenzymes; D010940:Plant Proteins; D011073:Polyamines; D005680:gamma-Aminobutyric Acid; D006631:Amine Oxidase (Copper-Containing)
4-Aminobutyrate; Abiotic stress; Apple fruit; Copper amine oxidase; Enzyme kinetics; Polyamines; Recombinant expression; Subcellular localization