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J Biol Chem, Vol. 274, Issue 50, 35483-35491, December 10, 1999

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^*

Patrik Raymond Jones^§¶, Birger Lindberg Møller^§¶**, and Peter Bordier Høj

From the  Department of Horticulture, Viticulture, and Oenology, the University of Adelaide, Waite Campus PMB1, Glen Osmond SA 5064, South Australia, Australia, the ^§ Plant Biochemistry Laboratory, Department of Plant Biology, The Royal Veterinary and Agricultural University, the ^¶ Center for Molecular Plant Physiology (Place), The Royal Veterinary and Agricultural University, 40 Thorvaldensvej, DK-1871 Frederiksberg C, Copenhagen, Denmark, and the  Australian Wine Research Institute, P. O. Box 197, Glen Osmond SA 5064, South Australia, Australia

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.

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^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EMBL Data Bank with accession number(s) AF199453.

Recipient of an Australian Postgraduate award.

^** To whom correspondence should be addressed. Tel.: 45-35283352; Fax: 45-35283333; E-mail: blm@kvl.dk.

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Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.

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