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PubMed2002

Over-expression of an Arabidopsis gene encoding a glucosyltransferase of indole-3-acetic acid: phenotypic characterisation of transgenic lines.

An analysis of the multigene family of Group 1 glucosyltransferases (UGTs) of Arabidopsis thaliana revealed a gene, UGT84B1, whose recombinant product glucosylated indole-3-acetic acid (IAA) in vitro. Transgenic Arabidopsis plants constitutively over-expressing UGT84B1 under the control of the CaMV 35S promoter have been constructed and their phenotype analysed. The transgenic lines displayed a number of changes that resembled those described previously in lines in which auxin levels were depleted. A root elongation assay was used as a measure of auxin sensitivity. A reduced sensitivity of the transgenic lines compared to wild-type was observed when IAA was applied. In contrast, application of 2,4-dichlorophenoxyacetic acid (2,4-D), previously demonstrated not to be a substrate for UGT84B1, led to a wild-type response. These data suggested that the catalytic specificity of the recombinant enzyme in vitro was maintained in planta. This was further confirmed when levels of IAA metabolites and conjugates were measured in extracts of the transgenic plants and 1-O-IAGlc was found to be elevated to approximately 50 pg mg-1 FW, compared to the trace levels characteristic of wild-type plants. Surprisingly, in the same extracts, levels of free IAA were also found to have accumulated to some 70 pg mg-1 FW compared to approximately 15 pg mg-1 FW in extracts of wild-type plants. Analysis of leaves at different developmental stages revealed the auxin gradient, typical of wild-type plants, was not observed in the transgenic lines, with free IAA levels in the apex and youngest leaves at a lower level compared to wild-type. In total, the data reveal that significant changes in auxin homeostasis can be caused by overproduction of an IAA-conjugating enzyme.

Arabidopsis↗

PubMed1997

Glucosyltransferase activity of commercial juice-processing enzyme preparations.

Of various commercial enzyme preparations examined, Cytolase M102 was found to contain the highest glucosyltransferase activity (55 U ml-1). It rapidly converted maltose to panose (Glc alpha-->6 Glc alpha l-->4 Glc) with a Vmax value of 5.8 mmol l-1 min-1 at 50 degrees C in 0.05 mol l-1 sodium acetate buffer (pH 4.4). The Km value of the enzyme for maltose was 750 mmol l-1. Yields of panose and glucose after 45 min of reaction, for example, were 47.2% and 52.8%, respectively, on the basis of the amount of maltose consumed.

Enzymes↗

PubMed1981

Deficiency of galactosylhydroxylysyl glucosyltransferase, an enzyme of collagen synthesis, in a family with dominant epidermolysis bullosa simplex.

Members of a family with dominant epidermolysis bullosa simplex were found to have a deficiency of galactosylhydroxylysyl glucosyltransferase (GGT), an enzyme catalyzing the glucosylation of galactosylhydroxylysyl residues in the biosynthesis of collagen. The enzyme's activity was low in serum, skin tissue, and cultured skin fibroblasts, although no abnormality was found in three other intracellular enzymes of collagen biosynthesis. Mixtures of serum samples from patients and healthy controls gave the expected GGT activity, indicating that the low values were not due to inhibitors. GGT deficiency was accompanied by decreased product formation in vivo, as shown by a markedly decreased urinary excretion of glucosylgalactosylhydroxylysine. Six of 12 affected members had definite GGT deficiency, and five had some evidence suggestive of this abnormality; 13 of 15 unaffected members had no such manifestations. No similar GGT deficiency was found in three other families with the same disease. We conclude that GGT deficiency may be etiologically related to this disease in some families, but that different defects must be the cause in other cases.

Adolescent↗

PubMed2001

Immunolocalization of UDP-glucose:glycoprotein glucosyltransferase indicates involvement of pre-Golgi intermediates in protein quality control.

The UDP-glucose:glycoprotein glucosyltransferase (GT) is a protein folding sensor and glycosyltransferase that constitutes an important component of the protein quality control machinery. With the use of quantitative immunogold electron microscopy, we established the subcellular distribution of GT in rat liver and pancreas and Drosophila melanogaster salivary gland as well as cell lines and correlated it with that of glucosidase II, calreticulin, and pre-Golgi intermediate markers. Labeling for GT, as well as for glucosidase II and calreticulin, was found in the endoplasmic reticulum (ER), including nuclear envelope and pre-Golgi intermediates located between ER and Golgi apparatus, and in the cell periphery. In the rough ER, labeling for GT was inhomogeneous, with variously sized labeled and unlabeled cisternal regions alternating, indicative of a meshwork of quality control checkpoints. Notably, labeling intensity for GT was highest in pre-Golgi intermediates, corresponding to twice that of rough ER, whereas the Golgi apparatus exhibited no specific labeling. These results suggest that protein quality control is not restricted to the ER and that the pre-Golgi intermediates, by virtue of the presence of GT, glucosidase II, and calreticulin, are involved in this fundamental cellular process.

Animals↗

PubMed1999

Isolation of a cytokinin gene, ZOG1, encoding zeatin O-glucosyltransferase from Phaseolus lunatus.

Zeatin is the most active and ubiquitous of the naturally occurring cytokinins. The O-glucoside of zeatin, found in all plants examined, is considered to be important in cytokinin transport, storage, and protection against cytokinin oxidases. The enzyme UDPglucose:zeatin O-glucosyltransferase (EC 2.4.1.203) was previously isolated from Phaseolus lunatus seeds. Immunoscreening of an expression library with monospecific antibody resulted in the isolation of a cDNA encoding the enzyme. The recombinant protein efficiently converts labeled zeatin to O-glucosylzeatin and has properties similar to the native enzyme. The cDNA of 1.5 kb contains an ORF encoding a 51. 4-kDa polypeptide of 459 amino acids. The sequence is unique based on a BLAST search of data bases. The genomic sequence, isolated with PCR using specific primers based on the cDNA sequence, does not contain introns. The cloning of this gene provides the tools for further study of the regulation of cytokinin metabolism and analysis of the precise role of O-glucosylzeatin in plant development.

Amino Acid Sequence↗

PubMed1996

Induction of ceramide glucosyltransferase activity in cultured human keratinocytes. Correlation with culture differentiation.

Ceramides are the major component of the extracellular lipids that comprise the epidermal permeability barrier. They are derived from glucosylceramides (GlcCer) upon their extrusion from lamellar granules into the extracellular space in the upper layers of the epidermis. To better understand the regulation of the unique pathway for ceramide production in epidermis, we have studied the activity of the enzyme responsible for GlcCer synthesis, ceramide glucosyltransferase (CerGlc transferase), during keratinocyte culture differentiation. Human keratinocyte cultures were expanded in low calcium keratinocyte growth medium (KGM) and then switched to either normal calcium KGM (nKGM) or "complete" Dulbecco's modified Eagle's medium/Ham's F-12 (3:1) supplemented with 10% fetal bovine serum (cDMEM). At 7 and 10 days after the medium switch, electron microscopy revealed that cDMEM cultures were more fully differentiated morphologically and contained numerous lamellar granules. The GlcCer/DNA content of cDMEM cultures increased to 6 times that of day 0 cultures and was nearly 4 times greater than that of nKGM cultures, whereas the total lipid/DNA content of cDMEM cultures increased to only 1.8 times that of day 0 cultures and was approximately 1.2 times that of nKGM cultures. CerGlc transferase activity/DNA increased 6 times in cDMEM cultures but <1.5 times in nKGM cultures. By contrast, beta-glucocerebrosidase activity, which is responsible for the conversion of GlcCer to ceramide, increased to a similar extent in both differentiating culture systems. Treatment of cultures with the reversible CerGlc transferase inhibitor, DL-threo-1-phenyl-2-(palmitoylamino)-3-morpholino-1-propanol, prevented the increase of GlcCer in cDMEM cultures, and blocked conversion of exogenously added ceramide to GlcCer. A low level of CerGlc transferase activity, relative to that in differentiated keratinocytes, was detected in cultures of other human cell types. These results indicate that CerGlc transferase activity is induced during epidermal differentiation and that regulation of this enzyme may be an important determinant of the specialized production and compartmentalization of epidermal sphingolipids.

4-Chloro-7-nitrobenzofurazan↗

PubMed1996

Purification and characterization of UDP-glucose:ceramide glucosyltransferase from rat liver Golgi membranes.

We present a method for solubilizing and purifying UDP-Glc:ceramide glucosyltransferase (EC 2.4.1.80; glucosylceramide synthase (GCS) from a rat liver and present data on its substrate specificity. A Golgi membrane fraction was isolated, washed with N-lauroylsarcosine, and subsequently treated with 3[3-cholamidopropyl)-dimethylammonio]-2-hydroxy-1-propanesulfonate to solubilize the enzyme. GCS activity was monitored throughout purification using UDP-Glc and a fluorescent ceramide analog as substrates. Purification of GCS was achieved via a two-step dye-agarose chromatography procedure using UDP-Glc to elute the enzyme. This resulted in an enrichment > 10,000-fold relative to the starting homogenate. The enzyme was further characterized by sedimentation on a glycerol gradient, I labeling, and SDS-polyacrylamide gel electrophoresis. which demonstrated that two polypeptides (60-70 kDa) corresponded closely with GCS activity. Purified GCS was found to require exogenous phospholipids for activity, and optimal results were obtained using dioleoyl phosphatidylcholine. Studies of the substrate specificity of the purified enzyme demonstrated that it was stereospecific and dependent on the nature and chain length of the N-acyl-spingosine or -sphinganine substrate. UDP-Glc was the preferred hexose donor, but TDP-glucose and CDP-glucose were also efficiently used. This study provides a basis for molecular characterization of this key enzyme in glycosphingolipid biosynthesis.

Animals↗

PubMed1997

Activating amphiphiles cause a conformational change of the 1,2-diacylglycerol 3-glucosyltransferase from Acholeplasma laidlawii membranes according to proteolytic digestion.

1,2-Diacylglycerol 3-glucosyltransferase synthesizes the major nonbilayer-prone lipid monoglucosyldiacylglycerol (MGlcDAG) in the membrane of Acholeplasma laidlawii, which is important for the spontaneous curvature, and is a regulatory site for the lipid surface charge density. A potential connection between activity and a conformational change of this enzyme, governed by essential lipid activators, was studied with purified MGlcDAG synthase in different lipid aggregates. Critical fractions of anionic phospholipids 1, 2-dioleoyl-phosphatidylglycerol (DOPG) and 1,2-dioleoyl-phosphatidylserine (DOPS) were essential for the restoration of enzyme activity, while the zwitterionic 1,2-dioleoyl-phosphatidylcholine (DOPC) and the uncharged diglucosyldiacylglycerol (DGlcDAG) were not. Proteolytic resistance had a very good correlation with the enzyme activity in various lipid-CHAPS mixed micelles. Anionic lipids DOPG and DOPS could protect the exposed MGlcDAG synthase from digestion, whereas DOPC and DGlcDAG could not. Similar features were observed in liposome bilayers. Likewise, the detergent dodecylphosphoglycerol (PGD), with a phosphatidylglycerol-like headgroup, could also stimulate the MGlcDAG synthase activity efficiently with a concomitant protection toward proteolytic digestion. Neither proteolytic resistance nor restored enzyme activity was observed using soluble glycerol 3-phosphate. It is concluded that in addition to critical amounts, both the negatively charged headgroup and hydrophobic chains of the activator amphiphiles, but not a certain aggregate curvature, seem necessary for a proper conformation and the resulting active state of the MGlcDAG synthase.

Acholeplasma↗

PubMed1998

The assembly system for the outer core portion of R1- and R4-type lipopolysaccharides of Escherichia coli. The R1 core-specific beta-glucosyltransferase provides a novel attachment site for O-polysaccharides.

The major core oligosaccharide biosynthesis operons from prototype Escherichia coli strains displaying R1 and R4 lipopolysaccharide core types were polymerase chain reaction-amplified and analyzed. Comparison of deduced products of the open reading frames between the two regions indicate that all but two share total similarities of 94% or greater. Core oligosaccharide structures resulting from nonpolar insertion mutations in each gene of the core OS biosynthesis operon in the R1 strain allowed assignment of all of the glycosyltransferase enzymes required for outer core assembly. The difference between the R1 and R4 core oligosaccharides results from the specificity of the WaaV protein (a beta1, 3-glucosyltransferase) in R1 and WaaX (a beta1, 4-galactosyltransferase) in R4. Complementation of the waaV mutant of the R1 prototype strain with the waaX gene of the R4 strain converted the core oligosaccharide from an R1- to an R4-type lipopolysaccharide core molecule. Aside from generating core oligosaccharide specificity, the unique beta-linked glucopyranosyl residue of the R1 core plays a crucial role in organization of the lipopolysaccharide. This residue provides a novel attachment site for lipid A-core-linked polysaccharides and distinguishes the R1-type LPS from existing models for enterobacterial lipopolysaccharides.

Amino Acid Sequence↗

PubMed1999

Modification of the T cell antigen receptor (TCR) complex by UDP-glucose:glycoprotein glucosyltransferase. TCR folding is finalized convergent with formation of alpha beta delta epsilon gamma epsilon complexes.

Most T lymphocytes express on their surfaces a multisubunit receptor complex, the T cell antigen receptor (TCR) containing alpha, beta, gamma, delta, epsilon, and zeta molecules, that has been widely studied as a model system for protein quality control. Although the parameters of TCR assembly are relatively well established, little information exists regarding the stage(s) of TCR oligomerization where folding of TCR proteins is completed. Here we evaluated the modification of TCR glycoproteins by the endoplasmic reticulum folding sensor enzyme UDP-glucose:glycoprotein glucosyltransferase (GT) as a unique and sensitive indicator of how TCR subunits assembled into multisubunit complexes are perceived by the endoplasmic reticulum quality control system. These results demonstrate that all TCR subunits containing N-glycans were modified by GT and that TCR proteins were differentially reglucosylated during their assembly with partner TCR chains. Importantly, these data show that GT modification of most TCR subunits persisted until assembly of CD3alpha beta chains and formation of CD3-associated, disulfide-linked alpha beta heterodimers. These studies provide a novel evaluation of the folding status of TCR glycoproteins during their assembly into multisubunit complexes and are consistent with the concept that TCR folding is finalized convergent with formation of alpha beta delta epsilon gamma epsilon complexes.

Animals↗

PubMed1999

Inhibitory effect of a self-derived peptide on glucosyltransferase of Streptococcus mutans. Possible novel anticaries measures.

Glucosyltransferase (GTF) plays an important role in the development of dental caries. We examined the possible presence of self-inhibitory segments within the enzyme molecule for the purpose of developing anticaries measures through GTF inhibition. Twenty-two synthetic peptides derived from various regions presumably responsible for insoluble-glucan synthesis were studied with respect to their effects on catalytic activity. One of them, which is identical in amino acid sequence to residues 1176-1194, significantly and specifically inhibited both sucrose hydrolysis and glucosyl transfer to glucan by GTF-I. Double-reciprocal analysis revealed that the inhibition is noncompetitive. Scramble peptides, composed of the identical amino acids in randomized sequence, had no effect on GTF-I activity. Furthermore, the peptide is tightly bound to the enzyme once complexed, even in the presence of sodium dodecyl sulfate (SDS). Kinetic analysis using an optical evanescent resonant mirror cuvette system demonstrated that the enzyme-peptide interaction was biphasic. These results indicate that the peptide directly interacts with the enzyme with high affinity and inhibits its activity in a sequence-specific manner. This peptide itself could possibly be an effective agent for prevention of dental caries, although its effectiveness may be improved by further modification.

Amino Acid Sequence↗

PubMed1999

Purification, cloning, and expression of a pathogen inducible UDP-glucose:Salicylic acid glucosyltransferase from tobacco.

Salicylic acid (SA) plays an important role in plant disease resistance. Inoculation of tobacco leaves with incompatible pathogens triggers the biosynthesis of SA which accumulates primarily as the SA 2-O-beta-D-glucoside (SAG) and glucosyl salicylate (GS). The tobacco UDP-glucose:salicylic acid glucosyltransferase (SA GTase) capable of forming both SAG and GS was purified, characterized, and partially sequenced. It has an apparent molecular mass of 48 kDa, a pH optimum of 7.0, and an isoelectric point at pH 4.4. UDP-glucose was the sole sugar donor for the enzyme. However, SA and several phenolics served as glucose acceptors. The apparent K(m) values for UDP-glucose and SA were 0.27 and 1-2 mM, respectively. Zn(2+) and UDP inhibited its activity. The corresponding cDNA clone which encoded a protein of 459 amino acids was isolated from an SA-induced tobacco cDNA library and overexpressed in Escherichia coli. The recombinant protein catalyzed the formation of SAG and GS, and exhibited a broad specificity to simple phenolics, similar to that of the purified enzyme. Northern blot analysis showed that the SA GTase mRNA was induced both by SA and incompatible pathogens. The rapid induction timing of the mRNA by SA indicates that it belongs to the early SA response genes.

Amino Acid Sequence↗

PubMed2000

Identification of essential amino acid residues in the Sinorhizobium meliloti glucosyltransferase ExoM.

ExoM is a beta(1-4)-glucosyltransferase involved in the assembly of the repeat unit of the exopolysaccharide succinoglycan from Sinorhizobium meliloti. By comparing the sequence of ExoM to those of other members of the Pfam Glyco Domain 2 family, most notably SpsA (Bacillus subtilis) for whom the three-dimensional structure has been resolved, three potentially important aspartic acid residues of ExoM were identified. Single substitutions of each of the Asp amino acids at positions 44, 96, and 187 with Ala resulted in the loss of mutant recombinant protein activity in vitro as well as the loss of succinoglycan production in an in vivo rescue assay. Mutants harboring Glu instead of Asp-44 or Asp-96 possessed no in vitro activity but could restore succinoglycan production in vivo. However, replacement of Asp-187 with Glu completely inactivated ExoM as judged by both the in vitro and in vivo assays. These results indicate that Asp-44, Asp-96, and Asp-187 are essential for the activity of ExoM. Furthermore, these data are consistent with the functions proposed for each of the analogous aspartic acids of SpsA based on the SpsA-UDP structure, namely, that Asp-44 and Asp-96 are involved in UDP substrate binding and that Asp-187 is the catalytic base in the glycosyltransferase reaction.

Amino Acid Sequence↗

PubMed2001

Association between the 15-kDa selenoprotein and UDP-glucose:glycoprotein glucosyltransferase in the endoplasmic reticulum of mammalian cells.

Mammalian selenocysteine-containing proteins characterized with respect to function are involved in redox processes and exhibit distinct expression patterns and cellular locations. A recently identified 15-kDa selenoprotein (Sep15) has no homology to previously characterized proteins, and its function is not known. Here we report the intracellular localization and identification of a binding partner for this selenoprotein which implicate Sep15 in the regulation of protein folding. The native Sep15 isolated from rat prostate and mouse liver occurred in a complex with a 150-kDa protein. The latter protein was identified as UDP-glucose:glycoprotein glucosyltransferase (UGTR), the endoplasmic reticulum (ER)-resident protein, which was previously shown to be involved in the quality control of protein folding. UGTR functions by glucosylating misfolded proteins, retaining them in the ER until they are correctly folded or transferring them to degradation pathways. To determine the intracellular localization of Sep15, we expressed a green fluorescent protein-Sep15 fusion protein in CV-1 cells, and this protein was localized to the ER and possibly other perinuclear compartments. We determined that Sep15 contained the N-terminal signal peptide that was essential for translocation and that it was cleaved in the mature protein. However, C-terminal sequences of Sep15 were not involved in trafficking and retention of Sep15. The data suggest that the association between Sep15 and UGTR is responsible for maintaining the selenoprotein in the ER. This report provides the first example of the ER-resident selenoprotein and suggests a possible role of the trace element selenium in the quality control of protein folding.

Amino Acid Sequence↗

PubMed2001

Tts, a processive beta-glucosyltransferase of Streptococcus pneumoniae, directs the synthesis of the branched type 37 capsular polysaccharide in Pneumococcus and other gram-positive species.

The type 37 capsule of Streptococcus pneumoniae is a homopolysaccharide built up from repeating units of [beta-d-Glcp-(1-->2)]-beta-d-Glcp-(1-->3). The elements governing the expression of the tts gene, coding for the glucosyltransferase involved in the synthesis of the type 37 pneumococcal capsular polysaccharide, have been studied. Primer extension analysis and functional tests demonstrated the presence of four new transcriptional start points upstream of the previously reported tts promoter (ttsp). Most interesting, three of these transcriptional start points are located in a RUP element thought to be involved in recombinational events (Oggioni, M. R., and Claverys, J. P. (1999) Microbiology 145, 2647-2653). Transformation experiments using either a recombinant plasmid containing the whole transcriptional unit of tts or chromosomal DNA from a type 37 pneumococcus showed that tts is the only gene required to drive the biosynthesis of a type 37 capsule in S. pneumoniae and other Gram-positive bacteria, namely Streptococcus oralis, Streptococcus gordonii, and Bacillus subtilis. The Tts synthase was overproduced in S. pneumoniae and purified as a membrane-associated enzyme. These membrane preparations used UDP-Glc as substrate to catalyze the synthesis of a high molecular weight polysaccharide immunologically identical to the type 37 capsule. In addition, UDP-Gal was also a substrate to produce type 37 polysaccharide since a strong UDP-Glc-4'-epimerase activity is associated to the membrane fraction of S. pneumoniae. These results indicated that Tts has a dual biochemical activity that leads to the synthesis of the branched type 37 polysaccharide.

Bacterial Proteins↗

PubMed2001

Sequence properties of the 1,2-diacylglycerol 3-glucosyltransferase from Acholeplasma laidlawii membranes. Recognition of a large group of lipid glycosyltransferases in eubacteria and archaea.

Synthesis of the nonbilayer-prone alpha-monoglucosyldiacylglycerol (MGlcDAG) is crucial for bilayer packing properties and the lipid surface charge density in the membrane of Acholeplasma laidlawii. The gene for the responsible, membrane-bound glucosyltransferase (alMGS) (EC ) was sequenced and functionally cloned in Escherichia coli, yielding MGlcDAG in the recombinants. Similar amino acid sequences were encoded in the genomes of several Gram-positive bacteria (especially pathogens), thermophiles, archaea, and a few eukaryotes. All of these contained the typical EX(7)E catalytic motif of the CAZy family 4 of alpha-glycosyltransferases. The synthesis of MGlcDAG by a close sequence analog from Streptococcus pneumoniae (spMGS) was verified by polymerase chain reaction cloning, corroborating a connection between sequence and functional similarity for these proteins. However, alMGS and spMGS varied in dependence on anionic phospholipid activators phosphatidylglycerol and cardiolipin, suggesting certain regulatory differences. Fold predictions strongly indicated a similarity for alMGS (and spMGS) with the two-domain structure of the E. coli MurG cell envelope glycosyltransferase and several amphipathic membrane-binding segments in various proteins. On the basis of this structure, the alMGS sequence charge distribution, and anionic phospholipid dependence, a model for the bilayer surface binding and activity is proposed for this regulatory enzyme.

Acholeplasma laidlawii↗

PubMed2002

Identification of amino acids important for the catalytic activity of the collagen glucosyltransferase associated with the multifunctional lysyl hydroxylase 3 (LH3).

Collagen glucosyltransferase (GGT) activity has recently been shown to be associated with human lysyl hydroxylase (LH) isoform 3 (LH3) (Heikkinen, J., Risteli, M., Wang, C., Latvala, J., Rossi, M., Valtavaara, M., Myllylä, R. (2000) J. Biol. Chem. 275, 36158-36163). The LH and GGT activities of the multifunctional LH3 protein modify lysyl residues in collagens posttranslationally to form hydroxylysyl and glucosylgalactosyl hydroxylysyl residues respectively. We now report that in the nematode, Caenorhabditis elegans, where only one ortholog is found for lysyl hydroxylase, the LH and GGT activities are also associated with the same gene product. The aim of the present studies is the identification of amino acids important for the catalytic activity of GGT. Our data indicate that the GGT active site is separate from the carboxyl-terminal LH active site of human LH3, the amino acids important for the GGT activity being located at the amino-terminal part of the molecule. Site-directed mutagenesis of a conserved cysteine at position 144 to isoleucine and a leucine at position 208 to isoleucine caused a marked reduction in GGT activity. These amino acids were conserved in C. elegans LH and mammalian LH3, but not in LH1 or LH2, which lack GGT activity. The data also reveal a DXD-like motif in LH3 characteristic of many glycosyltransferases and the mutagenesis of aspartates of this motif eliminated the GGT activity. Reduction in GGT activity was not accompanied by a change in the LH activity of the molecule. Thus GGT activity can be manipulated independently of LH activity in LH3. These data provide the information needed to design knock-out studies for investigation of the function of glucosylgalactosyl hydroxylysyl residues of collagens in vivo.

Amino Acid Sequence↗

PubMed2002

Glucose conjugation of anthranilate by the Arabidopsis UGT74F2 glucosyltransferase is required for tryptophan mutant blue fluorescence.

Plant mutants with defects in intermediate enzymes of the tryptophan biosynthetic pathway often display a blue fluorescent phenotype. This phenotype results from the accumulation of the fluorescent tryptophan precursor anthranilate, the bulk of which is found in a glucose-conjugated form. To elucidate factors that control fluorescent tryptophan metabolites, we conducted a genetic screen for suppressors of blue fluorescence in the Arabidopsis trp1-100 mutant, which has a defect in the second enzymatic step of the tryptophan pathway. This screen yielded loss-of-function mutations in the UDP-glucosyltransferase gene UGT74F2. The bacterially expressed UGT74F2 enzyme catalyzed a conjugation reaction, with free anthranilate and UDP-glucose as substrates, that yielded the same fluorescent glucose ester compound as extracted from the trp1-100 mutant. These results indicate that sugar conjugation of anthranilate by UGT74F2 allows its stable accumulation in plant tissues. A highly related Arabidopsis enzyme UGT74F1 could also catalyze this reaction in vitro and could complement the ugt74F2 mutation when overexpressed in vivo. However, the UGT74F1 gene is expressed at a lower level than the UGT74F2 gene. Therefore, even though UGT74F1 and UGT74F2 have redundant conjugating activities toward anthranilate, UGT74F2 is the major source of this activity in the plant.

Amino Acid Sequence↗