title: Purification and characterization of a betanidin glucosyltransferase from Amaranthus tricolor L catalyzing non-specific biotransformation of flavonoids.
abstract: Betacyanins are the major pigments present in Amaranthus tricolor, a leafy vegetable consumed globally. The terminal glycosylation of the aglycone betanidin is an important step in the biosynthesis of this natural red antioxidant pigment. A betanidin 5-O-glucosyltransferase (BGT) was fully purified to 134 folds (specific activity, 265.2 nkat mg(-1)) from the red amaranth by ammonium sulfate precipitation followed by hydrophobic interaction, anion exchange and size exclusion chromatography. Homogeneity of the purified protein was confirmed by 2-dimensional polyacrylamide gel electrophoresis (2D PAGE). The molecular weight of the enzyme determined by liquid chromatography-mass spectrometry (LC-MS) was found to be 62.8 kDa. Furthermore, the enzyme glycosylated flavonoids (kaempferol and quercetin) but not anthocyanidins, presence of which is mutually exclusive to betacyanin accumulating plants. The apparent Km (344±2.34 μM) and Vmax (17.24 μM min(-1)) of the enzyme were determined by LC-MS/MS. Peptide mass fingerprinting of the purified protein showed 38.4% coverage of peptide masses with anthocyanidin 3-O-glucosyltransferase from Zea mays. Study on this purified enzyme, for the first time, revealed its role of glycosylation in biosynthesis of betacyanin in A. tricolor and indicates promiscuous substrate-specificity.
authors.affiliation: Plant Biotechnology Laboratory, Department of Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur, West Bengal, India.
authors.forename: Shibendu Sekhar
authors.initials: SS
authors.lastname: Das
authors.forename: Samiran S
authors.initials: SS
authors.lastname: Gauri
authors.forename: Biswapriya Biswavas
authors.initials: BB
authors.lastname: Misra
authors.forename: Mousumi
authors.initials: M
authors.lastname: Biswas
authors.forename: Satyahari
authors.initials: S
authors.lastname: Dey
mesh_terms: D027721:Amaranthus / Q000737:chemistry / Q000201:enzymology*; D050859:Betacyanins / Q000378:metabolism*; D001711:Biotransformation; D002853:Chromatography, Liquid; D015180:Electrophoresis, Gel, Two-Dimensional; D005419:Flavonoids / Q000378:metabolism*; D005964:Glucosyltransferases / Q000302:isolation & purification* / Q000378:metabolism; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D013058:Mass Spectrometry; D008970:Molecular Weight; D010449:Peptide Mapping; D018515:Plant Leaves / Q000737:chemistry / Q000378:metabolism; D010940:Plant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012492:Salts; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D050859:Betacyanins; D005419:Flavonoids; D010940:Plant Proteins; D012492:Salts; D005964:Glucosyltransferases; C110839:betanidin 5-O-glucosyltransferase
keywords: Amaranthus tricolor L; BGT; Betanidin glucosyltransferase; Flavonoids; GT; LC–MS; Liquid chromatography–mass spectrometry; PMF; Peptide mass fingerprinting; betanidin glucosyltransferase; glycosyltransferase; liquid chromatography–mass spectrometry; peptide mass fingerprinting