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Phytochemistry\ Vol[ 38\ No[ 1\ pp[ 296Ð207\ 0887
Þ 0887 Elsevier Science Ltd[ All rights reserved
Printed in Great Britain
Pergamon
\
9920Ð8311:87:,Ðsee front matter
PII] S992Ð0831"18#79914Ð19
ENZYMOLOGY OF UDP!GLUCOSE]SINAPIC ACID
GLUCOSYLTRANSFERASE FROM BRASSICA NAPUS
IN HONOUR OF PROFESSOR G[ H[ NEIL TOWERS 64TH BIRTHDAY
SHAWN X[ WANGꢀ and BRIAN E[ ELLIS$
Department of Plant Science\ University of British Columbia\ Vancouver\ B[C[ V5T 0Z3 Canada
"Received 17 January 0887^ received in revised form 06 March 0887#
Key Word Index*Brassica napus^ Cruciferae^ UDP!glucose]sinapic acid glucosyltransferase^
sinapine^ enzymology
Abstract*UDP!glucose]sinapic acid glucosyltransferase "SGT^ EC 1[3[0[019# was puri_ed from 59!h!old
seedlings of Brassica napus[ The puri_ed SGT appears to be a cytosolic monomeric polypeptide with a Mr of
31 kDa and a pI of 4[ Kinetic analysis suggested that the catalytic mechanism used by SGT best _ts a {{random
biÐbi|| model\ with a Km "UDP!glucose# of 1[3 mM and Km "sinapic acid# of 9[05 mM[ SGT also catalyzes the reverse
reaction in vitro\ using UDP and sinapoylglucose to form UDP!glucose[ No cofactors are required for enzyme
activity\ but reducing agents and glycerol are required to stabilize the activity[ The enzyme is strongly inhibited
by p!OH!mercuribenzoic acid\ UDP\ TDP\ Zn¦¦\ Cu¦¦ and Hg¦¦[ Þ 0887 Elsevier Science Ltd[ All rights
reserved
INTRODUCTION
carota\ where its product is believed to serve as an
intermediate in the synthesis of a sinapoyl antho!
cyanin derivative accumulated by the carrot cells ð6Ł[
However\ as a class\ glucosyltransferases are not
abundant proteins in plant tissues\ and only a small
number of nucleotide sugar!dependent glu!
cosyltransferases have been puri_ed to varying
degrees ð7Ð01Ł[
Because of its participation in two di}erent path!
ways that operate at di}erent stages of plant devel!
opment\ and in di}erent tissues\ the mechanisms
which regulate SGT expression are of interest[ As a
key enzyme in sinapine biosynthesis\ SGT is also a
potential target for genetic engineering approaches to
creation of an agronomically!desirable low!sinapine
Brassica genotype[ Here\ we report the puri_cation
and characterization of SGT from seedlings of B[
napus "canola#[
Sinapine "O!sinapoyl choline# is known to be a
remarkable chemotaxonomic character in many cru!
cifers\ especially in Brassica and closely!related species
ð0Ł[ In edible oilseed Brassica "canola# it accumulates
in the mature seeds to levels that limit the utilization
of the protein!rich post!crushing meal as an animal
feed supplement ð1Ł[ Among the enzymes required in
sinapine biosynthesis and metabolism\ UDPG]sinapic
acid glucosyl transferase "SGT#\ which catalyzes the
formation of 0!O!sinapoyl!b!D!glucose from sinapic
acid and UDP!glucose "UDPG#\ is notable because it
is required at two points[ Its product\ sinapoylglucose\
is an essential substrate both for sinapine formation
in the seed\ and for sinapoylmalate formation in the
leaves of the plant ð2Ł[
SGT activity has been demonstrated previously in
leaf tissue of B[ oleracea ð3Ł\ and in seeds and young
seedlings of Raphanus sativus ð0Ł\ and it has been par!
tially puri_ed from seedlings of R[ sativus ð0\ 4\ 5Ł[
Outside the Brassicaceae family\ SGT activity has also
been detected in extracts of cultured cells of Daucus
RESULTS AND DISCUSSION
Inducibility of SGT
SGT activity in B[ napus changes dramatically dur!
ing the growth cycle of the plant ð02Ł[ The underlying
regulatory mechanisms are unknown\ but could
include both developmental and environmental cues[
One possible control factor might be changes in the
size of the intracellular pool of sinapate\ arising from
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