Eng-Kiat Lim

Higher plant glycosyltransferases

SummaryUridine diphosphate (UDP) glycosyltransferases (UGTs) mediate the transfer of glycosyl residues from activated nucleotide sugars to acceptor molecules (aglycones), thus regulating properties of the acceptors such as their bioactivity, solubility and transport within the cell and throughout the organism. A superfamily of over 100 genes encoding UGTs, each containing a 42 amino acid consensus sequence, has been identified in the model plant Arabidopsis thaliana. A phylogenetic analysis of the conserved amino acids encoded by these Arabidopsis genes reveals the presence of 14 distinct groups of UGTs in this organism. Genes encoding UGTs have also been identified in several other higher plant species. Very little is yet known about the regulation of plant UGT genes or the localization of the enzymes they encode at the cellular and subcellular levels. The substrate specificities of these UGTs are now beginning to be established and will provide a foundation for further analysis of this large enzyme superfamily as well as a platform for future biotechnological applications.

A class of plant glycosyltransferases involved in cellular homeostasis

Many small lipophilic compounds in living cells can be modified by glycosylation. These processes can regulate the bioactivity of the compounds, their intracellular location and their metabolism. The glycosyltransferases involved in biotransformations of small molecules have been grouped into Family 1 of the 69 families that are classified on the basis of substrate recognition and sequence relatedness. In plants, these transfer reactions generally use UDP‐glucose with acceptors that include hormones such as auxins and cytokinins, secondary metabolites such as flavonoids, and foreign compounds including herbicides and pesticides. In mammalian organisms, UDP‐glucuronic acid is typically used in the transfer reactions to endogenous acceptors, such as steroid and thyroid hormones, bile acids and retinoids, and to xenobiotics, including nonsteroidal anti‐inflammatory drugs and dietary metabolites. There is widespread interest in this class of enzyme since they are known to function both in the regulation of cellular homeostasis and in detoxification pathways. This review outlines current knowledge of these glycosyltransferases drawing on information gained from studies of plant and mammalian enzymes.

Characterization and Engineering of the Bifunctional N- and O-Glucosyltransferase Involved in Xenobiotic Metabolism in Plants.

The glucosylation of pollutant and pesticide metabolites in plants controls their bioactivity and the formation of subsequent chemical residues. The model plant Arabidopsis thaliana contains >100 glycosyltransferases (GTs) dedicated to small-molecule conjugation and, whereas 44 of these enzymes catalyze the O-glucosylation of chlorinated phenols, only one, UGT72B1, shows appreciable N-glucosylating activity toward chloroanilines. UGT72B1 is a bifunctional O-glucosyltransferase (OGT) and N-glucosyltransferase (NGT). To investigate this unique dual activity, the structure of the protein was solved, at resolutions up to 1.45 Å, in various forms including the Michaelis complex with intact donor analog and trichlorophenol acceptor. The catalytic mechanism and basis for O/N specificity was probed by mutagenesis and domain shuffling with an orthologous enzyme from Brassica napus (BnUGT), which possesses only OGT activity. Mutation of BnUGT at just two positions (D312N and F315Y) installed high levels of NGT activity. Molecular modeling revealed the connectivity of these residues to H19 on UGT72B1, with its mutagenesis exclusively defining NGT activity in the Arabidopsis enzyme. These results shed light on the conjugation of nonnatural substrates by plant GTs, highlighting the catalytic plasticity of this enzyme class and the ability to engineer unusual and desirable transfer to nitrogen-based acceptors.

Regioselectivity of glucosylation of caffeic acid by a UDP-glucose:glucosyltransferase is maintained in planta.

Caffeic acid is a phenylpropanoid playing an important role in the pathways leading to lignin synthesis and the production of a wide variety of secondary metabolites. The compound is also an antioxidant and has potential utility as a general protectant against free radicals. Three glucosylated forms of caffeic acid are known to exist: the 3- O - and 4- O -glucosides and the glucose ester. This study describes for the first time a glucosyltransferase [UDP-glucose:glucosyltransferase (UGT)] that is specific for the 3-hydroxyl, and not the 4-hydroxyl, position of caffeic acid. The UGT sequence of Arabidopsis, UGT71C1, has been expressed as a recombinant fusion protein in Escherichia coli, purified and assayed against a range of substrates in vitro. The assay confirmed that caffeic acid as the preferred substrate when compared with other hydroxycinnamates, although UGT71C1 also exhibited substantial activity towards flavonoid substrates, known to have structural features that can be recognized by many different UGTs. The expression of UGT71C1 in transgenic Arabidopsis was driven by the constitutive cauliflower mosaic virus 35 S (CaMV35S) promoter. Nine independent transgenic lines were taken to homozygosity and characterized by Northern-blot analysis, assay of enzyme activity in leaf extracts and HPLC analysis of the glucosides. The level of expression of UGT71C1 was enhanced considerably in several lines, leading to a higher level of the corresponding enzyme activity and a higher level of caffeoyl-3- O -glucoside. The data are discussed in the context of the utility of UGTs for natural product biotransformations.

Identification and characterisation of Arabidopsis glycosyltransferases capable of glucosylating coniferyl aldehyde and sinapyl aldehyde

This study describes the substrate recognition profile of UGT72E1, an UDP–glucose:glycosyltransferase of Arabidopsis thaliana that is the third member of a branch of glycosyltransferases, capable of conjugating lignin monomers and related metabolites. The data show that UGT72E1, in contrast to the two closely related UGTs 72E2 and 72E3, is specific for sinapyl and coniferyl aldehydes. The biochemical properties of UGT72E1 are characterised, and are compared with that of UGT72E2, which is capable of glycosylating the aldehydes as well as coniferyl and sinapyl alcohols.

Discovery of New Biocatalysts for the Glycosylation of Terpenoid Scaffolds

The synthesis of terpenoid glycosides typically uses a chemical strategy since few biocatalysts have been identified that recognise these scaffolds. In this study, a platform of 107 recombinant glycosyltransferases (GTs), comprising the multigene family of small molecule GTs of Arabidopsis thaliana have been screened against a range of model terpenoid acceptors to identify those enzymes with high activity. Twenty-seven GTs are shown to glycosylate a diversity of mono-, sesqui- and diterpenes, such as geraniol, perillyl alcohol, artemisinic acid and retinoic acid. Certain enzymes showing substantial sequence similarity recognise terpenoids containing a primary alcohol, irrespective of the linear or cyclical structure of the scaffold; other GTs glycosylate scaffolds containing secondary and tertiary alcohols; the carboxyl group of other terpenoids also represents a feature that is recognized by GTs previously known to form glucose esters with many different compounds. These data underpin the rapid prediction of potential biocatalysts from GT sequence information. To explore the potential of GTs as biocatalysts, their use for the production of terpenoid glycosides was investigated by using a microbial-based whole-cell biotransformation system capable of regenerating the cofactor, UDP-glucose. A high cell density fermentation system was shown to produce several hundred milligrams of a model terpenoid, geranyl-glucoside. The activities of the GTs are discussed in relation to their substrate recognition and their utility in biotransformations as a complement or alternative to chemical synthesis.

A Kinetic Analysis of Regiospecific Glucosylation by Two Glycosyltransferases of Arabidopsis thaliana DOMAIN SWAPPING TO INTRODUCE NEW ACTIVITIES

Plant Family 1 glycosyltransferases (GTs) recognize a wide range of natural and non-natural scaffolds and have considerable potential as biocatalysts for the synthesis of small molecule glycosides. Regiospecificity of glycosylation is an important property, given that many acceptors have multiple potential glycosylation sites. This study has used a domain-swapping approach to explore the determinants of regiospecific glycosylation of two GTs of Arabidopsis thaliana, UGT74F1 and UGT74F2. The flavonoid quercetin was used as a model acceptor, providing five potential sites for O-glycosylation by the two GTs. As is commonly found for many plant GTs, both of these enzymes produce distinct multiple glycosides of quercetin. A high performance liquid chromatography method has been established to perform detailed steady-state kinetic analyses of these concurrent reactions. These data show the influence of each parameter in determining a GT product formation profile toward quercetin. Interestingly, construction and kinetic analyses of a series of UGT74F1/F2 chimeras have revealed that mutating a single amino acid distal to the active site, Asn-142, can lead to the development of a new GT with a more constrained regiospecificity. This ability to form the 4 ′-O-glucoside of quercetin is transferable to other flavonoid scaffolds and provides a basis for preparative scale production of flavonoid 4 ′-O-glucosides through the use of whole-cell biocatalysis.

Metabolism of the Folate Precursor p-Aminobenzoate in Plants GLUCOSE ESTER FORMATION AND VACUOLAR STORAGE

Plants produce p-aminobenzoate (pABA) in chloroplasts and use it for folate synthesis in mitochondria. In plant tissues, however, pABA is known to occur predominantly as its glucose ester (pABA-Glc), and the role of this metabolite in folate synthesis has not been defined. In this study, the UDP-glucose:pABA acyl-glucosyltransferase (pAGT) activity in Arabidopsis extracts was found to reside principally (95%) in one isoform with an apparent Km for pABA of 0.12 mm. Screening of recombinant Arabidopsis UDP-glycosyltransferases identified only three that recognized pABA. One of these (UGT75B1) exhibited a far higher kcat/Km value than the others and a far lower apparent Km for pABA (0.12 mm), suggesting its identity with the principal enzyme in vivo. Supporting this possibility, ablation of UGT75B1 reduced extractable pAGT activity by 95%, in vivo [14C]pABA glucosylation by 77%, and the endogenous pABA-Glc/pABA ratio by 9-fold. The Keq for the pABA esterification reaction was found to be 3 × 10-3. Taken with literature data on the cytosolic location of pAGT activity and on cytosolic UDP-glucose/UDP ratios, this Keq value allowed estimation that only 4% of cytosolic pABA is esterified. That pABA-Glc predominates in planta therefore implies that it is sequestered away from the cytosol and, consistent with this possibility, vacuoles isolated from [14C]pABA-fed pea leaves were estimated to contain≥88% of the [14C]pABA-Glc formed. In total, these data and the fact that isolated mitochondria did not take up [3H]pABA-Glc, suggest that the glucose ester represents a storage form of pABA that does not contribute directly to folate synthesis.

Bone marrow-derived and resident liver macrophages display unique transcriptomic signatures but similar biological functions

Graphical abstract

The Synthesis of Small-Molecule Rhamnosides through the Rational Design of a Whole-Cell Biocatalysis System

Glycosyltransferases (GTs) offer a simple and green chemical approach to synthesising glycoconjugates of lipophilic small molecules, such as plant and microbial natural products. Through the use of these enzymes, regioselective and enantioselective glycosylation becomes readily feasible, by-passing the need to rely on more complex, blocking and deblocking chemical routes of synthesis. More importantly, as demonstrated by Samain and co-workers, engineering bacteria with GTs allows complex carbohydrates to be produced at low cost. Other recent studies have shown that small molecules added to bacterial cultures expressing recombinant plant GTs are taken up by the bacterial cells and glycosylated with the cells’ endogenous sugar donors before secretion of the glycosides into the culture medium. Thus, product recovery from the whole-cell biocatalysis system is simple, the process can be readily scaled up, and the addition of supplementary donor sugars is not required. To date, the glycoconjugates of small molecules made in the Escherichia coli whole-cell system have been glucosides; 5] this is hardly surprising given that the major sugar donor available to the plant GTs in the bacterial cells is UDP-glucose (UDP-Glc). There is evidence to indicate, however, that small molecules can be conjugated to a wider range of sugars in plants, including rhamnose (Rha), galactose (Gal) and glucuronic acid (GlcA), and that these glycosides can have unique and different bioactivities from the glucosides. In particular, interest in the rhamnosylation of dietary nutraceuticals has increased in recent years due to the chemoprotectant effects of the ramnosidic linkage in relation to the hydrolysis of glucosides by human intestinal glucosidases and in relation to their novel biological activities. In plants, UDP-Rha is a ubiquitous sugar donor, as shown by the many diverse polysaccharides of the plant cell wall that are rhamnosylated. Plant GTs are thought to use this sugar nuACHTUNGTRENNUNGcleotide in preference to dTDP-Rha, which is typically used by bacterial enzymes. However, enzymatic rhamnosylation of plant natural products has not been explored extensively due to the fact that UDP-Rha is not commercially available and its chemical synthesis is multistep, time-consuming and low-yielding. Our aim in this study has been to investigate whether bacterial cells can be designed to provide Rha nucleotides for GT bioconversions in whole-cell biocatalysis. Whilst little is known of UDP-Rha synthesis in plants, the genome of Arabidopsis thaliana contains three genes with significant homology to those in micro-organisms that encode enzymes able to convert dTDP-Glc to dTDP-Rha. As illustrated in Scheme 1, the Nterminal domains of the proteins encoded by each of the plant genes RHM1 (At1g78570), RHM2 (At1g53500) and RHM3 (At3g14790; GeneBank Accessions AY042833, AY328518 and AY078958, respectively) contain a sequence similar to the bacterial 4,6-dehydratase, whilst their C-terminal domains resemble the bacterial 3,5-epimerase and the bacterial 4-keto-reductase. This sequence homology strongly suggested the plant enzymes might be capable of synthesising UDP-Rha from UDPGlc. In turn, engineering E. coli with a recombinant RHM gene might provide a novel means of generating UDP-Rha in the bacterial cell that could act as a sugar donor for biotransformations of small-molecule natural products. To examine the catalytic activity of each of the RHM proteins, their corresponding cDNAs were amplified from an Arabidopsis root cDNA library and were subcloned into a pGEX-2T vector for the production of recombinant protein. In the presence of UDP-Glc and the cofactor NADPH, the recombinant RHM proteins were found to be capable of synthesising UDPRha in vitro (Table 1). When dTDP-Glc was used an alternative