The UDP-glucose:p-hydroxymandelonitrile-O-glucosyltransferase that catalyzes the last step in synthesis of the cyanogenic glucoside dhurrin in Sorghum bicolor. Isolation, cloning, heterologous expression, and substrate specificity.
The final step in the biosynthesis of the cyanogenic glucoside dhurrin in Sorghum bicolor is the transformation of the labile cyanohydrin into a stable storage form by O-glucosylation of (S)-p-hydroxymandelonitrile at the cyanohydrin function. The UDP-glucose:p-hydroxymandelonitrile-O-glucosyltransferase was isolated from etiolated seedlings of S. bicolor employing Reactive Yellow 3 chromatography with UDP-glucose elution as the critical step. Amino acid sequencing allowed the cloning of a full-length cDNA encoding the glucosyltransferase. Among the few characterized glucosyltransferases, the deduced translation product showed highest overall identity to Zea mays flavonoid-glucosyltransferase (Bz-Mc-2 allele). The substrate specificity of the enzyme was established using isolated recombinant protein. Compared with endogenous p-hydroxymandelonitrile, mandelonitrile, benzyl alcohol, and benzoic acid were utilized at maximum rates of 78, 13, and 4%, respectively. Surprisingly, the monoterpenoid geraniol was glucosylated at a maximum rate of 11% compared with p-hydroxymandelonitrile. The picture that is emerging regarding plant glucosyltransferase substrate specificity is one of limited but extended plasticity toward metabolites of related structure. This in turn ensures that a relatively high, but finite, number of glucosyltransferases can give rise to the large number of glucosides found in plants.
Jones
P R
PR
Department of Horticulture, the University of Adelaide, Waite Campus PMB1, Glen Osmond SA 5064, South Australia, Australia.
Moller
B L
BL
Hoj
P B
PB
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D002523:Edible Grain / Q000201:enzymology* / Q000235:genetics; D005964:Glucosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009570:Nitriles / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D003313:Zea mays / Q000201:enzymology
D017931:DNA Primers; D018076:DNA, Complementary; D009570:Nitriles; D011994:Recombinant Proteins; D005964:Glucosyltransferases; C402689:cyanohydrin beta-glucosyltransferase; C011220:dhurrin