Identification and Biochemical Characterization of anArabidopsis Indole-3-acetic Acid Glucosyltransferase

Jackson, Rosamond G.; Lim, Eng‐Kiat; Li, Yang; Kowalczyk, Mariusz; Sandberg, Göran; Hoggett, J. G.; Ashford, David A.; Bowles, Dianna J.

doi:10.1074/jbc.m006185200

Cited by 254 publications

(230 citation statements)

References 39 publications

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“…Similarly, indole-3-acetic acid (IAA)-Glc synthesized by indole-3-acetic acid glucosyltransferase serves as an activated form of IAA, which is used in a transesterification reaction to form IAA-inositol (Michalczuk and Bandurski, 1982;Jackson et al, 2001Jackson et al, , 2002Ljung et al, 2002). This latter reaction is analogous to sinapoylmalate biosynthesis and preliminary indications suggest that it may be catalyzed by an SCPL enzyme (Kowalczyk et al, 2003).…”

Section: Candidate Reactions For Scpl Acyltransferases Can Be Found Tmentioning

confidence: 99%

“…In the course of evolutionary history, the genes that encode these proteins have diversified to coordinate these metabolic pathways unique to plants. Examples that have been particularly well studied include the Cyt P450-dependent monooxygenases, MYB transcription factors, terpene synthases, and UDPglucosyltransferases (Meyer et al, 1996;Mizutani et al, 1998;Jackson et al, 2001;Li et al, 2001b;Lim et al, 2001;Stracke et al, 2001;Aubourg et al, 2002;Jackson et al, 2002;Naur et al, 2003). SCPL proteins appear to represent a similar case of recruitment and diversification in plant lineages that has received limited attention to date.…”

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confidence: 99%

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An Expression and Bioinformatics Analysis of the Arabidopsis Serine Carboxypeptidase-Like Gene Family

2005

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The Arabidopsis (Arabidopsis thaliana) genome encodes a family of 51 proteins that are homologous to known serine carboxypeptidases. Based on their sequences, these serine carboxypeptidase-like (SCPL) proteins can be divided into several major clades. The first group consists of 21 proteins which, despite the function implied by their annotation, includes two that have been shown to function as acyltransferases in plant secondary metabolism: sinapoylglucose:malate sinapoyltransferase and sinapoylglucose:choline sinapoyltransferase. A second group comprises 25 SCPL proteins whose biochemical functions have not been clearly defined. Genes encoding representatives from both of these clades can be found in many plants, but have not yet been identified in other phyla. In contrast, the remaining SCPL proteins include five members that are similar to serine carboxypeptidases from a variety of organisms, including fungi and animals. Reverse transcription PCR results suggest that some SCPL genes are expressed in a highly tissue-specific fashion, whereas others are transcribed in a wide range of tissue types. Taken together, these data suggest that the Arabidopsis SCPL gene family encodes a diverse group of enzymes whose functions are likely to extend beyond protein degradation and processing to include activities such as the production of secondary metabolites.Serine carboxypeptidases (SCPs) are members of the a/b hydrolase family of proteins, which make use of a Ser-Asp-His catalytic triad to cleave the carboxyterminal peptide bonds of their protein or peptide substrates (Hayashi et al., 1973(Hayashi et al., , 1975Bech and Breddam, 1989;Liao and Remington, 1990;Liao et al., 1992;Ollis et al., 1992). Proteins that contain this catalytic triad and are otherwise homologous to SCPs have been found in a variety of organisms (Doi et al., 1980;Kim and Hayashi, 1983;Breddam, 1986;Baulcombe et al., 1987;Galjart et al., 1988;Bradley, 1992;Degan et al., 1994;Endrizzi et al., 1994;Wajant et al., 1994;Jones et al., 1996;Li and Steffens, 2000). Many of these serine carboxypeptidase-like (SCPL) proteins have been annotated as peptidases based only on this shared sequence similarity; however, studies have shown that some of them function not as peptidases, but as acyltransferases and lyases (Wajant et al., 1994;Lehfeldt et al., 2000;Li and Steffens, 2000;Shirley et al., 2001). For example, several SCPL proteins have been shown to catalyze the production of plant secondary metabolites involved in herbivory defense and UV protection (Wajant et al., 1994;Lehfeldt et al., 2000;Li and Steffens, 2000), including the hydroxynitrile lyase from Sorghum bicolor (SbHNL) necessary for cyanogenesis, and the acyltransferases in wild tomato (Lycopersicon pennellii) that catalyze the formation 2,3,4-isobutyryl-Glc (Wajant et al., 1994;Lehfeldt et al., 2000). Two enzymes in Arabidopsis (Arabidopsis thaliana) responsible for sinapate ester biosynthesis are also SCPL proteins. Sinapoylglucose:malate sinapoyltransferase (SMT) catalyzes the formation of s...

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Section: Candidate Reactions For Scpl Acyltransferases Can Be Found Tmentioning

confidence: 99%

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confidence: 99%

An Expression and Bioinformatics Analysis of the Arabidopsis Serine Carboxypeptidase-Like Gene Family

2005

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“…The major clade is vastly expanded and specific to plants, and is generally thought to be involved in biosynthesis of the vast array of glycosidic plant natural products. As documented for UGT85B1, plant UGTs involved in natural product synthesis generally exhibit a broad substrate specificity with respect to the aglycon (sugar acceptor; Jones et al, 1999;Taguchi et al, 2000;Jackson et al, 2001;Lim et al, 2001aLim et al, , 2001bLim et al, , 2003aLim et al, , 2003bVogt, 2002;Hansen et al, 2003;Paquette et al, 2003). Most animal UGTs utilize UDP-GlcUA as a sugar donor, whereas plant UGTs typically utilize UDP-Glc.…”

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confidence: 99%

Determination of Catalytic Key Amino Acids and UDP Sugar Donor Specificity of the Cyanohydrin Glycosyltransferase UGT85B1 from Sorghum bicolor. Molecular Modeling Substantiated by Site-Specific Mutagenesis and Biochemical Analyses

Thorsøe¹,

Bak²,

Olsen³

et al. 2005

Plant Physiology

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Plants produce a plethora of structurally diverse natural products. The final step in their biosynthesis is often a glycosylation step catalyzed by a family 1 glycosyltransferase (GT). In biosynthesis of the cyanogenic glucoside dhurrin in Sorghum bicolor, the UDPglucosyltransferase UGT85B1 catalyzes the conversion of p-hydroxymandelonitrile into dhurrin. A structural model of UGT85B1 was built based on hydrophobic cluster analysis and the crystal structures of two bacterial GTs, GtfA and GtfB, which each showed approximately 15% overall amino acid sequence identity to UGT85B1. The model enabled predictions about amino acid residues important for catalysis and sugar donor specificity. p-Hydroxymandelonitrile and UDP-glucose (Glc) were predicted to be positioned within hydrogen-bonding distance to a glutamic acid residue in position 410 facilitating sugar transfer. The acceptor was packed within van der Waals distance to histidine H23. Serine S391 and arginine R201 form hydrogen bonds to the pyrophosphate part of UDP-Glc and hence stabilize binding of the sugar donor. Docking of UDP sugars predicted that UDP-Glc would serve as the sole donor sugar in UGT85B1. This was substantiated by biochemical analyses. The predictive power of the model was validated by site-directed mutagenesis of selected residues and using enzyme assays. The modeling approach has provided a tool to design GTs with new desired substrate specificities for use in biotechnological applications. The modeling identified a hypervariable loop (amino acid residues 156-188) that contained a hydrophobic patch. The involvement of this loop in mediating binding of UGT85B1 to cytochromes P450, CYP79A1, and CYP71E1 within a dhurrin metabolon is discussed.A glycosyltransferase (GT) is an enzyme that attaches a sugar molecule to a specific acceptor and thereby creates a glycosidic bond. GTs are found in all phylae. The nature of the acceptor molecules used is highly diverse, whereas the donor molecules typically are restricted to monosaccharides with either D-or L-configuration linked to a nucleotide (UDP/GDP/ CMP/(d)TDP; Leloir, 1964). Glycosylation reactions constitute an integral part of both primary and secondary metabolism. The final step in plant natural product synthesis is often a glycosylation reaction that serves to stabilize, solubilize, and provide a safe storage form of potentially toxic aglycones. In Sorghum bicolor, the GT UGT85B1 catalyzes glucosylation of Tyr-derived p-hydroxymandelonitrile to yield the cyanogenic glucoside dhurrin (Jones et al., 1999). In planta, neither p-hydroxymandelonitrile nor its degradation products are detectable, indicating that the glucosylation reaction is highly efficient and channeled (Møller and Conn, 1980). This observation has been substantiated by metabolome and transcriptome analyses of transgenic Arabidopsis (Arabidopsis thaliana) plants expressing the entire dhurrin pathway (Tattersall et al., 2001;Jørgensen et al., 2005;Kristensen et al., 2005), as well as by confocal laser-scanning microscopy studies ...

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“…Although closely related homologs exist in plants, as indicated by the presence of some 100 open reading frames in Arabidopsis encoding polypeptides containing a C-terminal consensus sequence common to all members of the UDP-glycosyltransferase superfamily Lim et al, 2001), much less is known about these enzymes than their mammalian counterparts. The majority of the plant enzymes are presumed to use UDP-Glc as the sugar donor and an increasing number have been purified and enzymologically characterized in the last several years (Ford et al, 1998;FraissinetTachet et al, 1998;Lee and Raskin, 1999;Vogt et al, 1999;Jackson et al, 2001), but their natural substrates and physiological roles largely remain to be determined. Notwithstanding our ignorance of these enzymes, however, it is suspected that one of the roles of plant UDP-glucosyltransferases is to target endogenous and exogenous toxins to the vacuole.…”

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confidence: 99%

Alternate Energy-Dependent Pathways for the Vacuolar Uptake of Glucose and Glutathione Conjugates

et al. 2002

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Through the development and application of a liquid chromatography-mass spectrometry-based procedure for measuring the transport of complex organic molecules by vacuolar membrane vesicles in vitro, it is shown that the mechanism of uptake of sulfonylurea herbicides is determined by the ligand, glucose, or glutathione, to which the herbicide is conjugated. ATP-dependent accumulation of glucosylated chlorsulfuron by vacuolar membrane vesicles purified from red beet (Beta vulgaris) storage root approximates Michaelis-Menten kinetics and is strongly inhibited by agents that collapse or prevent the formation of a transmembrane H+gradient, but is completely insensitive to the phosphoryl transition state analog, vanadate. In contrast, ATP-dependent accumulation of the glutathione conjugate of a chlorsulfuron analog, chlorimuron-ethyl, is incompletely inhibited by agents that dissipate the transmembrane H+ gradient but completely abolished by vanadate. In both cases, however, conjugation is essential for net uptake because neither of the unconjugated parent compounds are accumulated under energized or nonenergized conditions. That the attachment of glucose to two naturally occurring phenylpropanoids, p-hydroxycinnamic acid and p-hydroxybenzoic acid via aromatic hydroxyl groups, targets these compounds to the functional equivalent of the transporter responsible for chlorsulfuron-glucoside transport, confirms the general applicability of the H+ gradient dependence of glucoside uptake. It is concluded that H+gradient-dependent, vanadate-insensitive glucoside uptake is mediated by an H+ antiporter, whereas vanadate-sensitive glutathione conjugate uptake is mediated by an ATP-binding cassette transporter. In so doing, it is established that liquid chromatography-mass spectrometry affords a versatile high-sensitivity, high-fidelity technique for studies of the transport of complex organic molecules whose synthesis as radiolabeled derivatives is laborious and/or prohibitively expensive.

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Identification and Biochemical Characterization of anArabidopsis Indole-3-acetic Acid Glucosyltransferase

Cited by 254 publications

References 39 publications

An Expression and Bioinformatics Analysis of the Arabidopsis Serine Carboxypeptidase-Like Gene Family

An Expression and Bioinformatics Analysis of the Arabidopsis Serine Carboxypeptidase-Like Gene Family

Determination of Catalytic Key Amino Acids and UDP Sugar Donor Specificity of the Cyanohydrin Glycosyltransferase UGT85B1 from Sorghum bicolor. Molecular Modeling Substantiated by Site-Specific Mutagenesis and Biochemical Analyses

Alternate Energy-Dependent Pathways for the Vacuolar Uptake of Glucose and Glutathione Conjugates

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