The Activity of ArabidopsisGlycosyltransferases toward Salicylic Acid, 4-Hydroxybenzoic Acid, and Other Benzoates
2002; Elsevier BV; Volume: 277; Issue: 1 Linguagem: Inglês
10.1074/jbc.m109287200
ISSN1083-351X
AutoresEng‐Kiat Lim, Charlotte J. Doucet, Yi Li, Luisa Elias, Dawn Worrall, Steven P. Spencer, Joe Ross, Dianna J. Bowles,
Tópico(s)Phytochemicals and Antioxidant Activities
ResumoBenzoates are a class of natural products containing compounds of industrial and strategic importance. In plants, the compounds exist in free form and as conjugates to a wide range of other metabolites such as glucose, which can be attached to the carboxyl group or to specific hydroxyl groups on the benzene ring. These glucosylation reactions have been studied for many years, but to date only one gene encoding a benzoate glucosyltransferase has been cloned. A phylogenetic analysis of sequences in theArabidopsis genome revealed a large multigene family of putative glycosyltransferases containing a consensus sequence typically found in enzymes transferring glucose to small molecular weight compounds such as secondary metabolites. Ninety of these sequences have now been expressed as recombinant proteins in Escherichia coli, and their in vitro catalytic activities toward benzoates have been analyzed. The data show that only 14 proteins display activity toward 2-hydroxybenzoic acid, 4-hydroxybenzoic acid, and 3,4-dihydroxybenzoic acid. Of these, only two enzymes are active toward 2-hydroxybenzoic acid, suggesting they are theArabidopsis salicylic acid glucosyltransferases. All of the enzymes forming glucose esters with the metabolites were located in Group L of the phylogenetic tree, whereas those formingO-glucosides were dispersed among five different groups. Catalytic activities were observed toward glucosylation of the 2-, 3-, or 4-hydroxyl group on the ring. To further explore their regioselectivity, the 14 enzymes were analyzed against benzoic acid, 3-hydroxybenzoic acid, 2,3-, 2,4-, 2,5-, and 2,6-dihydroxybenzoic acid. The data showed that glycosylation of specific sites could be positively or negatively influenced by the presence of additional hydroxyl groups on the ring. This study provides new tools for biotransformation reactions in vitro and a basis for engineering benzoate metabolism in plants. Benzoates are a class of natural products containing compounds of industrial and strategic importance. In plants, the compounds exist in free form and as conjugates to a wide range of other metabolites such as glucose, which can be attached to the carboxyl group or to specific hydroxyl groups on the benzene ring. These glucosylation reactions have been studied for many years, but to date only one gene encoding a benzoate glucosyltransferase has been cloned. A phylogenetic analysis of sequences in theArabidopsis genome revealed a large multigene family of putative glycosyltransferases containing a consensus sequence typically found in enzymes transferring glucose to small molecular weight compounds such as secondary metabolites. Ninety of these sequences have now been expressed as recombinant proteins in Escherichia coli, and their in vitro catalytic activities toward benzoates have been analyzed. The data show that only 14 proteins display activity toward 2-hydroxybenzoic acid, 4-hydroxybenzoic acid, and 3,4-dihydroxybenzoic acid. Of these, only two enzymes are active toward 2-hydroxybenzoic acid, suggesting they are theArabidopsis salicylic acid glucosyltransferases. All of the enzymes forming glucose esters with the metabolites were located in Group L of the phylogenetic tree, whereas those formingO-glucosides were dispersed among five different groups. Catalytic activities were observed toward glucosylation of the 2-, 3-, or 4-hydroxyl group on the ring. To further explore their regioselectivity, the 14 enzymes were analyzed against benzoic acid, 3-hydroxybenzoic acid, 2,3-, 2,4-, 2,5-, and 2,6-dihydroxybenzoic acid. The data showed that glycosylation of specific sites could be positively or negatively influenced by the presence of additional hydroxyl groups on the ring. This study provides new tools for biotransformation reactions in vitro and a basis for engineering benzoate metabolism in plants. UDP-glycosyltransferase high pressure liquid chromatography benzoic acid hydroxybenzoic acid dihydroxybenzoic acid indole-3-acetic acid Glycosyltransferases catalyze the transfer of sugar from nucleoside diphosphate donors to a wide range of acceptor molecules (1Hostel W. The Biochemistry of Plants. 7. Academic Press, Inc., New York1981: 725-753Google Scholar). In recent years, the enzymes have been classified in a number of different ways including overall sequence homology (2Campbell J.A. Davies G.J. Bulone V. Henrissat B. Biochem. J. 1997; 326: 929-942Crossref PubMed Scopus (624) Google Scholar) and the presence of a variety of common motifs (3Bairoch A. Nucleic Acids Res. 1991; 19: 2241-2245Crossref PubMed Scopus (518) Google Scholar, 4Hughes J. Hughes M.A. DNA Seq. 1994; 5: 41-49Crossref PubMed Scopus (164) Google Scholar, 5Mackenzie P.I. Owen I.S. Burchell B. Bock K.W. Bairoch A. Bélanger A. Fournel-Gigleux S. Green M. Hum D.W. Iyanagi T. Lancet D. Louisot P. Magdalou J. Chowdhury J.R. Ritter J.K. Schachter H. Tephly T.R. Tipton K.F. Nebert D.W. Pharmacogenetics. 1997; 7: 255-269Crossref PubMed Scopus (992) Google Scholar, 6Busch C. Hofmann F. Selzer J. Munro S. Jeckel D. Aktories K. J. Biol. Chem. 1998; 273: 19566-19572Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 7Henrissat B. Davies G.J. Plant Physiol. 2000; 124: 1515-1519Crossref PubMed Scopus (226) Google Scholar). Analysis of sequence homology has led to the classification of 54 families with a ubiquitous distribution throughout living organisms (7Henrissat B. Davies G.J. Plant Physiol. 2000; 124: 1515-1519Crossref PubMed Scopus (226) Google Scholar). One of the largest families is Family 1, in which many of its members contain a consensus sequence found toward the C termini of the proteins thought to be related to nucleoside diphosphate-sugar binding (8Ross J. Li Y. Lim E.-K. Bowles D.J. Genome Biol. 2001; 2 (reviews): 3004.1-3004.6Crossref Google Scholar). In a study of mammalian enzymes, the consensus has been used to define a large multigene family of UDP-glucuronosyltransferases (5Mackenzie P.I. Owen I.S. Burchell B. Bock K.W. Bairoch A. Bélanger A. Fournel-Gigleux S. Green M. Hum D.W. Iyanagi T. Lancet D. Louisot P. Magdalou J. Chowdhury J.R. Ritter J.K. Schachter H. Tephly T.R. Tipton K.F. Nebert D.W. Pharmacogenetics. 1997; 7: 255-269Crossref PubMed Scopus (992) Google Scholar). In plant species, many enzymes are known that also contain this consensus sequence, but in contrast to the mammalian enzymes, the plant enzymes generally use UDP-glucose as the donor in the transfer reaction (1Hostel W. The Biochemistry of Plants. 7. Academic Press, Inc., New York1981: 725-753Google Scholar, 9Jones P. Vogt T. Planta. 2001; 213: 164-174Crossref PubMed Scopus (318) Google Scholar).The plant UDP-glucosyltransferases (UGTs)1 containing the consensus sequence function in a variety of processes including hormonal homeostasis, pathways of secondary metabolism, and detoxification of xenobiotics, such as herbicides and pesticides (9Jones P. Vogt T. Planta. 2001; 213: 164-174Crossref PubMed Scopus (318) Google Scholar, 10Kleczkowski K. Schell J. Crit. Rev. Plant Sci. 1995; 14: 283-298Crossref Scopus (86) Google Scholar, 11Wetzel A. Sandermann H. Arch. Biochem. Biophys. 1994; 314: 323-328Crossref PubMed Scopus (28) Google Scholar, 12Davis D.G. Olson P.A. Swanson H.R. Frear D.S. Plant Sci. 1991; 74: 73-80Crossref Scopus (12) Google Scholar). Transfer of a sugar onto the acceptor molecule can lead to changes in the activity of the acceptor as well as changes in its subcellular localization. For example, glucosylation of plant hormones such as auxins, abscisic acid, and gibberellins inactivates their biological activity (10Kleczkowski K. Schell J. Crit. Rev. Plant Sci. 1995; 14: 283-298Crossref Scopus (86) Google Scholar). Once conjugated, the glycosylated acceptor can exit the cytosol and enter a membrane compartment through recognition by transporters, such as those identified in the tonoplast membrane of the vacuole, which is considered to be the major compartment for storage of the glucoconjugates (reviewed in Ref.13Rea P.A. J. Exp. Bot. 1999; 50: 895-913Crossref Google Scholar).Recently, we have used the consensus sequence to screen the genome data base of Arabidopsis to identify the number of putative enzymes of this class in a single plant species (14Li Y. Baldauf S. Lim E.-K. Bowles D.J. J. Biol. Chem. 2001; 276: 4338-4343Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar). We discovered the existence of a large multigene family composed of 107 sequences (8Ross J. Li Y. Lim E.-K. Bowles D.J. Genome Biol. 2001; 2 (reviews): 3004.1-3004.6Crossref Google Scholar). As one means of gaining an understanding of their potential functions, we have expressed the sequences in Escherichia coli and screened recombinant proteins for catalytic activity in vitro. This has led to the identification of genes encoding enzymes that glucosylate the monolignol precursors of lignin (15Lim E.-K. Li Y. Parr A. Jackson R.G. Ashford D.A. Bowles D.J. J. Biol. Chem. 2001; 276: 4344-4349Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar) and two auxins, indole-3-acetic acid (IAA) and indole butyric acid (16Jackson R.G. Lim E.-K. Li Y. Kowalczyk M. Sandberg G. Hoggett J. Ashford D.A. Bowles D.J. J. Biol. Chem. 2001; 276: 4350-4356Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar). The studies have established a foundation for understanding the physiological role of the enzymes, their substrates, and their productsin planta. For example, overexpression of the gene encoding UGT84B1, forming the glucose ester of IAA and indole butyric acidin vitro, produced transgenic Arabidopsis lines displaying a typical auxin-deficient phenotype. Application of 2,4-D, an auxin homologue not recognized as a substrate by the UGT, recovered the transgenic lines to a wild-type phenotype. 2R. G. Jackson, M. Kowalczyk, Y. Li, G. Higgins, J. Ross, G. Sandberg, and D. J. Bowles, submitted for publication.2R. G. Jackson, M. Kowalczyk, Y. Li, G. Higgins, J. Ross, G. Sandberg, and D. J. Bowles, submitted for publication.This study now extends the analysis of Arabidopsis UGTs to a new class of substrates, the benzoates (see Fig. 1). There is considerable interest in these compounds, both from the perspective of their functions in the plant, such as the role of salicylic acid in defense responses (reviewed in Ref. 18Alvarez M.E. Plant Mol. Biol. 2000; 44: 429-442Crossref PubMed Scopus (257) Google Scholar), and from their potential utility in biotechnological applications, such as the use of 4-hydroxybenzoic acid (4-HBA) as a polyester precursor and 3,4-dihydroxybenzoic acid (3,4-DHBA) as an antioxidant (19Dong D. Jiang S. Ni Y. Jiang B. Eur. Polym. J. 2001; 37: 611-617Crossref Scopus (11) Google Scholar, 20Gaisser S. Heide L. Phytochemistry. 1996; 41: 1065-1072Crossref Scopus (71) Google Scholar, 21Nakamura Y. Torikai K. Ohigashi H. Free Radic. Biol. Med. 2001; 30: 967-978Crossref PubMed Scopus (39) Google Scholar, 22Tyrakowska B. Soffers A.E.M.F. Szymusiak H. Boeren S. Boersma M.G. Lemańska K. Vervoort J. Rietjiens I.M.C.M. Free Radic. Biol. Med. 1999; 27: 1427-1436Crossref PubMed Scopus (88) Google Scholar, 23Rice-Evans C.A. Miller N.J. Paganga G. Free Radic. Biol. Med. 1996; 20: 933-956Crossref PubMed Scopus (7395) Google Scholar). Benzoates also offer an excellent opportunity to explore the regioselectivity of glucosylation given that hydroxylation can occur at any position on the benzene ring and a wide variety of compounds are available for use as potential substrates. The position of hydroxyl groups is known to influence many properties of the molecules, such as their polarity and their ability to scavenge free radicals (23Rice-Evans C.A. Miller N.J. Paganga G. Free Radic. Biol. Med. 1996; 20: 933-956Crossref PubMed Scopus (7395) Google Scholar, 24Chan W.-S. Wen P.-C. Chiang H.-C. Anticancer Res. 1995; 15: 703-708PubMed Google Scholar, 25Chimi H. Cillard J. Chillard P. Rahmani M. J. Am. Oil Chem. Soc. 1991; 68: 307-312Crossref Scopus (282) Google Scholar).Glucosylation at the carboxyl group of the aglycone forms a glucose ester, whereas glucosylation at any one of the hydroxyl positions on the ring forms the respective O-glucoside. Naturally occurring compounds are known in which the ortho (2-OH, 6-OH), meta (3-OH, 5-OH), and para (4-OH) hydroxyl groups are glucosylated (26Lee H.-I. Raskin I. Biochem. Cell Biol. 1998; 88: 692-697Google Scholar, 27Lee H.-I. Raskin I. J. Biol. Chem. 1999; 274: 36637-36642Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 28Yamanaka M. Shimomura K. Sasaki K. Yoshihira K. Ishimaru K. Phytochemistry. 1995; 40: 1149-1150Crossref Scopus (25) Google Scholar, 29Sakushima A. Coskun M. Maoka T. Phytochemistry. 1995; 40: 257-261Crossref Scopus (96) Google Scholar, 30Klick S. Herrmann K. Phytochemistry. 1988; 27: 2177-2180Crossref Scopus (76) Google Scholar, 31Kent P.W. Brunet P.C.J. Tetrahedron. 1959; 7: 252-256Crossref Scopus (24) Google Scholar). Glucose esters of aglycones are considered to be biosynthetic intermediates in which the high energy of the glucose ester drives transfer of the aglycone onto a further acceptor (32Mock H.P. Strack D. Phytochemistry. 1993; 32: 575-579Crossref Scopus (70) Google Scholar). For example, in phenylpropanoid metabolism inArabidopsis, there is good evidence that formation of sinapoyl glucose ester is an intermediate step in the synthesis of sinapoylmalate and sinapoylcholine (33Sharma V. Strack D. Planta. 1985; 163: 563-568Crossref PubMed Scopus (41) Google Scholar, 34Strack D. Knogge W. Dahlbender B. Z. Naturforsch. 1983; 38: 1-27Google Scholar, 35Strack D. Planta. 1982; 155: 31-36Crossref PubMed Scopus (51) Google Scholar, 36Strack D. Z. Pflanzenphysiol. 1977; 84: 139-145Crossref Google Scholar). Similarly, in maize the glucose ester of IAA is an intermediate in the formation of IAA-myo-inositol (37Michalczuk L. Bandurski R.S. Biochem. J. 1982; 207: 273-281Crossref PubMed Scopus (50) Google Scholar). In contrast, O-glucosides are thought to be more stable forms of the aglycones and often are those that are transported either from the cytosol into membrane compartments or throughout the plant (1Hostel W. The Biochemistry of Plants. 7. Academic Press, Inc., New York1981: 725-753Google Scholar). In this context, inhibition of the synthesis of the naphthoquinone pigment shikonin leads to abnormally high levels of 4-HBA in the cytosol that is subsequently detoxified by glucosylation and transported into the vacuole (38Bechthold A. Berger U. Heide L. Arch. Biochem. Biophys. 1991; 288: 39-47Crossref PubMed Scopus (23) Google Scholar, 39Heide L. Nishioka N. Fukui H. Tabata M. Phytochemistry. 1989; 28: 1873-1877Crossref Scopus (82) Google Scholar, 40Yazaki K. Fukui H. Tabata M. Phytochemistry. 1986; 25: 1629-1632Crossref Scopus (47) Google Scholar, 41Yazaki K. Inushima K. Kataoka M. Tabata M. Phytochemistry. 1995; 38: 1127-1130Crossref Scopus (49) Google Scholar).In this study we have screened the activity of 90 recombinant UGTs toward a panel of three model benzoates chosen for their biological relevance and position of their hydroxyl groups on the ring. The 14 UGTs that displayed activities toward these three compounds have been further analyzed against additional substrates (Fig.1) to confirm the regioselectivity of their catalytic activities.DISCUSSIONPlant glucosyltransferases such as those involved in cytoplasmic glycosylation typically transfer glucose from UDP-glucose to small acceptor molecules. There is considerable interest currently in this class of enzymes because they offer a simple means of regioselective glycosylation that is extremely difficult to achieve by chemical synthesis. The enzymes also offer a potential means to engineer pathways of secondary metabolism in plants and thereby to use the synthetic capacity of crops to manipulate novel industrial products. Despite these opportunities, comparatively few glycosyltransferases have been purified from plant extracts, and even fewer numbers have been cloned (reviewed in Refs. 8Ross J. Li Y. Lim E.-K. Bowles D.J. Genome Biol. 2001; 2 (reviews): 3004.1-3004.6Crossref Google Scholar and 9Jones P. Vogt T. Planta. 2001; 213: 164-174Crossref PubMed Scopus (318) Google Scholar). Traditionally, the route to gaining glycosyltransferase genes has been purification of the enzyme and use of its sequence. This approach can involve many years of work, particularly when the enzymes are present in negligible amounts in the plant. Nevertheless, despite these obstacles, the genes corresponding to salicylic acid glucosyltransferase (27Lee H.-I. Raskin I. J. Biol. Chem. 1999; 274: 36637-36642Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar), flavonol-3-O-glucosyltransferase (44Miller K.D. Guyon V. Evans J.N.S. Shuttleworth W.A. Taylor L.P. J. Biol. Chem. 1999; 274: 34011-34019Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar), and betanidin-5-O-glucosyltransferase (45Vogt T. Grimm R. Strack D. Plant J. 1999; 19: 509-519Crossref PubMed Scopus (111) Google Scholar) are good examples of where the approach has led to considerable benefits.An alternative approach is to apply techniques of bioinformatics to screen genomic and EST data bases to identify sequences that may correspond to those encoding functional enzymes. This approach is made easier with the completion of a genome-sequencing program of many organisms. In this context, analysis of the completed genome ofArabidopsis revealed 107 sequences containing a consensus motif that suggested they correspond to glycosyltransferases (8Ross J. Li Y. Lim E.-K. Bowles D.J. Genome Biol. 2001; 2 (reviews): 3004.1-3004.6Crossref Google Scholar, 14Li Y. Baldauf S. Lim E.-K. Bowles D.J. J. Biol. Chem. 2001; 276: 4338-4343Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar) such as those that conjugate hormones, secondary metabolites, and xenobiotics. As a means of defining whether these sequences corresponded to functional enzymes and identifying their potential substrates, recombinant proteins produced in E. coli were screened for catalytic activities in vitro. This strategy led to the first identification of genes encoding glucosyltransferases of the lignin precursors including coniferyl alcohol and sinapyl alcohol (15Lim E.-K. Li Y. Parr A. Jackson R.G. Ashford D.A. Bowles D.J. J. Biol. Chem. 2001; 276: 4344-4349Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). The studies have also demonstrated that functional annotation in multigene families can be misleading both through misannotation and through the lack of predictability of function through sequence homology. For example, the IAA glucosyltransferase ofArabidopsis did not correspond to any of the sequences previously annotated with this activity, including that with the greatest sequence homology to the maize iaglu sequence (16Jackson R.G. Lim E.-K. Li Y. Kowalczyk M. Sandberg G. Hoggett J. Ashford D.A. Bowles D.J. J. Biol. Chem. 2001; 276: 4350-4356Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar).In the present study we have applied the strategy of in vitro screening for catalytic activities toward another class of natural products, the benzoates. This class includes many compounds of interest, both for their known activities in the plant and their potential applications. For example, 2-HBA, commonly known as salicylic acid, is important in plant defense responses and is known to up- and down-regulate a wide spectrum of genes (46Glazebrook J. Curr. Opin. Plant Biol. 2001; 4: 301-308Crossref PubMed Scopus (570) Google Scholar). Glucosylation of salicylic acid in planta has been well characterized, although the function(s) of the glucoside and glucose ester remain obscure (26Lee H.-I. Raskin I. Biochem. Cell Biol. 1998; 88: 692-697Google Scholar, 27Lee H.-I. Raskin I. J. Biol. Chem. 1999; 274: 36637-36642Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). It has been speculated that glucosylation represents detoxification of salicylic acid. Another benzoate, 4-HBA, has attracted considerable interest due to its potential as a polyester precursor (19Dong D. Jiang S. Ni Y. Jiang B. Eur. Polym. J. 2001; 37: 611-617Crossref Scopus (11) Google Scholar). The compound is also the precursor in planta of shikonin, a natural pigment of commercial relevance (for review, see Ref. 47Papageorgiou V.P. Assimopoulou A.N. Couladouros E.A. Hepworth D. Nicolaou K.C. Angew. Chem. Int. Ed. 1999; 38: 270-300Crossref PubMed Scopus (524) Google Scholar). Whereas 4-HBA is a cytoplasmic component, its O-glucoside is located in the vacuole (41Yazaki K. Inushima K. Kataoka M. Tabata M. Phytochemistry. 1995; 38: 1127-1130Crossref Scopus (49) Google Scholar). This change in subcellular compartmentation is considered to be part of the detoxification process but, significantly, is also a potential mechanism for increasing yield through the removal of product from the cytoplasmic reaction mixture. 3,4-DHBA is yet another commercially interesting benzoate because of its antioxidant activity (21Nakamura Y. Torikai K. Ohigashi H. Free Radic. Biol. Med. 2001; 30: 967-978Crossref PubMed Scopus (39) Google Scholar). Glucosylation is also relevant to this application, given observations that glucosides of antioxidant aglycones have increased bioavailability in the diet (48Nishimura T. Kometani T. Takii H. Terada Y. Okada S. J. Ferment. Bioeng. 1995; 80: 18-23Crossref Scopus (33) Google Scholar, 49Hollman P.C.H. van Trijp J.M.P. Buysman M.N.C.P. van der Gaag M.S. Mengelers M.J.B. de Vries J.H.M. Katan M.B. FEBS Lett. 1997; 418: 152-156Crossref PubMed Scopus (698) Google Scholar). The glycosyltransferases involved in the conjugation of these three compounds have been studied over many years. In the case of 2-HBA, the enzyme has been purified to homogeneity from tobacco extracts and used to identify its corresponding gene. The recombinant protein was assayedin vitro and shown to form the glucose ester in preference to the O-glucoside (27Lee H.-I. Raskin I. J. Biol. Chem. 1999; 274: 36637-36642Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). For 4-HBA, an enzyme has been purified from L. erythrorhizon, but as yet the gene has not been cloned (50Li S.-M. Wang Z.-X. Heide L. Phytochemistry. 1997; 46: 27-32Crossref Scopus (11) Google Scholar). Work on glucosyltransferases of 3,4-DHBA is even less developed; the product has been identified in plant extracts, but the enzyme has not been studied.Using the three benzoates in a preliminary screen of 90 recombinant proteins from the Arabidopsis multigene family, we have now identified 14 genes encoding enzymes with catalytic activity toward one or more of these substrates. The study has highlighted a regioselectivity exhibited by the plant glycosyltransferases that is maintained in vitro. For example, only two of the enzymes can glucosylate the 2-OH position of the benzene ring, UGT74F1 and UGT74F2. Although one of the two, UGT74F2, can also glucosylate the carboxyl group of salicylic acid, UGT74F1 is highly specific forO-glucoside formation. Interestingly, if the tobacco UGT of salicylic acid is used to search the phylogenetic tree ofArabidopsis sequences, the branch of the tree that is identified is precisely that containing UGT74F1 and UGT74F2 (data not shown). This correct prediction of function from sequence homology contrasts with that described earlier for the IAA glucosyltransferase (16Jackson R.G. Lim E.-K. Li Y. Kowalczyk M. Sandberg G. Hoggett J. Ashford D.A. Bowles D.J. J. Biol. Chem. 2001; 276: 4350-4356Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar). By comparing the biochemistry of the catalytic activity of the tobacco and Arabidopsis UGTs toward salicylic acid, it is possible that UGT74F2 is the orthologue of the tobacco gene.In contrast to the selectivity of UGT74F1 and UGT74F2 for the 2-OH position, many more enzymes were found to be selective for either the 3-OH position or the 4-OH position. In the case ofO-glucoside synthesis as well as the synthesis of glucose esters, the in vitro activity of the enzymes was found to be strongly influenced by the relative position of additional hydroxyl groups to the glucosylation site. The influence could be positive or negative, since examples for each were observed.This study demonstrates the utility of a screening strategy to identify novel glycosyltransferases that are active toward substrates of importance. As such, a foundation is established for using the enzymes as regioselective biocatalysts or further refining their catalytic activities by forced evolution of their corresponding genes. Their use in biotransformation is made more feasible by methods newly available for high yielding enzyme-catalyzed synthesis of the UDP-glucose substrate (51Ma X. Stöckigt J. Carbohydr. Res. 2001; 333: 159-163Crossref PubMed Scopus (29) Google Scholar). Similarly, a knowledge of catalytic activity in vitro also provides a new foundation for understanding the role of the enzymes in the plant. In this context, overexpression of the IAA glucosyltransferase proved sufficient to deregulate auxin homeostasis.2 In a similar manner, it should now be possible to determine the consequences of deregulating salicylic acid homeostasis in Arabidopsis and to study changes in the progress of plant defense responses. Also, importantly, combining overexpression of bacterial enzymes and the relevant plant glucosyltransferases such as in the synthesis and conjugation of 4-HBA (17Siebert M. Sommer S. Li S.-M. Wang Z.-X. Severin K. Heide L. Plant Physiol. 1996; 112: 811-819Crossref PubMed Scopus (79) Google Scholar, 43Sommer S. Heide L. Plant Cell Physiol. 1998; 39: 1240-1244Crossref PubMed Scopus (19) Google Scholar, 52Sommer S. Köhle A. Yazaki K. Shimomura K. Bechtold A. Heide L. Plant Mol. Biol. 1999; 39: 683-693Crossref PubMed Scopus (41) Google Scholar) may well offer a mechanism of engineering both the production of higher levels of the metabolite and increasing its stable storage in the plant. Glycosyltransferases catalyze the transfer of sugar from nucleoside diphosphate donors to a wide range of acceptor molecules (1Hostel W. The Biochemistry of Plants. 7. Academic Press, Inc., New York1981: 725-753Google Scholar). In recent years, the enzymes have been classified in a number of different ways including overall sequence homology (2Campbell J.A. Davies G.J. Bulone V. Henrissat B. Biochem. J. 1997; 326: 929-942Crossref PubMed Scopus (624) Google Scholar) and the presence of a variety of common motifs (3Bairoch A. Nucleic Acids Res. 1991; 19: 2241-2245Crossref PubMed Scopus (518) Google Scholar, 4Hughes J. Hughes M.A. DNA Seq. 1994; 5: 41-49Crossref PubMed Scopus (164) Google Scholar, 5Mackenzie P.I. Owen I.S. Burchell B. Bock K.W. Bairoch A. Bélanger A. Fournel-Gigleux S. Green M. Hum D.W. Iyanagi T. Lancet D. Louisot P. Magdalou J. Chowdhury J.R. Ritter J.K. Schachter H. Tephly T.R. Tipton K.F. Nebert D.W. Pharmacogenetics. 1997; 7: 255-269Crossref PubMed Scopus (992) Google Scholar, 6Busch C. Hofmann F. Selzer J. Munro S. Jeckel D. Aktories K. J. Biol. Chem. 1998; 273: 19566-19572Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 7Henrissat B. Davies G.J. Plant Physiol. 2000; 124: 1515-1519Crossref PubMed Scopus (226) Google Scholar). Analysis of sequence homology has led to the classification of 54 families with a ubiquitous distribution throughout living organisms (7Henrissat B. Davies G.J. Plant Physiol. 2000; 124: 1515-1519Crossref PubMed Scopus (226) Google Scholar). One of the largest families is Family 1, in which many of its members contain a consensus sequence found toward the C termini of the proteins thought to be related to nucleoside diphosphate-sugar binding (8Ross J. Li Y. Lim E.-K. Bowles D.J. Genome Biol. 2001; 2 (reviews): 3004.1-3004.6Crossref Google Scholar). In a study of mammalian enzymes, the consensus has been used to define a large multigene family of UDP-glucuronosyltransferases (5Mackenzie P.I. Owen I.S. Burchell B. Bock K.W. Bairoch A. Bélanger A. Fournel-Gigleux S. Green M. Hum D.W. Iyanagi T. Lancet D. Louisot P. Magdalou J. Chowdhury J.R. Ritter J.K. Schachter H. Tephly T.R. Tipton K.F. Nebert D.W. Pharmacogenetics. 1997; 7: 255-269Crossref PubMed Scopus (992) Google Scholar). In plant species, many enzymes are known that also contain this consensus sequence, but in contrast to the mammalian enzymes, the plant enzymes generally use UDP-glucose as the donor in the transfer reaction (1Hostel W. The Biochemistry of Plants. 7. Academic Press, Inc., New York1981: 725-753Google Scholar, 9Jones P. Vogt T. Planta. 2001; 213: 164-174Crossref PubMed Scopus (318) Google Scholar). The plant UDP-glucosyltransferases (UGTs)1 containing the consensus sequence function in a variety of processes including hormonal homeostasis, pathways of secondary metabolism, and detoxification of xenobiotics, such as herbicides and pesticides (9Jones P. Vogt T. Planta. 2001; 213: 164-174Crossref PubMed Scopus (318) Google Scholar, 10Kleczkowski K. Schell J. Crit. Rev. Plant Sci. 1995; 14: 283-298Crossref Scopus (86) Google Scholar, 11Wetzel A. Sandermann H. Arch. Biochem. Biophys. 1994; 314: 323-328Crossref PubMed Scopus (28) Google Scholar, 12Davis D.G. Olson P.A. Swanson H.R. Frear D.S. Plant Sci. 1991; 74: 73-80Crossref Scopus (12) Google Scholar). Transfer of a sugar onto the acceptor molecule can lead to changes in the activity of the acceptor as well as changes in its subcellular localization. For example, glucosylation of plant hormones such as auxins, abscisic acid, and gibberellins inactivates their biological activity (10Kleczkowski K. Schell J. Crit. Rev. Plant Sci. 1995; 14: 283-298Crossref Scopus (86) Google Scholar). Once conjugated, the glycosylated acceptor can exit the cytosol
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