Sandra L. Baldauf

Current understanding of the higher order systematics of eukaryotes relies largely on analyses of the small ribosomal subunit RNA (SSU rRNA). Independent testing of these results is still limited. We have combined the sequences of four of the most broadly taxonomically sampled proteins available to create a roughly parallel data set to that of SSU rRNA. The resulting phylogenetic tree shows a number of striking differences from SSU rRNA phylogeny, including strong support for most major groups and several major supergroups.

Most cultivated and characterized eukaryotes can be confidently assigned to one of eight major groups. After a few false starts, we are beginning to resolve relationships among these major groups as well. However, recent developments are radically revising this picture again, particularly (i) the discovery of the likely antiquity and taxonomic diversity of ultrasmall eukaryotes, and (ii) a fundamental rethinking of the position of the root. Together these data suggest major gaps in our understanding simply of what eukaryotes are or, when it comes to the tree, even which end is up.

A class of UDP-glycosyltransferases (UGTs) defined by the presence of a C-terminal consensus sequence is found throughout the plant and animal kingdoms. Whereas mammalian enzymes use UDP-glucuronic acid, the plant enzymes typically use UDP-glucose in the transfer reactions. A diverse array of aglycones can be glucosylated by these UGTs. In plants, the aglycones include plant hormones, secondary metabolites involved in stress and defense responses, and xenobiotics such as herbicides. Glycosylation is known to regulate many properties of the aglycones such as their bioactivity, their solubility, and their transport properties within the cell and throughout the plant. As a means of providing a framework to start to understand the substrate specificities and structure-function relationships of plant UGTs, we have now applied a molecular phylogenetic analysis to the multigene family of 99 UGT sequences inArabidopsis. We have determined the overall organization and evolutionary relationships among individual members with a surprisingly high degree of confidence. Through constructing a composite phylogenetic tree that also includes all of the additional plant UGTs with known catalytic activities, we can start to predict both the evolutionary history and substrate specificities of new sequences as they are identified. The tree already suggests that while the activities of some subgroups of the UGT family are highly conserved among different plant species, others subgroups shift substrate specificity with relative ease. A class of UDP-glycosyltransferases (UGTs) defined by the presence of a C-terminal consensus sequence is found throughout the plant and animal kingdoms. Whereas mammalian enzymes use UDP-glucuronic acid, the plant enzymes typically use UDP-glucose in the transfer reactions. A diverse array of aglycones can be glucosylated by these UGTs. In plants, the aglycones include plant hormones, secondary metabolites involved in stress and defense responses, and xenobiotics such as herbicides. Glycosylation is known to regulate many properties of the aglycones such as their bioactivity, their solubility, and their transport properties within the cell and throughout the plant. As a means of providing a framework to start to understand the substrate specificities and structure-function relationships of plant UGTs, we have now applied a molecular phylogenetic analysis to the multigene family of 99 UGT sequences inArabidopsis. We have determined the overall organization and evolutionary relationships among individual members with a surprisingly high degree of confidence. Through constructing a composite phylogenetic tree that also includes all of the additional plant UGTs with known catalytic activities, we can start to predict both the evolutionary history and substrate specificities of new sequences as they are identified. The tree already suggests that while the activities of some subgroups of the UGT family are highly conserved among different plant species, others subgroups shift substrate specificity with relative ease. UDP-glycosyltransferase open reading frame polymerase chain reaction Glycosyltransferases are found in all living organisms, catalyzing the transfer of a glycosyl moiety from an activated donor to an acceptor molecule, forming a glycosidic bond. These glycosyl transfer reactions have been highlighted as the most important biotransformation on earth, since in quantitative terms they account for the assembly and degradation of the bulk of biomass (1Campbell J.A. Davies G.J. Bulone V. Henrissat B. Biochem. J. 1997; 326: 929-942Crossref PubMed Scopus (624) Google Scholar).A unique signature motif has been identified in the amino acid sequence of many of these glycosyltransferases, leading to their classification into a single UDP-glycosyltransferase (UGT)1 superfamily (2Mackenzie P.I. Owens I.S. Burchell B. Bock K.W. Bairoch A. Belanger 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). Of these, the mammalian UGTs using UDP-glucuronic acid have attracted considerable attention in pharmaceutical and clinical research due to their central role in the metabolism and detoxification of foreign chemicals such as carcinogens and hydrophobic drugs (3de Wildt S.N. Kearns G.L. Leeder J.S. van den Anker J.N. Clin. Pharmacokinet. 1999; 36: 439-452Crossref PubMed Scopus (353) Google Scholar, 4Nebert D.W. Biochem. Pharmacol. 1994; 47: 25-37Crossref PubMed Scopus (208) Google Scholar).Plant UGTs are involved in a parallel range of activities, the majority of which use UDP-glucose in the transfer reaction. These reactions are known to have a number of important consequences. First, compounds can be activated or inactivated by their conjugation to glucose. For example, glucose esters are high energy compounds that are known to act as biosynthetic intermediates for further reactions involving the aglycones (5Mock H. Strack D. Phytochemistry. 1993; 32: 575-579Crossref Scopus (70) Google Scholar). In contrast, many of the plant hormones are known to be inactivated following glucosylation (6Sembdner G. Atzorn R. Schneider G. Plant Mol. Biol. 1994; 26: 1459-1481Crossref PubMed Scopus (102) Google Scholar, 7Kleckowski K. Schell J. Crit. Rev. Plant Sci. 1995; 14: 283-298Crossref Scopus (86) Google Scholar). Second, glucosylation alters the solubility of compounds by increasing their hydrophilic properties and providing access to active membrane transport systems that recognize the glucosides but not the aglycones (8Hostel W. The Biochemistry of Plants. 7. Academic Press, Inc., New York1981: 725-753Google Scholar).As a consequence of these events, glucosylation plays a crucial role in the maintenance of cellular homeostasis in plants through regulating the level, activity, and location of key cellular metabolites. Despite this general importance and the likely large number of these enzymes, given the diversity of substrates, plant UGTs have not been studied systematically. Rather, individual UGTs have been purified on the basis of a particular catalytic activity (9–20). The disadvantage of this approach is that the relationships of different UGTs cannot be defined easily, and, in consequence, predictions of catalytic activities based on structure-function relatedness cannot be made.Genome sequencing programs offer a new route into understanding multigene families both within a single species and across different species. In this study, we have used the data available from theArabidopsis genome sequencing program to start to build a foundation for understanding the UGT multigene family. This analysis focuses on the phylogeny and evolution of UGTs and complements parallel investigations into substrate specificity using recombinant proteins corresponding to known UGT sequences (21Lim E.-K. Li Y. Parr A. Jackson R. Ashford D.A. Bowles D.J. J. Biol. Chem. 2001; 276: 4344-4349Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 22Jackson 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).Note Added in ProofAn additional 18 complete UGT sequences have been identified in Arabidopsis genome data base subsequent to the submission of this manuscript. These do not change the composition of the groups defined in this manuscript, and they add an additional two groups to the tree. Glycosyltransferases are found in all living organisms, catalyzing the transfer of a glycosyl moiety from an activated donor to an acceptor molecule, forming a glycosidic bond. These glycosyl transfer reactions have been highlighted as the most important biotransformation on earth, since in quantitative terms they account for the assembly and degradation of the bulk of biomass (1Campbell J.A. Davies G.J. Bulone V. Henrissat B. Biochem. J. 1997; 326: 929-942Crossref PubMed Scopus (624) Google Scholar). A unique signature motif has been identified in the amino acid sequence of many of these glycosyltransferases, leading to their classification into a single UDP-glycosyltransferase (UGT)1 superfamily (2Mackenzie P.I. Owens I.S. Burchell B. Bock K.W. Bairoch A. Belanger 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). Of these, the mammalian UGTs using UDP-glucuronic acid have attracted considerable attention in pharmaceutical and clinical research due to their central role in the metabolism and detoxification of foreign chemicals such as carcinogens and hydrophobic drugs (3de Wildt S.N. Kearns G.L. Leeder J.S. van den Anker J.N. Clin. Pharmacokinet. 1999; 36: 439-452Crossref PubMed Scopus (353) Google Scholar, 4Nebert D.W. Biochem. Pharmacol. 1994; 47: 25-37Crossref PubMed Scopus (208) Google Scholar). Plant UGTs are involved in a parallel range of activities, the majority of which use UDP-glucose in the transfer reaction. These reactions are known to have a number of important consequences. First, compounds can be activated or inactivated by their conjugation to glucose. For example, glucose esters are high energy compounds that are known to act as biosynthetic intermediates for further reactions involving the aglycones (5Mock H. Strack D. Phytochemistry. 1993; 32: 575-579Crossref Scopus (70) Google Scholar). In contrast, many of the plant hormones are known to be inactivated following glucosylation (6Sembdner G. Atzorn R. Schneider G. Plant Mol. Biol. 1994; 26: 1459-1481Crossref PubMed Scopus (102) Google Scholar, 7Kleckowski K. Schell J. Crit. Rev. Plant Sci. 1995; 14: 283-298Crossref Scopus (86) Google Scholar). Second, glucosylation alters the solubility of compounds by increasing their hydrophilic properties and providing access to active membrane transport systems that recognize the glucosides but not the aglycones (8Hostel W. The Biochemistry of Plants. 7. Academic Press, Inc., New York1981: 725-753Google Scholar). As a consequence of these events, glucosylation plays a crucial role in the maintenance of cellular homeostasis in plants through regulating the level, activity, and location of key cellular metabolites. Despite this general importance and the likely large number of these enzymes, given the diversity of substrates, plant UGTs have not been studied systematically. Rather, individual UGTs have been purified on the basis of a particular catalytic activity (9–20). The disadvantage of this approach is that the relationships of different UGTs cannot be defined easily, and, in consequence, predictions of catalytic activities based on structure-function relatedness cannot be made. Genome sequencing programs offer a new route into understanding multigene families both within a single species and across different species. In this study, we have used the data available from theArabidopsis genome sequencing program to start to build a foundation for understanding the UGT multigene family. This analysis focuses on the phylogeny and evolution of UGTs and complements parallel investigations into substrate specificity using recombinant proteins corresponding to known UGT sequences (21Lim E.-K. Li Y. Parr A. Jackson R. Ashford D.A. Bowles D.J. J. Biol. Chem. 2001; 276: 4344-4349Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 22Jackson 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). Note Added in ProofAn additional 18 complete UGT sequences have been identified in Arabidopsis genome data base subsequent to the submission of this manuscript. These do not change the composition of the groups defined in this manuscript, and they add an additional two groups to the tree. An additional 18 complete UGT sequences have been identified in Arabidopsis genome data base subsequent to the submission of this manuscript. These do not change the composition of the groups defined in this manuscript, and they add an additional two groups to the tree. We thank the Arabidopsis Biological Resource Center for providing all expressed sequence tag clones. We also thank Dr. Joe Ross for critical reading of the manuscript and helpful discussions. Supplementary Material Download .pdf (.05 MB) Help with pdf files Download .pdf (.05 MB) Help with pdf files