3 Centre for Novel Agricultural Products, Department of Biology, University of York, York YO10 5DD, United Kingdom
4 Department of Biology, University of York, York YO10 5DD, United Kingdom
5 Gene Research Center, Shinshu University, Ueda, Nagano 386-8567, Japan
Received on April 19, 2002; revised on September 24, 2002; accepted on September 24, 2002
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Abstract |
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Key words: Arabidopsis / glucosyltransferases / hydroxycoumarins / phylogeny / plant secondary metabolites
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Introduction |
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Interestingly, the action of UGTs can result in the formation of either a glucose ester or a glucoside. The former is a high-energy compound acting as biosynthetic intermediate in which the aglycone can be further transferred onto a second acceptor. The classic example of this event is the glucosylation of sinapic acid, yielding sinapoylglucose, which acts as an intermediate in the formation of sinapoylmalate and sinapoylcholine (Tkotz and Strack, 1980; Strack et al., 1983
). Similarly, the coumaroyl moiety of 1-O-p-coumaroylglucose is transferred to quinic acid, forming p-coumaroylquinic acid (Kojima and Villegas, 1984
). However, for many glucose esters, the second acceptor remains unknown. In contrast to the biosynthetic role of glucose esters, most glucosides are thought to represent detoxification compounds, although monolignol glucosides have long been thought to represent precursors of lignin (reviewed in Whetten et al., 1998
). Whether the glucose ester or the glucoside is formed, both modifications are considered to provide access to membrane-bound transporters and exit pathways from the cytosol, such as to the cell wall or to the vacuole (reviewed in Jones and Vogt, 2001
).
Although the number of glucosylated small-molecular-weight compounds in plants is vast and their roles in cellular function are well recognized, progress on the identification and characterization of the enzymes involved is slow. Often the proteins are nonabundant, and purification to homogeneity has proven difficult to achieve. This has hampered the cloning of their respective genes using traditional approaches. Genome sequencing programs have provided a new opportunity to solve these problems, because the databases can be searched for sequences that contain motifs characteristic of proteins with particular properties. Cloning these sequences and expressing recombinant proteins in vitro enables a large-scale screen of catalytic activities to be undertaken, thereby rapidly identifying enzymes of importance.
Using this genomic approach, a number of UGTs previously intractable to biochemical analyses have been identified for the first time and characterized for their in vitro activity. This includes UGTs for the monolignols, coniferyl and sinapyl alcohol, as well as those for the sinapoylglucose intermediate (Lim et al., 2001). Similarly, the Arabidopsis gene encoding an enzyme glucosylating indole-3-acetic acid has been characterized (Jackson et al., 2001
, 2002
), and two enzymes specific for salicylic acid have been identified (Lim et al., 2002
).
The availability of such a large, multigene family of UGT sequences can provide the basis for a structural genomic approach in which three-dimensional structures across the family can be compared to their catalytic activities. As yet, however, no structure for any of the enzymes in this family has been elucidated. Nevertheless, the phylogenetic tree established through linear sequence comparison (Li et al., 2001) does provide some basis from which to explore molecular evolution of the gene family. This study addresses substrate recognition and regioselectivity of glycosylation by the UGTs using model hydroxycoumarin subtrates.
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Results |
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However, due to the highly divergent nature of the UGT88A1 sequence and the inconclusive bootstrap support for its placement (79/65, neighbor joining/parsimony), two other possibilities cannot be ruled out. One alternative is that UGT88A1 branched off before the split of the UGT72 and UGT71 families, making it the outgroup to both. In this case, the interpretation of the tree is similar to that already discussed, except that the regioselectivity switching event would have occurred after the split of the two families (Figure 3B). Alternatively, UGT88A1 could belong to the branch with the UGT72 family; the regioselectivity switching event would then have occurred shortly after the branch including the UGT72 family and UGT88A1 separated from UGT71 family, with the ancestor's regioselectivity towardesculetin unknown (Figure 3C).
Regardless of the timing of the first switching event, the second regioselectivity switching event in group E happened either in the common ancestor of UGT72B2 and UGT72B3 or at some stage along the unique line leading to UGT72B3, as indicated in Figure 3A. This event switched the regioselectivity of UGT72B3 from the 6-OH position to the 7-OH position of esculetin. The exact interpretation depends on the regioselectivity of UGT72B2, which has not been obtained in soluble form. The availability of UGT72B2 will help confirm the regioselectivity switching event in this branch.
Data from groups H and L are shown in Figures 4 and 5. When activity was observed toward the model substrates, the UGTs were found to glucosylate scopoletin and esculetin only at the 7-OH position. Interpretation of negative results in group H is difficult because no other catalytic activity of these proteins has as yet been observed. However, in group L, there is clear evidence for loss of activity toward hydroxycoumarins in at least one evolutionary event leading to UGT84A2 (Figure 5), because this enzyme has known activity toward phenylpropanoids (Lim et al., 2001).
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Discussion |
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We use a genomic approach to study this multigene family of UGTs in Arabidopsis. The sequences have been cloned, expressed as recombinant proteins in Escherichia coli, and used to screen for catalytic activity in vitro toward substrates of importance. This has led to the identification of genes encoding UGTs specific for hydroxycinnamates, benzoates, and plant hormones (Lim et al., 2001, 2002
; Jackson et al., 2001
). Despite the recent successes, substrates for only
18 UGTs out of the 107 have been identified so far. Interestingly, from these limited studies, there are data showing that linear sequence homology correlates with similarity in substrate recognition. For example, the four UGTs 84A1A4 all recognize the carboxyl group of cinnamic acids and form a long deep branch in group L (Lim et al., 2001
). Similarly, UGT74F1 and UGT74F2, again forming a long deep branch in group L, are the only two enzymes that recognize salicylic acid (Lim et al., 2002
). Although both of these enzymes recognize the same substrate, they exhibit different regioselectivity of glucosylation with UGT74F1 glucosylating the 2-OH of salicylic acid to form an O-glucoside, whereas UGT74F2 transfers glucose onto the carboxyl group to produce a glucose ester.
To explore the molecular evolution of substrate recognition and regioselectivity of glucosylation in more depth, this study uses model substrates with features recognized by many different UGTs spread across the entire family. Earlier work had shown that a number of UGTs could recognize the hydroxycoumarin scopoletin (Lim et al., 2001). This suggested that scopoletin could be a useful model substrate. Esculetin, another hydroxycoumarin, has one additional OH group, making it possible to analyze both substrate recognition and regioselectivity on the ring. Surprisingly, as summarized in Figure 6, as many as three major phylogenetic groups (groups D, E, and L) all show activity toward the two hydroxycoumarins. It is unlikely that all of these UGTs will catalyze the glucosylation of these compounds in vivo. However, the results of this study imply that there are at least three ancestral UGTs that once recognized substrates with features common to hydroxycoumarins. During evolution, new sequences evolved to recognize structurally related metabolites. Although we know that these include the monolignols, hydroxycinnamates, benzoates, and auxins (Lim et al., 2001
, 2002
; Jackson et al., 2001
), the precise identity of other endogenous metabolites is yet to be revealed.
Conclusions on the correlation between sequence similarities and substrate recognition have been drawn from positive results. Negative results are more difficult to interpret because there are at least two possible explanations. Individuals of an entire group may be all catalytically inactive. Alternatively, the group members simply may not recognize the features of hydroxycoumarins, but instead recognize quite different compounds. It may be relevant that all of the compounds we have analyzed so far are metabolites on the shikimate pathway.
In addition to overall substrate recognition, an interesting observation has emerged concerning regioselectivity of glucosylation in group E. Following an ancestral change in regioselectivity between 6- and 7-OH glucosylation, all individuals of the two separate branches retain the different regioselectivity. In a much more recent evolutionary event, another switching event has occurred once (Figures 3 and 6).
Data gained in this study of a multigene family in Arabidopsis show a correlation between phylogenetic relatedness and substrate recognition. The question arises as to whether there is a similar correlation when UGTs from different plant species are analyzed. Two recent reviews have summarized plant UGTs and their catalytic activities (reviewed in Jones and Vogt, 2001; Keegstra and Raikhel, 2001
). When these sequences are included in the Arabidopsis phylogenetic tree, the eight UGTs known to recognize substrates structurally similar to the hydroxycoumarins are all located in the same three major groups D, E, and L (Li et al., 2001
; Ross et al., 2001
). It will be interesting to examine UGTs from rice, of which the genome has been sequenced (Yu et al., 2002
; Goff et al., 2002
), to determine whether substrate recognition again remains conserved across the major phylogenetic groups.
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Materials and methods |
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Glucosyltransferase activity assay
Recombinant UGTs were purified as GST fusion proteins from E. coli carrying the expression constructs following the methods described previously (Lim et al., 2001). Each glucosyltransferase activity assay mix (200 µl) contained 1 µg of recombinant protein, 50 mM Tris-HCl, pH 7.0, 14 mM 2-mercaptoethanol, 5 mM UDP-glucose, and 1 mM phenolic substrate. The reaction was carried out at 30°C for 30 min and was stopped by the addition of 20 µl trichloroacetic acid (240 mg/ml), quick-frozen, and stored at -20°C prior to the reverse-phase HPLC analysis. The specific enzyme activity was expressed as nmol of phenolic compound glucosylated per second (nkat) by 1 mg of protein in 30 min of reaction time.
HPLC analysis
Reverse-phase HPLC (SpectraSYSTEM HPLC systems and UV6000LP Photodiode Array Detector, ThermoQuest, Manchester, UK) analyses were carried out using a Columbus 5 µ C18 column (250x4.6 mm, Phenomenex, Macclesfield, UK). 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: esculetin, 296\ nm, 1020% acetonitrile; scopoletin,
296\ nm, 1030% acetonitrile. The retention time (Rt) of the glucose conjugates analyzed was as follows: esculin, Rt=9.7 min; cichoriin, Rt=9.6 min; scopolin, Rt=9.9 min.
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Acknowledgements |
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Footnotes |
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1 To whom correspondence should be addressed; e-mail:djb32{at}york.ac.uk
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Abbreviations |
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References |
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