The in vitro substrate regiospecificity of recombinant UGT85B1, the cyanohydrin glucosyltransferase from Sorghum bicolor.
The in vitro substrate specificity of UDP-glucose:p-hydroxymandelonitrile-O-glucosyltransferase from Sorghum bicolor (UGT85B1) was examined using a range of potential acceptor molecules, including cyanohydrins, terpenoids, phenolics, hexanol derivatives and plant hormones. Qualitative enzyme activity assays employing 20 different putative substrates were performed and 15 proved to be glucosylated using recombinant UGT85B1 isolated from Escherichia coli. K(m) and k(cat) values were determined for nine of these substrates including mandelonitrile, geraniol, nerol and beta-citronellol, 2-hydroxy-3-methoxybenzyl alcohol, 1-hexanol, cis-3-hexen-1-ol, 3-methyl-3-buten-1-ol and 3-methyl-2-buten-1-ol. UGT85B1 has a rather broad substrate specificity in vitro but shows regiospecificity, demanding the presence of a sterically unhindered hydroxyl group e.g. as part of a cyanohydrin function, as a primary alcohol or as a phenolic hydroxyl group and being influenced by the stereochemistry and/or interactive chemistry of the substituents on the hydroxyl-bearing carbon atom.
Hansen
Karina Sinding
KS
Plant Biochemistry Laboratory, Department of Plant Biology, Royal Veterinary and Agricultural University, Thorvaldsensvej 40, and Center of Molecular Plant Physiology (PlaCe), DK-1871 Frederiksberg C, Copenhagen, Denmark.
Kristensen
Charlotte
C
Tattersall
David Bruce
DB
Jones
Patrik Raymond
PR
Olsen
Carl Erik
CE
Bak
Søren
S
Møller
Birger Lindberg
BL
D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D005964:Glucosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D000441:Hexanols / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D009570:Nitriles / Q000378:metabolism; D010636:Phenols / Q000737:chemistry / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000201:enzymology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D045868:Sorghum / Q000201:enzymology*; D013237:Stereoisomerism; D013379:Substrate Specificity; D013729:Terpenes / Q000737:chemistry / Q000378:metabolism; D014532:Uridine Diphosphate Glucose / Q000378:metabolism
D029681:Arabidopsis Proteins; D000441:Hexanols; D009570:Nitriles; D010636:Phenols; D011994:Recombinant Proteins; D013729:Terpenes; D005964:Glucosyltransferases; C402689:cyanohydrin beta-glucosyltransferase; C011220:dhurrin; D014532:Uridine Diphosphate Glucose