Purification and characterization of an O-methyltransferase capable of methylating 2-hydroxy-3-alkylpyrazine from Vitis vinifera L. (cv. Cabernet Sauvignon)

An S-adenosyl-L-methionine-dependent O-methyltransferase capable of methylating 2-hydroxy-3-alkylpyrazine (HP) was purified 7,300-fold to apparent homogeneity with an 8.2% overall recovery from Vitis vinifera L. (cv. Cabernet Sauvignon) through a purification procedure including column chromatograph...

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Bibliographic Details
Published in:Bioscience, biotechnology, and biochemistry biotechnology, and biochemistry, 2001-10, Vol.65 (10), p.2213-2219
Main Authors: Hashizume, K. (National Research Inst. of Brewing, Higashihiroshima, Hiroshima (Japan)), Tozawa, K, Hiraga, Y, Aramaki, I
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Analytical, structural and metabolic biochemistry
Biological and medical sciences
Chromatography, DEAE-Cellulose
Chromatography, Gel
Chromatography, High Pressure Liquid
COLUMN CHROMATOGRAPHY
ELECTROPHORESIS
Electrophoresis, Polyacrylamide Gel
Enzymes and enzyme inhibitors
Fundamental and applied biological sciences. Psychology
Hydrogen-Ion Concentration
hydroxypyrazine
Kinetics
methoxypyrazine
METHYLATION
Molecular Sequence Data
MOLECULAR WEIGHT
O-methyltransferase
Protein O-Methyltransferase - isolation & purification
Protein O-Methyltransferase - metabolism
PURIFICATION
PYRAZINES
Pyrazines - metabolism
Sequence Homology, Amino Acid
TRANSFERASES
Vitis - enzymology
VITIS VINIFERA
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Summary:An S-adenosyl-L-methionine-dependent O-methyltransferase capable of methylating 2-hydroxy-3-alkylpyrazine (HP) was purified 7,300-fold to apparent homogeneity with an 8.2% overall recovery from Vitis vinifera L. (cv. Cabernet Sauvignon) through a purification procedure including column chromatography on DEAE-Sepharose FF, Ether-5PW, hydroxyapatite, G2000SW subXL, and DEAE-5PW. The relative molecular mass of the native enzyme estimated on gel permeation chromatography was 85kDa, and the subunit molecular mass was estimated to be 41kDa on SDS-poIyacrylamide gel electrophoresis. The enzyme also methylates caffeic acid. The V sub(max) for IBHP and caffeic acid were 0.73 and 175 pkatals/mg, respectively, and the respective K sub(m) for IBHP and caffeic acid were 0.30 and 0.032mM. The optimum pH for IBHP (8.5) was different from that for caffeic acid (7.5).
ISSN:0916-8451
1347-6947
DOI:10.1271/bbb.65.2213