From the Department of Biology, University of York,
York YO10 5DD, United Kingdom and the § Institute of Food
Research, Norwich Research Park, Colney,
Norwich NR4 7UA, United Kingdom
Received for publication, August 10, 2000, and in revised form, September 25, 2000
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ABSTRACT |
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Sinapic acid is a major phenylpropanoid in
Brassicaceae providing intermediates in two distinct metabolic pathways
leading to sinapoyl esters and lignin synthesis. Glucosyltransferases play key roles in the formation of these intermediates, either through
the production of the high energy compound
1-O-sinapoylglucose leading to sinapoylmalate and
sinapoylcholine or through the production of sinapyl
alcohol-4-O-glucoside, potentially leading to the syringyl units found in lignins. While the importance of these
glucosyltransferases has been recognized for more than 20 years, their
corresponding genes have not been identified. Combining sequence
information in the Arabidopsis genomic data base with
biochemical data from screening the activity of recombinant proteins
in vitro, we have now identified five gene sequences
encoding enzymes that can glucosylate sinapic acid, sinapyl alcohol,
and their related phenylpropanoids. The data provide a foundation
for future understanding and manipulation of sinapate metabolism and
lignin biology in Arabidopsis.
Plasticity, both in terms of development and metabolism, is a key
feature of plants, most probably arising through the need of sedentary
organisms to respond rapidly to prevailing environmental conditions.
Phenylpropanoid metabolism is one example of extreme plasticity in
which reactions can lead to a wide diversity of products functioning
either in their own right or acting as gateways into different
metabolic pathways (1). Lignin polymers are major end products of
phenylpropanoid metabolism, providing compressive strength and water
resistance to the protein-polysaccharide matrix of plant cell walls and
conferring general protection against microbial attack (2, 3). Other
pathways arising from phenylpropanoids include the synthesis and
modification of flavonoids, such as those acting as free radical
scavengers, signaling molecules, and anti-microbial compounds (4), and
the benzoate pathways leading to compounds such as salicylic acid
(5).
Among the many different gene functions contributing to this plasticity
are those that correspond to modifying enzymes such as the multigene
families encoding P450 hydroxylases (6), methyltransferases (7), and
the glycosyltransferases (8). Typically in plants, the group of
glycosyltransferases (UDP-glucosyltransferase;
UGTs)1 involved in these
modifications are characterized by the presence of a signature motif
located toward the C terminus of the polypeptide (9, 10). The reactions
most often involve the transfer of glucose from UDP-glucose to the
second substrate, leading either to the formation of a glucose ester or
to the formation of a glucoside. Whereas glucose esters have long been
recognized to be high energy compounds acting as transient
intermediates in the formation of other metabolites (11), glucosides
have increased water solubility, provide access to membrane transport
systems, and can act as storage forms of the aglycone (8).
A classic example of these different consequences of glucosylation
is the formation of the glucose ester and glucoside of sinapic acid and
the glucoside of sinapyl alcohol. Brassicaceae such as
Arabidopsis predominantly accumulate sinapoyl esters. 1-O-sinapoylglucose (a glucose ester) is the intermediate in
the synthesis of sinapoylmalate, which is a putative UV protectant located in leaf epidermis of the plant (12, 13) and sinapoylcholine (14). Sinapoylcholine is made only during seed development and is
degraded during germination to provide sinapic acid for the seedling
which is converted again via 1-O-sinapoylglucose to
sinapoylmalate (15). In contrast to the role of the glucose ester of
sinapic acid, formation of the more soluble glucoside of sinapic acid (sinapoyl-4-O-glucoside) may be related to storage or
detoxification, involving removal of the metabolite from the cytoplasm
through transfer into the vacuole (8). Sinapyl
alcohol-4-O-glucoside, also known as syringin, is considered
to be involved in lignin synthesis, since it is thought that
glucosylation of the three monolignols (sinapyl alcohol, coniferyl
alcohol, and p-coumaryl alcohol) may aid transport of the
monomers out of the cell for polymerization into lignin in
muro (3). Recently, a specific glucosidase of coniferin (coniferyl
alcohol-4-O-glucoside) has been localized at the
differentiating xylem, providing some support for these events in
lignin assembly (16).
One way of investigating the role of these three glucosylation
reactions in planta is to manipulate the level of expression of the genes encoding the respective UGTs and to analyze the phenotypic consequences. Despite considerable interest in the enzymes over many
years, the proteins have not been purified to homogeneity as yet, nor
have their genes been cloned. This study describes an alternative
approach, where we have used information from the genomic data bases of
Arabidopsis to identify sequences containing the UGT
signature motif (10). In parallel to a phylogenetic analysis of these
sequences, we have screened recombinant proteins for their activities
in vitro toward sinapates and their related phenylpropanoids. The data in this study describe the identification of
five genes that show relevant specificities in vitro,
thereby providing a new foundation for defining their roles in the plant.
Chemicals--
The majority of the chemicals and phenolic
compounds (Fig. 1) used in this study were purchased from Sigma.
Coniferyl aldehyde, sinapyl aldehyde, and sinapyl alcohol were
purchased from Apin Chemicals Ltd. p-Coumaryl aldehyde and
p-coumaryl alcohol were supplied courtesy of John Ralph
(Department of Forestry, University of Wisconsin).
Construction of GST-UGT Expression Plasmids--
DNA fragments
corresponding to putative UGT sequences with no introns (10) were
amplified from Arabidopsis thaliana Columbia genomic DNA by
polymerase chain reaction. For those sequences containing introns,
full-length expressed sequence tags obtained from the
Arabidopsis Biological Resource Center stock center were used to construct the expression plasmids. Specific oligomer sets were
designed according to the sequences of the UGT genes (10). The
polymerase chain reactions were set up following the conditions described previously (17). The polymerase chain reaction products were
electroeluted from 1% DNA agarose gel. After purification with
phenol/chloroform, the DNA fragments were subcloned into the
appropriate restriction sites on the multiple cloning site of the GST
gene fusion vector pGEX-2T (Amersham Pharmacia Biotech).
Recombinant UGT Purification--
Recombinant UGTs were
expressed as fusion proteins, each containing a GST fusion partner at
the N terminus. To prepare large quantities of recombinant proteins,
Escherichia coli strain XL1-Blue was grown at 20 °C in
500 ml of 2× YT medium containing 50 µg/ml ampicillin until the
A600 reached 1.0. The culture was then incubated with 1 mM
isopropyl-1-thio-
The protein assays were carried out with Bio-Rad Protein Assay Dye
using bovine serum albumin as reference. The purified recombinant proteins were also analyzed by SDS-polyacrylamide gel electrophoresis following the methods described by Sambrook et al. (19).
Glucosyltransferase Activity Assay--
The assay mix (200 µl)
contained 0.2 µg of recombinant protein, 14 mM
2-mercaptoethanol, 5 mM UDP-glucose, and 1 mM
phenylpropanoid substrate. Initial screening for activity of the 36 proteins against each of the 11 potential substrates was carried out at
pH 7.0 (100 mM Tris-HCl) and 30 °C for 30 min. For
detailed kinetic analysis of the enzymes showing significant activity,
reactions leading to 4-O-glucosides were carried out at pH
7.0/20 °C/30 min, and those leading to glucose esters were carried
out at pH 6.0 (potassium phosphate)/20 °C/30 min due to their pH
optima and linearity of the reactions. Reactions were stopped by the
addition of 20 µl of trichloroacetic acid (240 mg/ml), quick-frozen,
and stored at HPLC Analysis--
Reverse phase HPLC (Waters Separator 2690 and
Waters Tunable Absorbance Detector 486, Waters Limited, Herts, UK)
using a Columbus 5-µm C18 column (250 × 4.60-mm, Phenomenex). A linear gradient of acetonitrile in
H2O (all solutions contained 0.1% trifluoroacetic acid) at
1 ml/min over 20 min was used to separate the glucose conjugates from
their aglycone. The HPLC methods were as follows: cinnamic acid,
1H NMR Analysis--
The glucosides for NMR analysis
were purified using the HPLC methods described above. The samples were
freeze-dried and resuspended in deuterated methanol. The NMR spectra
were acquired on a Bruker AMX 500-MHz NMR spectrometer at 22 °C. The
data were processed and analyzed using Bruker XWIN-NMR software,
version 2.6.
Computer Analysis of Sequence--
The sequence analyses were
carried out using Genetics Computer Group software (Wisconsin
package, version 10.1).
Preparation of Recombinant Proteins--
Screening the
GenBankTM data base with the UGT signature motif has
revealed a large multigene family of putative UGT sequences in
Arabidopsis (10, 20, 21). The sequences were named following the standardized system of the UGT Nomenclature Committee (22) and were
classified into subgroups based on homology comparisons, which were
confirmed through detailed phylogenetic analysis (10).
To gain insight into the biochemical properties of the gene products,
36 sequences were used to produce recombinant fusion proteins with GST
in E. coli. The relatedness of the sequences chosen for
expression and the purified recombinant proteins used for the
biochemical assays are shown in Fig. 2. Following purification using
glutathione Sepharose as an affinity matrix, some of these fusions
proved to be unstable, releasing GST (26 kDa) as a separate polypeptide, a common observation with this fusion system (23).
Screening for Glucosyltransferase Activity--
The 36 recombinant
proteins were screened for glucosyltransferase activity using
UDP-glucose as the sugar donor and each of 11 closely related
phenylpropanoids (as shown in Fig. 1) as
substrates under identical assay conditions. When the reaction mixes
were analyzed using HPLC, only five proteins (UGT84A1, UGT84A2,
UGT84A3, UGT72E2, and UGT72E3) showed significant activity toward the
cinnamic acids and alcohols; 25 proteins showed no activity toward any of the substrates, and a further six displayed only trace activities and are not described in detail. None of the 36 proteins were able to
glucosylate the aldehydes (data not shown). Results from this screening
are summarized in Fig. 2. The control,
using GST alone, was unable to glucosylate any of the phenylpropanoids
(data not shown). Of the 25 recombinant proteins that showed no
activity toward any of the substrates tested in Fig. 1, 10 sequences
showed some activities toward scopoletin, implying that the proteins were catalytically active in vitro (data not shown). As yet,
no substrate has been identified for the remaining 15 proteins. While these were purified from soluble fractions of the E. coli
lysate, it remains unknown whether they will be catalytically active
under the conditions used in the in vitro assays.
Characterization of Glucose Ester and 4-O-Glucoside Reaction
Products--
Glucosylation of the cinnamic acids can lead to the
formation of glucose esters or glucosides, whereas glucosylation of
the cinnamyl alcohols only forms glucosides (Fig. 1). Using
sinapic acid and sinapyl alcohol as examples, the two types of reaction product could be distinguished by alkaline hydrolysis and HPLC. 1-O-Sinapoylglucose and sinapoyl-4-O-glucoside
are the glucose ester and the 4-O-glucoside of sinapic acid
formed by UGT84A2 and UGT72E2, respectively. Due to the high free
energy of hydrolysis (11), 1-O-sinapoylglucose is not stable
in the presence of 1 N NaOH. In contrast,
sinapoyl-4-O-glucoside is not affected by the alkaline
conditions (Fig. 3A).
Glucosylation of sinapyl alcohol produces syringin (sinapyl
alcohol-4-O-glucoside). As shown in Fig. 3B, this
product is also stable in 1 N
NaOH. The identities of the three glucose conjugates were further
confirmed by NMR analysis (Table I) using NMR spectra assigned
according to published information (24, 25).
UGTs Catalyzing the Formation of Cinnamate Glucose Esters in
Vitro--
From the initial screening as shown in Fig. 2, three
enzymes, UGT84A1, UGT84A2, and UGT84A3 showed significant activity in forming glucose ester conjugates with the cinnamic acids in
vitro. The specificity and kinetics of these enzymes were analyzed
in detail. As shown in Fig. 4, UGT84A2
clearly shows the highest specificity for sinapic acid, has a low
Km toward this substrate, and is virtually inactive
toward other cinnamic acids. Both UGT84A1 and UGT84A3 can glucosylate
sinapic acid, but they have a higher Km than UGT84A2
and are also active toward other substrates. UGT84A1 is the only enzyme
that shows significant activity and has high affinity toward caffeic
acid. While UGT84A1 also displays a strong activity toward
p-coumaric acid, the affinity of the enzyme toward the
substrate is low. UGT84A3 similarly has a broad enzyme activity toward
a number of substrates, but a comparison of the Michaelis-Menten
kinetics suggests that ferulic acid may be the preferred substrate
under the conditions used.
UGTs Catalyzing the Formation of Cinnamate Glucosides in
Vitro--
The two enzymes, UGT72E2 and UGT72E3, which produced
4-O-glucosides, were active only against the four substrates
shown in Fig. 5. Whereas UGT72E2 and
UGT72E3 glucosylate sinapyl alcohol at high levels of specific
activity, only UGT72E2 showed activity with coniferyl alcohol. From a
comparison of the Michaelis-Menten kinetics, UGT72E2 is likely to
glucosylate sinapyl alcohol, whereas UGT72E3 may be the enzyme
responsible for conversion of sinapic acid into its glucoside.
Our aim in this study has been to identify enzymes capable of
glucosylating sinapic acid and sinapyl alcohol in vitro, as a foundation for going on to study their role in the formation of
sinapoylmalate and lignin in planta. We have used
Arabidopsis thaliana as the model, since the availability of
gene sequence information in the data bases has enabled us to gain
insight rapidly into a family of sequences containing a UGT signature
motif (10). Phylogenetic analysis of the Arabidopsis UGT
multigene family has revealed 12 groups (10) that in turn have provided
a predictive framework for screening recombinant proteins for catalytic
activities of interest. The data described in this study highlights the
efficiency of such an approach, since we have identified five UGTs
expressing the relevant activities when assayed in
vitro.
Sinapoylmalate has been suggested to act as a foliar UV protectant in
Arabidopsis (12, 13), although as yet there is no direct
supportive evidence. The biosynthetic pathway leading to sinapoylmalate
in the Brassicaceae is well characterized biochemically (15), and
Arabidopsis genes encoding the enzymes upstream and downstream of UGT involvement have been identified by mutational analysis (26, 27). Study of the fah1 mutant showed that the seedlings were more susceptible than wild type to UV stress (12). Since
the FAH1 locus encodes ferulate-5-hydroxylase, a cytochrome P450-dependent monooxygenase responsible for the formation
of 5-hydroxyferulic acid, the precursor of sinapic acid (28), the data
implied that the product of the reaction, 5-hydroxyferulic acid, or
metabolites downstream of 5-hydroxyferulic acid, such as sinapic acid
and sinapoylmalate, were involved in UV protection. However, recent
analyses of Arabidopsis overexpressing FAH1 have shown no
accumulation of sinapoylmalate (29), suggesting that levels of FAH1 do
not control flux through this part of the cinnamate pathway. Since the
glucose ester is the direct precursor of sinapoylmalate, manipulation
of UGT levels involved in its formation may provide a better tool with
which to investigate the potential link between sinapoylmalate and UV protection.
The kinetic data we have gained from the in vitro assays now
suggest that UGT84A2 corresponds to the UGT responsible for synthesis of the glucose ester intermediate in the sinapoylmalate pathway. The
specificity of this UGT toward sinapic acid is surprisingly high and
contrasts with that shown for UGT84A1 and UGT84A3 that can form the
glucose ester of sinapic acid but can also glucosylate other cinnamic
acids. As yet, we do not know the relationship of these in
vitro analyses to physiological events in the plant. For example,
it is not known whether the same UGT is involved in glucosylation of
sinapic acid in the leaves and in the developing seed. In the leaves, a
glucose ester is converted to sinapoylmalate, whereas in seeds, a
glucose ester is converted to sinapoylcholine (14). Gene knock-outs in
UGT84A1, UGT84A2, or UGT84A3 and
metabolite profiling of the transgenic plants will provide important
insights into these possibilities. Similarly, the cellular specificity and regulation of expression of the three genes will also provide a
context for understanding the role of the gene products in the Arabidopsis plant.
A common feature of the three genes UGT84A1,
UGT84A2, and UGT84A3 is that they encode UGTs
that all form glucose esters. Interestingly, the sequences are located
within the same branch of the multigene family (Fig. 2; Ref. 10).
Another sequence closely related on the basis of homology and located
in the same branch is UGT84A4, but in contrast, the recombinant enzyme
only showed trace activity in forming glucose esters with cinnamic
acids when assayed under identical conditions in vitro. A
similar trace activity toward cinnamic acids was also observed with
UGT84B1 (Fig. 2), but other studies have shown this UGT to be highly
specific in forming the glucose ester of the structurally related
metabolite, indole-3-acetic acid (30).
Whereas sinapate ester metabolism is of almost exclusive relevance to
plant species of the Brassicaceae, lignin synthesis and metabolism
impact more generally on our understanding of plant cell walls and
their determining role in development and defense responses. Many
recent reviews have addressed the potential role of the glucosides of
the monolignols in lignin assembly (reviewed in Ref. 3). Typically, the
glucosides are considered to represent the transport forms of the
monolignols, with their respective UGTs and glucosidases acting
sequentially in the assembly process. In conifers, the existence of
coniferin (coniferyl alcohol-4-O-glucoside) is well
established, and recently the gene encoding a coniferin-specific glucosidase has been identified and shown to release the aglycone in vitro (16). To date, however, no gene encoding UGTs of
monolignols has been identified from any plant species, and the
biochemical work that has been undertaken for more than 20 years has
involved partially purified protein fractions (31).
The data described in this report now provide new tools with which to
understand the role of UGTs in lignin synthesis. The Arabidopsis genes UGT72E2 and UGT72E3
encode enzymes that can glucosylate coniferyl alcohol and sinapyl
alcohol in vitro. Analysis of their cell-specific expression
and regulation, together with metabolite profiling following knock-outs
or overexpression of the genes, will provide important contributions to
the ongoing debates surrounding lignin biology. For example, if the
glucosides of the monolignols are essential precursors to lignin, then
knocking out UGT72E2 or UGT72E3 should give
impact on the composition of the lignin synthesized.
As of March 1, 2000, 98 sequences corresponding to putative UGTs have
been identified in the Arabidopsis genome (10), suggesting that in the total genome there may be as many as 120 sequences containing the UGT signature motif. Surprisingly, phylogenetic analysis
of these additional sequences indicates that none is closely related to
those encoding the UGTs involved in glucose ester and
4-O-glucoside formation that are described in this study (10). While many early studies have biochemically analyzed UGT activities purified or partially purified from a wide range of different plant families (32-42), there have been no previous attempts to study this multigene family in a single species and therefore no
possibility of directly comparing relative activities across family
members. Access to the kinetic analysis of many family members from a
single species can provide increased confidence of the range of
substrates that may be used by each of these enzymes in
vitro and thereby a foundation for exploring their substrates in vivo and their physiological roles in the plant. Of equal
relevance is the wider use of UGTs for industrial applications. Studies building on known substrate specificities across the multigene family
and their experimental modification by processes such as DNA shuffling
can now provide a new platform for designing biotransformations in vivo and in vitro.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside for 24 h
at 20 °C to induce synthesis of the GST-UGT fusion proteins. Cells
were harvested (5000 × g for 5 min), resuspended (5 ml
of ice-cold phosphate-buffered saline), osmotically shocked (18), and
centrifuged again (40,000 × g for 5 min). The
supernatant was mixed with 100 µl of 50% glutathione-coupled
Sepharose (Amersham Pharmacia Biotech), the beads were washed with PBS,
and adsorbed proteins were eluted with 20 mM reduced form
glutathione, 100 mM Tris-HCl, pH 8.0, 120 mM
NaCl according to the manufacturer's instructions.
20 °C prior to the reverse phase HPLC analysis. The
specific enzyme activity was expressed as nmol of phenylpropanoids
glucosylated/s (nkat) by 1 mg of protein in 30 min of reaction time.
Alkaline hydrolysis was carried out in 0.1 N NaOH at
room temperature for 1 h and neutralized by 3 M
sodium acetate, pH 5.2.
288 nm, 10-55% acetonitrile; p-coumaric
acid,
311 nm, 10-25% acetonitrile; caffeic acid,
311 nm, 10-16% acetonitrile; ferulic acid,
311
nm, 10-35% acetonitrile; sinapic acid,
306 nm, 10-40% acetonitrile; p-coumaryl aldehyde,
315
nm, 10-46% acetonitrile; coniferyl aldehyde,
283
nm, 10-47% acetonitrile; sinapyl aldehyde,
280
nm, 10-47% acetonitrile; p-coumaryl alcohol,
283 nm, 10-27% acetonitrile; coniferyl alcohol,
306 nm, 10-25% acetonitrile; sinapyl alcohol,
285 nm, 10-25% acetonitrile. The retention time (Rt) of the glucose conjugates analyzed was as
follows: cinnamoylglucose, Rt = 12.3 min;
p-coumaroylglucose, Rt = 10.6 min;
caffeoylglucose, Rt = 8.5 min; feruloylglucose,
Rt = 10.3 min; sinapoylglucose,
Rt = 9.7 min; caffeoyl-4-O-glucoside,
Rt = 6.8 min; feruloyl-4-O-glucoside, Rt = 7.8 min; sinapoyl-4-O-glucoside,
Rt = 8.2 min; coniferin, Rt = 8.2 min; syringin, Rt = 9.1 min.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (23K):
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Fig. 1.
Phenylpropanoids analyzed as potential
substrates for the 36 recombinant UGTs. The phenylpropanoids
assayed in this study are involved in lignin biosynthesis. An
asterisk marks the hydroxyl group that can be glucosylated
to form a glucose ester, whereas a dagger labels the
O-glucosidic linkage site.
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[in a new window]
Fig. 2.
Sequences of Arabidopsis
encoding putative UGTs and their corresponding fusion
proteins. The amino acid sequences of 36 putative UGTs were
aligned (10) with the prefix UGT omitted for clarity. Corresponding
GST-UGT fusion proteins were purified from E. coli and were
analyzed using 10% (w/v) SDS-polyacrylamide gel electrophoresis. The
proteins were visualized with Coomassie staining. Large quantities of
these recombinant proteins were purified and incubated individually
with the 11 substrates shown in Fig. 1. Each assay contained 0.2 µg
of recombinant protein, 5 mM UDP-glucose, 1 mM
phenylpropanoid substrate, and 100 mM Tris-HCl, pH 7.0. The
mix was incubated at 30 °C for 30 min and was analyzed by reverse
phase HPLC. Proteins forming glucose esters ( ) and glucosides (
)
are highlighted, together with significant (
) or trace (
) enzyme
activity, which is defined as conversion of >5% or <5% activity,
respectively, relative to the maximum conversion (100%) observed for
each substrate.
View larger version (22K):
[in a new window]
Fig. 3.
Alkaline hydrolysis of the glucose conjugates
of sinapic acid and sinapyl alcohol. Each assay contained 0.2 µg
of recombinant protein, 5 mM UDP-glucose, 1 mM
phenylpropanoid substrate, and 100 mM Tris-HCl, pH 7.0. Following preincubation at 30 °C for 30 min, the reaction mix was
transferred to room temperature for 1 h in the presence (+) or
absence ( ) of 1 N NaOH. All of the reaction mixtures were
analyzed by reverse phase HPLC. GST protein was used as negative
control to show that this fusion partner does not catalyze the
glucosylation reaction. A, sinapic acid was used as
substrate in the assay. B, sinapyl alcohol was used as
substrate in the assay.
1H NMR spectral data of sinapic acid and sinapyl alcohol
glucose conjugates
scale with tetramethylsilane as an
internal standard. The position on the aromatic ring begins with the
carbon joining the propanoic acid or the propanol group. s,
singlet; d, doublet; dd, doublet of doublets;
dt, doublet of triplets; m, multiplet;
J, coupling
constant.
View larger version (31K):
[in a new window]
Fig. 4.
Characterization of the UGTs forming glucose
esters with phenylpropanoids. Each assay contained 0.2 µg of
recombinant protein, 5 mM UDP-glucose, 1 mM
phenylpropanoid substrate, and 100 mM potassium phosphate,
pH 6.0. The mix was incubated at 20 °C for 30 min. The reaction was
stopped by the addition of 20 µl of trichloroacetic acid (240 mg/ml)
and was analyzed by reverse phase HPLC subsequently. The results
represent the mean of three replicates ± S.D. The
Michaelis-Menten kinetics of recombinant glucosyltransferase activity
were measured over a range of 0-1 mM phenylpropanoid in
the presence of 10 mM UDP-glucose. ND, not
determined.
View larger version (19K):
[in a new window]
Fig. 5.
Characterization of the UGTs forming
4-O-glucosides with phenylpropanoids. Each assay
contained 0.2 µg of recombinant protein, 5 mM
UDP-glucose, 1 mM phenylpropanoid substrate, and 100 mM Tris-HCl, pH 7.0. The mix was incubated at 30 °C for
30 min. The reaction was stopped by the addition of 20 µl of
trichloroacetic acid (240 mg/ml) and was analyzed by reverse phase HPLC
subsequently. The results represent the mean of three replicates ± S.D. The Michaelis-Menten kinetics of recombinant
glucosyltransferase activity were measured over a range of 0-1
mM phenylpropanoid in the presence of 10 mM
UDP-glucose. ND, not determined.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Professor John Ralph and Professor Clint Chapple for helpful discussions. Heather Fish is thanked for help with NMR studies.
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FOOTNOTES |
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* The research was funded in part by Biotechnology and Biological Sciences Research Council Grant 87/P12844 (to D. J. B.).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 GenBankTM/EMBL Data Bank with accession number(s) AB018119 (UGT72E2), AF077407 (UGT72E3), Z97339 (UGT84A1), AB019232 (UGT84A2), and Z97339 (UGT84A3).
¶ To whom correspondence should be addressed. Tel.: 44-1904-434334; Fax: 44-1904-434336; E-mail: djb32@york.ac.uk.
Published, JBC Papers in Press, October 20, 2000, DOI 10.1074/jbc.M007263200
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ABBREVIATIONS |
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The abbreviations used are: UGT, UDP-glucosyltransferase; GST, glutathione S-transferase; HPLC, high pressure liquid chromatography.
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REFERENCES |
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