Cloning and functional characterisation of two regioselective flavonoid glucosyltransferases from Beta vulgaris.
Two full-length cDNAs encoding flavonoid-specific glucosyltransferases, UGT73A4 and UGT71F1, were isolated from a cDNA library of Beta vulgaris (Amaranthaceae) cell suspension cultures. They displayed high identity to position-specific betanidin and flavonoid glucosyltransferases from Dorotheanthus bellidiformis (Aizoaceae) and to enzymes with similar substrate specificities from various plant families. The open reading frame of the sequences encode proteins of 476 (UGT73A4) and 492 (UGT71F1) amino acids with calculated molecular masses of 54.07kDa and 54.39kDa, and isoelectric points of 5.8 and 5.6, respectively. Both enzymes were functionally expressed in Escherichia coli as His- and GST-tagged proteins, respectively. They exhibited a broad substrate specificity, but a distinct regioselectivity, glucosylating a variety of flavonols, flavones, flavanones, and coumarins. UGT73A4 showed a preference for the 4'- and 7-OH position in the flavonoids, whereas UGT71F1 preferentially glucosylated the 3- or the 7-OH position. Glucosylation of betanidin, the aglycone of the major betacyanin, betanin, in B. vulgaris was also observed to a low extent by both enzymes. Several O-glycosylated vitexin derivatives isolated from leaves of young B. vulgaris plants and rutin obtained from B. vulgaris tissue culture are discussed as potential endogenous products of UGT73A4 and UGT71F1. The results are analyzed with regard to evolution and specificity of plant natural product glucosyltransferases.
Isayenkova
Judith
J
Department of Secondary Metabolism, Leibniz Institute of Plant Biochemistry, Weinberg 3, D-06120 Halle (Saale), Germany.
Wray
Victor
V
Nimtz
Manfred
M
Strack
Dieter
D
Vogt
Thomas
T
D001483:Base Sequence; D027461:Beta vulgaris / Q000201:enzymology*; D015152:Blotting, Northern; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D019143:Evolution, Molecular; D005419:Flavonoids / Q000378:metabolism*; D005964:Glucosyltransferases / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D021241:Spectrometry, Mass, Electrospray Ionization; D013056:Spectrophotometry, Ultraviolet; D013237:Stereoisomerism; D013379:Substrate Specificity
D017931:DNA Primers; D018076:DNA, Complementary; D005419:Flavonoids; D012333:RNA, Messenger; D011994:Recombinant Proteins; D005964:Glucosyltransferases