Plant Secondary Metabolism Linked Glycosyltransferases: an Update on Expanding Knowledge and Scopes

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JBA-07035; No of Pages 26 Biotechnology Advances xxx (2016) xxx–xxx

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Research review paper Plant secondary metabolism linked glycosyltransferases: An update on expanding knowledge and scopes

Pragya Tiwari a, Rajender Singh Sangwan a,b,NeelamS.Sangwana,⁎ a Department of Metabolic and Structural Biology, CSIR-Central Institute of Medicinal and Aromatic Plants (CSIR-CIMAP), P.O. CIMAP, Lucknow 226015,India b Center of Innovative and Applied Bioprocessing (CIAB), A National Institute under Department of Biotechnology, Government of India, C-127, Phase-8, Industrial Area, S.A.S. Nagar, Mohali 160071, Punjab, India article info abstract

Article history: The multigene family of enzymes known as glycosyltransferases or popularly known as GTs catalyze the addition Received 6 October 2015 of carbohydrate moiety to a variety of synthetic as well as natural compounds. Glycosylation of plant secondary Received in revised form 6 February 2016 metabolites is an emerging area of research in drug designing and development. The unsurpassing complexity Accepted 19 March 2016 and diversity among natural products arising due to glycosylation type of alterations including Available online xxxx glycodiversification and glycorandomization are emerging as the promising approaches in pharmacological studies. While, some GTs with broad spectrum of substrate specificity are promising candidates for glycoengineering Keywords: fi Catalytic mechanism while others with stringent speci city pose limitations in accepting molecules and performing catalysis. With the Drug designing rising trends in diseases and the efficacy/potential of natural products in their treatment, glycosylation of plant Glycoconjugates secondary metabolites constitutes a key mechanism in biogeneration of their glycoconjugates possessing medic- Glycosyltransferases inal properties. The present review highlights the role of glycosyltransferases in plant secondary metabolism Metabolic engineering with an overview of their identification strategies, catalytic mechanism and structural studies on plant GTs. Fur- Plant secondary metabolism thermore, the article discusses the biotechnological and biomedical application of GTs ranging from detoxification of xenobiotics and hormone homeostasis to the synthesis of glycoconjugates and crop engineering. The future directions in glycosyltransferase research should focus on the synthesis of bioactive glycoconjugates via metabolic engineering and manipulation of enzyme's active site leading to improved/desirable catalytic properties. The multiple advantages of glycosylation in plant secondary metabolomics highlight the increasing significance of the GTs, and in near future, the enzyme superfamily may serve as promising path for progress in expanding drug targets for pharmacophore discovery and development. © 2016 Published by Elsevier Inc.

Contents

1. Introduction...... 0 1.1. Plantsecondarymetaboliteglycosylationmetabolism:anintroduction...... 0 2. Plantsecondarymetabolismglycosyltransferases:anoverview...... 0 3. PlantGTsinCAZYdatabase...... 0 4. IdentiﬁcationandisolationofGTs...... 0 4.1. Transcriptomicsand/ormetabolomicsstrategies...... 0 4.2. FunctionalanalysisofUGTgenesthroughclassicalapproaches...... 0 4.3. Bioinformaticstudies...... 0 4.4. Biochemicalmethods...... 0 4.5. Molecularbiologystudies...... 0 5. Kineticparametersofcatalysisandsubstratepreferences...... 0 6. Family1GTs:themechanismofcatalysis...... 0 7. Crystalstructureofglycosyltransferases...... 0

Abbreviations: GTs, glycosyltransferases; PSPG, plant secondary product glycosyltransferases; UGT, UDP dependent glycosyltransferase; TOGT, Tobacco-O-glucosyltransferase; ATTED- II, database of gene coexpression in Arabidopsis; AtUGT72B1, Arabidopsis thaliana UGT72B1; MtUGT71G1, Medicago truncatula UGT71G1; MtUGT85H2, Medicago truncatula UGT85H2; VvGT1, Vitis vinifera ﬂavonoid 3-O-glucosyltransferase; vvUFGT, Vitis vinifera anthocyanidin glucosyltransferase. ⁎ Corresponding author. E-mail addresses: [email protected], [email protected] (N.S. Sangwan).

7.1. GT-Afamily...... 0 7.2. GT-Bfamily...... 0 8. 3Dstructureofplantglycosyltransferases...... 0 8.1. Flavonoid/triterpene GT from M. truncatula ...... 0 8.2. Flavonoid specific glycosyltransferase (WsFGT) from W. somnifera ...... 0 8.3. Flavonoid glucosyltransferase (CaUGT3) from C. roseus ...... 0 9. BiotechnologicalandbiomedicalapplicationsofGTs...... 0 9.1. Roleindefensemechanism...... 0 9.2. Synthesisofvaluableglycoconjugates...... 0 9.3. GTsinvolvedinhormonalregulation...... 0 9.4. GTs involved in modification of xenobiotics and detoxificationofpollutants...... 0 9.5. GTsinvolvedinsecondarymetabolitebiosynthesis...... 0 9.6. Stabilizationofsecondarymetabolites...... 0 9.7. Plant-microbeinteractions...... 0 9.8. Metabolicengineeringofcrops...... 0 9.9. PharmacologicalstudiesusingUGTs...... 0 10. Futureprospectsinglycosyltransferaseresearch...... 0 Acknowledgments...... 0 References...... 0

1. Introduction of a sugar moiety to low molecular weight secondary metabolites influ- ences acceptor's properties like solubility, stability, bioactivity, subcellu- Glycosyltransferases (GTs) catalyze stereospecific and regiospecific lar localization and binding properties with other molecules leading to transfer of nucleotide diphosphate-activated sugars to a wide and reduced toxicity of endogenous and exogenous substances (Lim et al., diverse range of molecules from proteins, lipids, nucleic acids to antibi- 2004). For example, terpenoids such as monoterpenols (geraniol and otics and other low molecular weight compounds known as secondary linalool) are toxic for the plant as such and are chemically hydrophic metabolites (Lairson et al., 2008; Weadge and Palcic, 2009). Glycosylation thus affecting their mobility and transport across tissues in plants. How- mechanism is the key modification step occurring in various biological ever, glycosylation results into the generation of a monoterpenol gluco- processes resulting in formation of myriad of plant secondary metabolites side which becomes transportable, less toxic, stable and also attains possessing glycodiversity. Together with hydroxylation, methylation and altered volatility affecting aroma (Bonishch et al., 2014). acylation reactions, glycosylation contributes to the complexity and di- Glycosyltransferases bear considerable importance and interest versity of plant secondary metabolites. As per the IUBMB guidelines, gly- owing to the fact that glycan moiety forms an integral and essential cosyltransferases have separate Enzyme Commission numbers and are component of natural products, conferring pharmacological properties classified on various parameters including similarities based on amino to the molecule leading to enhanced bioavailability, reduced toxicity acids (Campbell et al., 1997; Coutinho et al., 2003), substrate specificity, and increased solubility. Although, the present trends have highlighted reaction mechanism (inversion or retention of anomeric carbon) the significant prospects of GTs in drug-designing and development, the (Coutinho et al., 2003), 3D structures (GT-A, GT-B or predicted GT-C) stringent specificity of some GTs limits glycodiversification and intro- and type of reaction. Till 2015, GTs have been classified into 97 families duces the need for GT engineering (Williams et al., 2007)whileothers (GT1-GT97, (http://www.cazy.org/GlycosylTransferases)withGT-1fam- are promiscuous tools in alterations involving glycosylation patterns. ily consisting of maximum candidates of UGT genes. 108 GT crystal struc- Emerging trends in glycoengineering have witnessed the manipulation tures are reported in Protein data bank comprising of 40 members of active sites in enzymes and site-directed mutagenesis with some suc- classified in GT-A sub-family, 58 members in GT-B sub-family, 2 mem- cess (Gutmann and Nidetzky, 2012; He et al., 2006; Modolo et al., 2009). bers in GT-C sub-family and 8 members have remained unclassified The present article provides an update on the knowledge and scopes of (http://www.rcsb.org/pdb). It is interesting to note that CAZY family the glycosyltransferase gene family and its functional aspects, more spe- shows a conserved mechanism of catalysis within its members and anal- cifically in plant secondary metabolism, methodologies employed for ysis of the primary sequence does not provide any information on enzy- their identification and isolation, multi-faceted role in biotechnological matic function (Breton et al., 2006). GTs have been further sub-divided and biomedical applications and the possible exploration of why the into four clans namely‐inverting GT-A fold (clan I) or GT-B fold (clan II) superfamily may serve as novel targets in drug discovery and develop- or retaining GT-A fold (clan III) or GT-B fold (clan IV). The four clans ment in future. include members of each CAZY family (Coutinho et al., 2003). (See Fig. 1.) Past decades have witnessed extensive progress in studies highlight- 1.1. Plant secondary metabolite glycosylation metabolism: an introduction ing the significance of GT superfamily but availability of limited biochemical data on individual member enzyme has hampered further Plant secondary metabolites form one of the most important group research for their functional understanding. Recent trends indicate the of metabolites finding extensive applications in food, nutraceuticals, identification and biochemical characterization of a number of GT medicine and pharmaceutical preparations. The plant secondary metab- genes with broad/specific functionality and their analysis at the tran- olism is in the global focal attention due to such growing applications of scriptome and metabolome levels has made significant contributions its products, and there is a clear uprising trend in studies pertaining to in studying the glycosylation mechanisms in planta. Large scale plant the pathways and biochemical reactions utilizing and biosynthesizing genome sequencing projects have contributed in deciphering the bio- plant secondary metabolites. Terpenoidal secondary metabolites are logical role of plant secondary metabolism glycosyltransferases in bio- biosynthesized through a complex network of reactions involving dif- synthesis of glycoconjugates of phytochemicals (Bhat et al., 2013; Ito ferential participation of MVA and DOXP pathways (Akhtar et al., et al., 2014), metabolic engineering of crops (Kristensen et al., 2005; 2012; Chaurasiya et al., 2012; Narnoliya et al., 2014). The wide variety Lim, 2005a; Weis et al., 2008) as well as prospective role in identifying of secondary metabolites have immense structural/chemical group di- key targets for drug designing and pharmacophore development versity such as terpenoids, alkaloids, phenylpropanoids, and steroids (Williams et al., 2007). Prior reports have suggested that incorporation and many of them are hydrophic and toxic to the producing cell.

Owing to this, most of them are stored in specific structures (Sharma et localizations within the cell as well as transported to modified struc- al., 2013; Yadav et al., 2014). Such secondary metabolites undergo var- tures such as trichomes (Sangwan et al. 2001; Yadav et al., 2014). ious important terminal transformations catalyzed by specificenzymes Most of the metabolites are synthesized, transported and accumulated so that their properties are modified in terms of volatility, toxicity and at secluded structural modifications such as glandular trichomes in mobility. Two of the such important transformations are catalyzed by plants (Shanker et al., 1999; Sharma et al., 2013; Bose et al., 2013; acyltransferases and glycosyltransferases in plants (Sharma et al., Odimegwu et al., 2013; Yadav et al., 2014). Glucosyltransferases in- 2009; Sharma et al., 2014; Tiwari et al., 2014). Acyltransferases are volved in plant secondary metabolism are those set of enzymes which major contributor in enhancing volatility through esterification where- bring out the addition of glycosyl moiety to hydrophobic as glycosyltransferases modify the properties and nature of aglycones phytoconstituents commonly referred as aglycones. Many of such reac- through glycosylation led lowering of volatility, if at all existed with tions are highly specific and diverse considering the nature of the the molecule (Sharma et al., 2009; Sharma et al., 2013). In this article, existing phytomolecules in nature (Singh et al., 2014). Such glycosylat- we have focused on the studies pertaining to glycosyltransferases in- ed derivatives of secondary metabolites not only exhibit tremendous volved in plant secondary metabolism. Many of the secondary metabo- structural diversity but also the pharmaceutical and medicinal signifi- lites are hydrophobic in nature and hence mobilized to targeted cance. (Singh et al., 2014).

2. Plant secondary metabolism glycosyltransferases: an overview ﬂavonoids constitute the largest family of secondary metabolites glycosylated by GTs, followed by phenolic GTs and hormone homeosta- The enzyme superfamily is ubiquitous, accounting for approximate- sis GTs, glycoconjugates of certain classes namely alkaloids and ly 1–2% genes in archaea, bacteria and eukaryotes (Lairson et al., 2008). cyanogenic compounds are relatively very few and still need to be Family 1 consists of maximum number of UGT candidates and placed in identiﬁed. clan II. The family 1 GTs exhibits inverting mechanism of catalysis and depict GT-B structural fold. Low molecular weight substances with 3. Plant GTs in CAZY database

\\OH,\\COOH,\\NH2,\\SH and C\\C functional groups are preferred for the enzymatic catalysis for single or multiple addition of a sugar moi- Carbohydrates are diverse molecules widely present in nature and ety (Lim and Bowles, 2004). UDP-glucose serves as the primary sugar perform a range of biological functions from structural components to donor in family 1 GT followed by UDP-galactose, UDP rhamnose, UDP carbon deposits and intra- and intercellular cell signaling between xylose and UDP-glucuronic acid respectively (Lim and Bowles, 2004). organisms. The Carbohydrate-Active Enzymes (CAZY) database was The category of sugar acceptors defines a wide range of plant secondary made accessable in 1999 and included a comprehensive database metabolites namely terpenes, sterols, phenolics, alkaloids, cyanohydrins linking the sequence, structure and function of the classified enzymes. and thiohydroximates (Vogt and Jones, 2000). The enormous diversity The CAZY database (CAZY, http://afmb.cnrs-mrs.fr/CAZY/) represents and complexity among the glycosides generated by the catalytic sugar more than 12,000 GT encoding sequences and includes the enzyme decoration suggest the presence of a class/family of GTs in plants but families which create, degrade or modify glycosidic bonds namely bio- on contrary, it has been seen that plant secondary metabolome defines synthesis of sugar conjugated molecules by glycosyltransferases and the occurrence of GTs. degradation by glycosidases, carbohydrate lyases and polysaccharide ly- Considering the structural aspects, a characteristic feature of plant ases, respectively (Lombard et al., 2014). The database provides online GTs is the presence of a consensus sequence, the plant secondary meta- access to the sequence-based classification of the gene families and bolic signature sequence (PSPG box), present in secondary metabolism their structural and functional information, upgraded time to time. GTs (Hughes and Hughes, 1994). The PSPG box is regarded as a con- Certain salient parameters define the features of the CAZY database served domain, essentially present in GTs involved in natural product and include the classification of the families based on significant glycosylation with only slight modifications and 60–80% sequence iden- amino-acid sequence homology, module based classification (CAZymes tity (Vogt and Jones, 2000). The N-terminal portion is more variable, are modular proteins) and the analysis of protein sequence released in suggesting that the catalytic domain might be involved in binding of di- GenBank systematically. However, in contrast to other enzymes namely verse sugar acceptors. It is interesting to note that family 1 GT is soluble proteases, esterases, DNAses etc. whose specificity cannot be estimated enzymes as compared to membrane-anchored mammalian GTs. An in from their sequence information, the broad substrate preference of silico motif diversity analysis has shown that the PSPG box was found CAZymes can be predicted from their family classification (Cantarel to be consistently present in all GT sequences at the C-terminal, discov- et al., 2012). Presently, the database includes genome information of ered through MEME tool. A wide range of sequences was analyzed for N2800 genomes classified in the respective kingdoms namely bacteria the presence of PSPG motif and phylogenetic evolution of the PSPG (2351), viruses (240), eukaryota (73) and archaea (158), respectively motif characteristic of plant secondary metabolic glycosyltransferases (Lombard et al., 2014). The methodology for gene sequence analysis was studied (Kumar et al., 2012). comprises of a variety of methods, tools and aids of bioinformatics in- The sugar dependent enzymes or Leloir enzymes possess cluding their combinations such as a combination of Hidden Markov remarkable functionality in glycosylation of diverse secondary Model (HMM) and Blast tools which compares protein models with se- metabolites resulting in generation of myriad of glycoconjugates quence information of catalytic and non-catalytic modules in CAZY da- (Tiwari et al., 2014a; Tiwari et al., 2014b). The addition of a carbohy- tabase, followed by a manual monitoring by experts. drate moiety to secondary metabolites such as flavonoids, terpenoids, Literature has suggested that a high percentage of plant GTs are in- phenolics, saponins, sterols and alkaloids results in positive implications volved in glycosylation of small molecules and classified in family 1 of on their properties thereby influencing their bioactivities. While, the CAZY database. The whole-genome sequencing of the model plant,

Fig. 1. Rooted phylogenetic tree constructed through neighbor joining method of MEGA 5.05 software highlighting the evolutionary relationship of GTs in plant secondary metabolism. The accession nos. of GTs involved in plant secondary metabolism in increasing order of evolution are as follows: glycosyltransferase UGT95A1 [Hieracium pilosella] (Accession no. 171906260), glucosyltransferase-like protein [Crocus sativus] (Accession no. 34015076), UDP-glucose:solanidine glucosyltransferase [Solanum tuberosum] (Accession no. 375004896), glycosyltransferase [Withania somnifera] (Accession no. 221228775), flavonoid glucosyltransferase [Allium cepa] (Accession no. 32816176), zeatin O-glucosyltransferase 3 [Arabidopsis thaliana] (Accession no. 46318045), ABA-glucosyltransferase [Vigna angularis] (Accession no. 18151384), ABA glucosyltransferase [Citrus sinensis] (Accession no. 367465462), UDP-glucose: chalcononaringenin 2′-O-glucosyltransferase [Dianthus caryophyllus] (Accession no. 52839682), tetrahydroxychalcone glucosyltransferase [Dianthus caryophyllus] (Accession no. 156138797), anthocyanin 3′-glucosyltransferase [Gentiana triflora] (Accession no. 27530875), phenylpropanoid:glucosyltransferase 2 [Nicotiana tabacum] (Accession no. 13492676), salicylate-induced glucosyltransferase [Nicotiana tabacum] (Accession no. 1685005), cyanohydrin UDP-glucosyltransferase UGT85K5 [Manihot esculenta] (Accession no. 346682867), UDP- glycosyltransferase 85C1 [Stevia rebaudiana] (Accession no. 37993673), UDP-glucose anthocyanin 5-Oglucosyltransferase [Medicago truncatula] (Accession no. 355499268), UDP-glucose glucosyltransferase [Rhodiola sachalinensis] (Accession no. 145280639), UDP-xylose phenolic glycosyltransferase [Solanum lycopersicum] (Accession no. 350534960), UDP-glycosyltransferase BMGT1 [Bacopa monnieri] (Accession no. 302310821), UDP-glucose glucosyltransferase [Fragaria × ananassa] (Accession no. 51705411), glucosyltransferase [Vitis vinifera](Acces- sion no. 363805188), limonoid UDP glucosyltransferase [Citrus maxima] (Accession no. 160690854), limonoid UDP glucosyltransferase [Citrus aurantium] (Accession no. 160690840), hydroxycinnamate glucosyltransferase [Brassica napus] (Accession no. 88999675), sinapate 1-glucosyltransferase [Brassica oleracea var. medullosa] (Accession no. 226533664), flavonoid glucosyltransferase [Crocus sativus] (Accession no. 222646154), monoterpene glucosyltransferase [Eucalyptus perriniana] (Accession no. 60650093), UDP-glucose:anthocyanin 5-O- glucosyltransferase [Perilla frutescens var. crispa] (Accession no. 4115559), UDP-glucose:flavonol 5-O-glucosyltransferase homolog [Solanum melongena] (Accession no. 112806966), DIMBOA UDP-glucosyltransferase BX9 [Zea mays] (Accession no. 226505740), UDP-glucose: anthocyanidin 3-O-glucosyltransferase [Freesia hybrid cultivar] (Accession no. 301353154), UDP glucose:flavonoid 3-O-glucosyltransferase [Ipomoea trifida] (Accession no. 32441911), UDP glucose-flavonoid 3-O-glucosyltransferase [Malus × domestica] (Accession no. 329790853), flavonoid 3-glucosyltransferase [Rosa hybrid cultivar] (Accession no. 327343824), flavonoid 3-O-glycosyltransferase [Litchi chinensis] (Accession no. 309951616), UDP-glucose:flavonoid-O-glucosyltransferase [Beta vulgaris] (Accession no. 46430997), glucosyltransferase [Phytolacca americana] (Accession no. 219566994), glycosyltransferase [Panax notoginseng] (Accession no. 332071130), UDP-glucose glucosyltransferase [Gardenia jasminoides] (Accession no. 342306020), tetrahydroxychalcone 2′-glucosyltransferase [Catharanthus roseus] (Accession no. 156138819), UDP-glucose:glucosyltransferase [Cucumis melo subsp. melo] (Accession no. 307136362), chalcone 4′-O-glucosyltransferase [Antirrhinum majus] (Accession no. 379067424), UDP-glucuronate:baicalein 7-O-glucuronosyltransferase [Scutellaria baicalensis] (Accession no. 37359710), UGT2 [Pueraria montana var. lobata](Acces- sion no. 216296852), UDP-glycose:flavonoid glycosyltransferase [Vigna mungo] (Accession no. 4115536), zeatin O-xylosyltransferase [Phaseolus vulgaris] (Accession no. 5802783), putative UDP-glucose:flavonoid glucosyltransferase [Ginkgo biloba] (Accession no. 378829085), flavonoid glycosyltransferase UGT94C2 [Veronica persica] (Accession no. 260279128), UDP- glucose:sesaminol 2′-O-glucoside-O-glucosyltransferase [Sesamum indicum] (Accession no. 165972256), UDP-glucose:flavonoid 3-O-glucosyltransferase [Prunus persica](Accessionno. 339715876), UDP-rhamnose:soyasaponin III-rhamnosyltransferase [Glycine max] (Accession no. 292684225), UDP rhamnose: anthocyanidin-3-glucoside rhamnosyltransferase [Petunia × hybrida](Accessionno.397567).

Arabidopsis thaliana have led to the comprehensive analysis of the GT glycoproteomic techniques namely Lectin-IGOT-LC/MS (an LC–MS superfamily in this plant. Sequence analysis methods have identified based approach) for β-1,4-galactosyltransferase-I (b4GalT-I), an iso- the presence of 120 GT sequences, grouped in family 1 in Arabidopsis ge- zyme of glycosyltransferase (Sugahara et al., 2012), Glycogene microar- nome. The identified genes in the Arabidopsis genome have the C- rays for studying differential expression of glycosyltransferases in terminal consensus and classified as UGT's with three exceptions corneas of mice (Saravanan et al., 2010), network-based approach to (Paquette et al., 2003). The family 1 GTs are further divided into two predict novel GTs (Sánchez-Rodríguez et al., 2014) and employing met- subsets: one subset consists of a PSPG conserved domain for nucleotide abolic quantitative trait loci (mQTL) analysis for the determination of sugar binding, present as well in mammalian GTs which glycosylate in- secondary metabolism associated genomic regions in fruit pericarp of ternal metabolites like steroids, dietary flavonoids and xenobiotics (Lim tomato (Alseekh et al., 2015). and Bowles, 2004). The other subset is relatively smaller, consisting of Furthermore, quantitative genetic approaches together with regula- GTs catalyzing sterol and glycerolipid glycosylation (Warnecke et al., tory network analysis were used for the identification of several key 1997). Microbial GTs leading to antibiotic biosynthesis namely genes in plant secondary metabolism in Zea mays (Wen et al., 2016). urdamycin (Hoffmeister et al., 2001), vancomycin (Zmijewski and The variation in plant secondary metabolites and the underlying genetic Briggs, 1989) and vicenistatin (Ogasawara et al., 2004) are classified constitution have been analyzed employing metabolic quantitative trait together in family 1 GTs. The glycosylation modification enhances the locus (QTL) studies. In one such study, the genetically inbred lines of hydrophilicity and stability of small molecules thereby influencing two populations were employed to determine the natural variation in their bioactivities (Thorson et al., 2001). Moreover, low molecular kernels of Z. mays. Furthermore, 57 QTL's were validated through ge- weight compounds are the substrate for family 1 GTs with the function- nome wide association studies (Wen et al., 2014) and a gene regulatory al groups \\COOH, \\NH2, \\OH, C\\Cand\\SH functional groups network was established for flavonoid biosynthetic pathway. Several present on substrate acting as probable glycosylation sites (Bowles et products of the respective genes were identified, a putative flavonol- al., 2006). The structural information on plant GTs is important to 3-O-glucosyltransferase (for vitexin biosynthesis) been one of them decode the functional mechanisms and their evolution, however the (Wen et al., 2016). availability of very few crystal structures of plant GTs namely Glycogenomics, a mass spectrometry-guided genome-mining meth- flavonoid/triterpene glycosyltransferase (UGT) from Medicago od comprises of a prospective approach for determination of microbial truncatula (Shao et al., 2005), flavonoid glycosyltransferase (WsFGT) glycosylated molecules. The method was employed for characterization from Withania somnifera (Jadhav et al., 2012)andflavonoid of glycosylated natural products and their biosynthetic network from glucosyltransferase (CaUGT3) from Catharanthus roseus (Masada et al., sequenced genome of microbes. The tandem mass spectrometry (MS) 2009)defines the major limitation. The structural information reveals technique was used to characterize N and O glycosyl group in the that plant GTs contain two Rossmann folds, where the activated donor sugar monomers and similarity to the respective secondary metabolism sugar binds in the C-terminal region whereas the acceptor binds in the gene was established through MS-glycogenetic code. Furthermore, the N-terminal region of the protein. The members of family 1 GTs partici- genes involved in aglycone biosynthesis (GNP) are classified (Kersten pate in diverse functions in plant secondary metabolism and play an et al., 2013). In addition to these, some other identification strategies important role in the growth and development of the plant (Wang are discussed in details for the identification and isolation of candidate and Hou, 2009). genes involved in glycosylation of plant secondary metabolism.

4. Identiﬁcation and isolation of GTs 4.1. Transcriptomics and/or metabolomics strategies

The breakthrough in whole genome sequencing and high- Large scale whole genome sequencing projects in the present era throughput screening and analysis of sequences have revolutionalized have been a breakthrough in the identification of new UGTs in the investigations on glycosyltransferases in recent years. It is believed A. thaliana. The decoding of complete genome sequence of A. thaliana, that plant glycosyltransferases are most abundant in nature as com- the model plant has revealed the presence of the glycosyltransferase pared to other organisms, considering the complex polysaccharide na- multigene family subject to initial analysis. The genome elucidation of ture of cell wall and glycosylation of secondary metabolites in plants Arabidopsis showed the presence of 100 putative GTs involved in the (Hansen et al., 2010). Various experimental methodologies in areas of glycosylation changes in plant secondary metabolites (Li et al., 2001). molecular biology, bioinformatics, biochemistry, DNA sequencing of ge- A superfamily of 119 UGTs was identified in Arabidopsis genome with nomes/transcriptomes, genome-wide association studies (GWAS) and the PSPG motif, the signature sequence conserved in members of family quantitative trait locus (QTL) mapping have facilitated the isolation, 1 GT respectively. The sequencing of whole plant genomes had uncov- cloning and analysis of genes encoding plant GTs. Earlier strategies in ered a huge family of UGT genes with novel functions. Transcriptome- isolation of these genes involved classical genetic approaches and bio- based strategies like transcriptome coexpression analysis, is a promising chemical studies such as isolation of mutants (Dooner and Nelson, technique in the identification of genes pertaining to secondary 1977) and enzyme purification (Vogt and Jones, 2000) which forms metabolism in plants. A set of regulatory genes regulate the activities the underlying platform in GT identification and research. Molecular bi- of genes operating in a specific metabolic pathway and coexpressed in ology techniques have facilitated the isolation and cloning of GT genes a specific organ or in a defined environmental condition (Yonekura- from microbes and plants into heterologous systems. Other identifica- Sakakibara, 2009). tion methods for UGT genes included homology-based screening of con- Flavonoids, the class of secondary metabolites have been extensively served domains or homologous genes for cDNA library screening studied for their glycosylation mechanism. The coexpression database (Martin et al., 2001a, 2001b). Additionally, biochemistry has ATTED-II (http://atted.jp) serves as a major resource for identification revolutionalized the entire GT research as the uncharacterized genes of new flavonoid encoding GTs in plants. Among 107 candidates in and the newly discovered ones are being increasingly characterized Arabidopsis, five UGTs were found to correlate with flavonoid biosyn- and their catalytic role in plants is being investigated and established. thetic genes with highest correlation with UGT89C1, for example two Other identification methods included the use of combinational genetic flavonoid UGTs were known namely flavonoid 3-O-glucosyltransferase libraries to establish N-linked glycosylation in yeast (Choi et al., 2003), and anthocyanin 5-O-glucosyltransferase (Tohge et al., 2005). use of web-based software for screening GT candidates SEARCHGTr Recently, genome-wide identification and expression profiling stud- (Kamra et al., 2005), development of rice databases for identification ies in developmental tissue stages revealed the presence of 96 UGT of GTs from rice (Cao et al., 2008) and glycogene database in Japan genes in Cicer arietinum genome. The analysis further highlighted the (JCGG-DB, http://riodb.ibase.aist.go.jp/rcmg/ggdb), utilization of differential expression pattern such as 84 CaUGTs showed high

Please cite this article as: Tiwari, P., et al., Plant secondary metabolism linked glycosyltransferases: An update on expanding knowledge and scopes, Biotechnol Adv (2016), http://dx.doi.org/10.1016/j.biotechadv.2016.03.006 6 P. Tiwari et al. / Biotechnology Advances xxx (2016) xxx–xxx expression levels while 12 CaUGTs revealed low levels of expression (2010) used a combination of multiple bioinformatic methods to iden- (Sharma et al., 2014). A transcriptome coexpression and independent tify new putative glycosyltransferases in A. thaliana.Theyusedseveral component analysis was undertaken to identify candidate genes in remote homology detection methods namely Profile Hidden Markov Arabidopsis utilizing 1388 microarray data available in public database. Models (HMM), Hydrophobic Cluster Analysis (HCA), BLAST and PSI- A. thaliana, a model plant extensively studied for flavonoid metabolism BLAST and 3D-Fold Recognition and discovered more than 150 protein and UGT79B1 and UGT84A2 were identified as anthocyanin sequences corresponding to glucosyltransferase class of enzymes. An glucosyltransferases. Out of the two genes, UGT79B1 exhibited similar example includes the accession ID At5g03795.1 which was discovered expression and correlation with anthocyanin biosynthetic genes while by employing all the three bioinformatic approaches and showed simi- UGT84A2 did not show significant correlation and was related to flavo- larity to exostosin protein family. The N-terminal domain catalyzing ad- nol biosynthetic genes, respectively (Yonekura-Sakakibara et al., 2012). dition of β-1, 4-glucuronic acid residues is classified in GT47 family Transcriptomic analysis was performed for the identification of MeJA- while the C-terminal domain introduce α1,4-N-acetylglucosaminyl res- inducible genes in Panax ginseng (Jung et al., 2014). The study constitut- idues to the acceptor is classified in GT64 family. Moreover, the study ed the sequencing and de novo assembly of P. ginseng transcriptome and highlighted the presence of GT signature sequence in DUF266 and two GTs namely PgUGT74AE2 and PgUGT94Q2 were characterized and DUF246 protein families although distantly related to CAZY families studied for their functional role in ginsenoside biosynthesis (Jung et al., GT14 and GT65, respectively. The data generated helped in the annota- 2014). tion of Arabidopsis genome, however the characterization of the identified genes accounts for the major challenge in glycobiology (Hansen 4.2. Functional analysis of UGT genes through classical approaches et al., 2008). Additionally, to identify new GTs not reported in CAZY database, Classical approaches like cloning, purification of protein, mutant multiple bioinformatic tools were developed. Transmembrane Hidden isolation and cDNA libraries screening using heterologous probes have Markov Model (TMHMM) 2.0 prediction server (Krogh et al., 2001) been used to identify the functions of UGTs (Vogt and Jones, 2000). was employed for screening of Arabidopsis proteome (26,095 protein The catalytic activity of UGTs was determined by heterologous expres- sequences) and identified 27 putative GT sequences. About 90% of the sion in Escherichia coli or yeast and in vitro enzymatic assays. This has re- protein sequences were filtered and 2611 sequences were submitted vealed that UGTs had broad substrate specificity in vitro (Hansen et al., to the SUPERFAMILY prediction server, a selection method used for pre- 2003). The enzyme is selective for a particular or few positions on the diction of GT related superfamilies. The probable sequences were run substrate (Lim et al., 2004). A technique to study the subcellular locali- through 3D-PSSM (Kelley et al., 2000) and GenTHREADER (McGuffin zation of the enzyme involved green fluorescent protein (GFP), fusion and Jones, 2003) (fold-recognition) servers. Another in silico strategy proteins showed that UGT85B1 constitutes complex of multienzymes depended on the increasing availability and elucidation of 3dimensional that biosynthesizes dhurrin, a cyanogenic glucoside (Kristensen et al., structures of GTs, utilizing the remote homology detection methods 2005). Recently, an up and down regulation strategy of gene expression (HMMer program (Eddy, 1998), PSI-BLAST and Structural overlap was used to determine the regulation of scopoletin levels by TOGT, phy- (Sov) parameter calculations) (Geourjon et al., 2001). Chemometrics toalexin accumulation subject to tobacco mosaic virus infection. More- and bioinformatics define powerful methods to analyze whole genomes over, oxidative stress in plants was found to increase with (identify GTs in Mycobacterium tuberculosis genome) (Wimmerova downregulation of TOGT indicated that accumulation of scopoletin is re- et al., 2003). Principal Component Analysis (PCA) forms an important sponsible for deactivation of oxygen formed due to oxidative stress statistical tool for analysis of data in large collection of data. (Chong et al., 2002; Gachon et al., 2004). 4.4. Biochemical methods 4.3. Bioinformatic studies Earlier, purification of the target enzyme, determination of substrate The progress in the area of bioinformatics made a significant contri- preferentiality and enzyme kinetics were the few available methods to bution in the identification of plant glycosyltransferases. Conserved identify and categorize new GTs. With the advent of high-throughput motifs were discovered through the use of various motif discovery technologies and discovery of novel GTs in plant genomes, the biochem- tools which showed that PSPG box is a conserved consensus sequence ical characterization of the enzymes forms an integral aspect in found in most of the GTs involved in glycosylation of secondary metab- glycobiology. Studies have made substantial contribution in delineating olites in plants. This was a good beginning in the identification of new the functional role in glucosylation of plant secondary metabolites GTs from a database. While some methods in GT identification utilized through characterization of the large repertoire of available GTs in motif search programs (Kikuchi and Narimatsu, 2006) others adopted public domain. With large complexity in enzymatic functions and BLAST approach to mine out new GTs (Cantarel et al., 2009; Campbell discovery of the broad as well as substrate-specific GT genes, the utiliza- et al., 1997). In 2006, under Glycogene project, Kikuchi and Narimatsu tion of biochemical techniques in glycobiology constitutes an integral developed a bioinformatic system for the identification and in silico approach in identification and validation of the uncharacterized cloning of human glycogens. The study identified and engineered 105 collection of GT clones. Wei et al. (2015) isolated four novel GTs namely glycogenes corresponding to human for heterologous expression and UGTPg100, UGTPg101, UGTPg102, and UGTPg103 (Yan et al., 2014)and 38 recombinant proteins were characterized for substrate specificity. established the catalytic activities of the corresponding enzymes. The The bioinformatic system comprised of multiple strategies including in metabolic pathway of ginsenosides (pharmacological value) biosynthe- silico cloning through Phrap and GENSCAN, using profile Hidden sis was elucidated through the synthesis of glycon gensenosides of Markov Model for clustering glucosyltransferases and deciphering protopanaxatriol (PPT) and PPT-type ginsenosides. Another study glycosyltransferase evolution (Kikuchi and Narimatsu, 2006). reported the purification of a UDP-glucose anthocyanin 3′-O- In silico tools determine the related expressed sequence tags (ESTs), glucosyltransferase from Gentiana triflora functionally involved in mod- cDNA or genes and molecular biology methods such as mining of com- ification of flower color (blue) and anthocyanin biosynthesis (Fukuchi- plete open reading frames of genes, their expression in heterologous Mizutani et al., 2003). Similar study by Ogata et al. (2001) reported the system (E.coli or yeast) and biochemical techniques like enzyme purifi- purification of an anthocyanin 5-O-glucosyltransferase from the flowers cation and characterization are sequentially performed. Several GTs for of Dahlia variabilis and studied its role in the biosynthesis of aliphatic plant hormones namely auxins, cytokinins, brassinosteroids (BR) and acylated anthocyanins in flower petals, respectively. Furthermore, abscisic acid (ABA) were identified through these methods (Hou et al., biochemical methods employed for GT identification led to the 2004; Poppenberger et al., 2005). A study by Hansen and colleagues isolation of key GT enzymes like a 2,4,5-trichlorophenol detoxifying O-

However, some challenges also exist in employing the biochemical Delineating enzyme kinetics with respect to Vmax,Km and Kcat as param- strategies for GT identification. These methods have limitation since eters of kinetics of their catalysis for a defined substrate would reveal many GTs are present in very minute quantities. GTs are very labile the biochemical characteristics of the enzyme and would further estab- and occur in relatively low quantitative abundance which make the pu- lish the biosynthetic/catalytic role of GTs in plant. Table 1 summarizes rification of the enzyme difficult. GTs in secondary metabolism, are sol- the literature available on biochemical and kinetic characterization of uble enzymes generally with a molecular mass between 45 kDa and GTs functionally involved in glycosylation of plant secondary 60 kDa. A combination of techniques including anion exchange, dye li- metabolites. gand chromatography and hydrophobic interactions (Vogt and Jones, 2000)isusedforproteinpurification. 6. Family 1 GTs: the mechanism of catalysis

4.5. Molecular biology studies Glycosyltransferase, the biocatalysts demonstrate natural course of evolution for catalyzing glycosylation reaction. Many GTs (microbial) Molecular biology techniques like polymerase chain reaction (PCR), find application in antibiotic glycosides and oligosaccharides biosynthe- cDNA cloning and heterologous expression of genes have opened new sis (Koizumi et al., 1998; Mendez and Salas, 2001) but the use of plant avenues in the identification and characterization of GT enzymes. GTs as biocatalyst was limited due to unavailability of plant GT Several enzymes involved in glucosylation of anthocyanidins and sequences (Lim et al., 2005). The identification of several plant GTs, flavonoids from various plant sources were identified by cDNA library recombinant expression of the protein and catalytic characterization screening and using GT probes for differentially expressed cDNAs. The have greatly enhanced our knowledge about the catalytic mechanism data obtained have greatly enhanced knowledge of natural of these enzymes. GTs which catalyze the glycosylation of plant second- product GTs and emphasize on acceptor specificity. For example ary metabolites are classified in family 1and designated as GTs for small p-hydroxymandelonitrile GT also accepts mandelonitrile (struc- molecules (Henrissat and Coutinho, 2001; Li et al., 2001). In plants, tural resemblance) and benzyl alcohol, exhibiting broad spectrum cytosolic UGTs glycosylate a wide array of natural products such as activity. The enzyme also shows activity for monoterpenoid geraniol terpenoids, flavonoids, phenylpropanoids, terpenoids and steroids as for benzyl alcohol (Jones et al., 1999). The salicylic acid glucosylating (Bowles et al., 2006). GT shows enzymatic preference for acyl group of p-hydroxybenzoic Plant GTs have been found to glycosylate both aglycone and glycone acid or benzoic acid compared to endogenous substrates (Lee and molecules. Some members of family 1 catalyze the sugar transfer to Raskin, 1999). These examples show the specificity of GTs for individual glycosides while some plant GTs glycosylate the aglycone moiety of hydroxyl groups. secondary metabolites, for example formation of quercetin-3,7-di-O- Several studies on GTs functionally involved in biosynthesis of glucoside (Lim et al., 2004). GTs classified in family 1 catalyze the O, S- secondary metabolites of therapeutic/food value are increasingly as well as N-linkages and can form different linkages with the particular discovered and explored for its commercial value. The complexity and substrate. For example, Arabidopsis GT 72B1 forms an N-glucosidic bond diversity of secondary products are enormously being valued for phar- with 3, 4-dichloroaniline (a pollutant) and an O-glucosidic bond with 3, macological significance, stress responses, detoxification of pollutants, 4-dihydroxybenzoic acid (Loutre et al., 2003; Xu et al., 2013). GTs which plant-microbe interactions, hormone regulation or pathway engineer- are phylogenetically related display regioselectivity but employ differ- ing to name a few. The use of homology based PCR screening approach ent sugar donors for glycosidic linkage formation. GTs add sugar resi- has led to the identification of several GT genes namely a sterol GT dues to diverse nucleophile acceptors namely carbon, oxygen, which catalyzes glucosylation of cholesterol (present in bacteria) and nitrogen and sulfur and except C-glycosylation GTs, most GTs are in- ergosterol (fungus) to stigmasterol (plants) thus paving way for volved in heteroatom glycosylation (O-glycosylation, N-glycosylation, glycol-engineering of sterol metabolism (Tiwari et al., 2014a, 2014b), and S-glycosylation) respectively (Chang et al., 2011). Examples include GT from Ipomea nil and its ability to glucosylate different phytohor- A. thaliana UGT72B1, which catalyzes O-glycosylation, N-glycosylation, mones (Suzuki et al., 2007), defense responses to stress conditions and S-glycosylation (Chang et al., 2011). Recently, a bi-functional C (Lim and Bowles, 2004; Kanoh et al., 2014), detoxification of pesticides and O glucosyltransferase was discovered from Z. mays catalyzing and herbicides (Wetzel and Sandermann, 1994), enhanced bioavailabil- glycosylation of flavonoid C and O glucosides (Ferreyra et al., 2013). ity (Thorson et al., 2001), detoxification of xenobiotics (Brazier et al., 2007) and UGT94F4 and UGT86C4, involved in the biosynthesis of 7. Crystal structure of glycosyltransferases picroside, an iridoid glycoside possessing pharmacological properties (Bhat et al., 2013). The methods in molecular biology offers a practical Although, in recent years, a number of GTs have been isolated from approach to delineate the functional role of glucosyltransferase in different sources but problems associated with their overexpression, plant secondary metabolism. purification and crystallization have made the studies on crystal structure difficult. The first crystal structure was reported in 1994 for T4- 5. Kinetic parameters of catalysis and substrate preferences glucosyltransferase, a bacteriophage (Vrielink et al., 1994). Information on GT structure is available for 17 distinct GT families consisting of both A vast set of plant secondary metabolic glycosyltransferases has inverting and retaining enzymes, available at Glyco3D site (http:// been characterized with respect to their biochemical and kinetic prop- www.cermav.cnrs.fr/glyco3d). Furthermore, the structural information erties. New researches on GTs have added to our knowledge on enzyme on GTs obtained in the last 4 years has provided important information activity and its catalytic mechanisms. The proteomic characterization of on GTs, their enzymatic mechanism of action and specificity (Gloster, the enzyme elucidates the actual biochemical parameters influencing 2014).

laect hsatcea:Twr,P,e l,Patscnaymtbls ikdgyoytaseae:A paeo xadn nweg and knowledge expanding on update An glycosyltransferases: linked metabolism secondary Plant al., et (2016), P., Adv Biotechnol Tiwari, scopes, as: article this cite Please Table 1 Kinetic parameters of catalysis and substrate preferences of plant secondary metabolic glycosyltransferases.

GT/plant system NCBI/Genbank/DDBJ/Uniprot Km Vmax Substrate Products Reference accession number

UGT74AC1 from Siraitia grosvenorii HQ259620 41.4 μM – Mogrol Mogroside IE Dai et al. (2015) 58.2 μM Quercetin Quercetin glycosides 54.7 μM Naringenin Naringenin glycosides − UGT85K11 and UGT94P1 from Camellia AB847092 UGT85K11 332.1 nkat mg 1 Monoterpene β-primeverosides Ohgami et al. (2015) sinensis AB847093 Geraniol protein Aromatic 44.2 μM Aliphatic alcohols UGTPg1, UGTPg100, UGTPg101, KP795113 KP795114 104 μM 386 Protopanaxadiol Ginsenosides Wei et al. (2015) UGTPg102 and UGTPg103 from KP795115 153 μM 426 Protopanaxatriol Panax ginseng KP795116 http://dx.doi.org/10.1016/j.biotechadv.2016.03.006 UGT80B1 from At1g43620 –– β-sitosterol Sitosterol glycoside Mishra et al. (2015) Arabidopsis thaliana UGT84A17 from DY801582 –– Caffeic acid Caffeoyl-glucose Babst et al. (2014) Populus trichocarpa 4-coumaric acid 4-coumaroyl-glucose 4-hydroxybenzoic acid 4-hydroxybenzoyl-glucose 2-coumaric acid Feruloyl-glucose Ferulic acid Cinnamoyl-glucose

Sinapic acid Benzoyl-glucose xxx (2016) xxx Advances Biotechnology / al. et Tiwari P. UGT74S1, UGT74T1, UGT89B3, JX011632 –– Secoisolariciresinol Secoisolariciresinol Ghose et al. (2014) UGT94H1, UGT712B1 from JX011633 monoglucoside Linum usitatissimum L. JX011634 Secoisolariciresinol JX011635 diglucoside JX011636 UGT708C1 and UGT708C2 from AB909375 –– 2-hydroxyﬂavanones 2-hydroxynaringenin C-glucoside Nagatomo et al. Fagopyrum esculentum M. AB909376 2-hydroxynaringenin 2-hydroxyeriodictyol C-glucoside (2014) 2-hydroxyeriodictyol 2-hydroxypinocembrin C-glucoside 2-hydroxypinocembrin Phloretin C-glucoside Dihydrochalcone (phloretin) Trihydroxyacetophenones Trihydroxyacetophenones SrUGT74G1 from AY345982.1 –– Steviolbioside Stevioside Guleria and Yadav Stevia rebaudiana (2014) PgUGT74AE2 and PgUGT94Q2 from JX898529 25 μM – Protopanaxadiol Ginsenoside Rg3 and Rd Jung et al. (2014) Panax ginseng JX898530 Protopanaxatriol TaUGT4 from tplb0040h20 –– Deoxynivalenol Deoxynivalenol 3-glucoside Xin et al. (2014) Triticum aestivum GsSGT from NS –– Sterols Steryl glucosides Tiwari et al. (2014b)

β –

Gymnema sylvestre R.Br. Cholesterol Cholesteryl -D glucoside xxx Ergosterol Ergosteryl β-D glucoside Stigmasterol Stigmasterol β-D glucoside PNgt1 and PNgt2 from Pharbitis nil AB757750 –– Flavonoids Skimmin Kanoh et al. (2014) AB757751 Umbelliferone 3-hydroxy flavone 3,6-dihydroxy flavone 3,7-dihydroxy flavone Benzaldehyde derivatives Vanillin Vanillyl alcohol Coumarins Scopoletin Esculetin AdGT4 from KF954944 –– Terpene alcohol/alcohol Terpene glycosides Yauk et al. (2014)) Actinidia deliciosa aglycones Hexanol (Z)-hex-3-enol − GhSGT1 and GhSGT2 from Gossypium JN004107 15.1 μM 0.56 pmol mg 1 β-sitosterol β-sitosterol glucoside Li et al. (2014) − hirsutum JN004108 12.6 μM min 1 0.90 UDP-glycosyltransferases from BRADI1G43600 –– Deoxynivalenol Deoxynivalenol-3-O-glucoside Schweiger et al. Brachypodium distachyon (2013) UGT6, UGT7 and UGT8 from AB591741 UGT6 7-Deoxyloganetic acid 7-deoxyloganic acid Asada et al. (2013) laect hsatcea:Twr,P,e l,Patscnaymtbls ikdgyoytaseae:A paeo xadn nweg and knowledge expanding on update An glycosyltransferases: linked metabolism secondary Plant al., et (2016), P., Adv Biotechnol Tiwari, scopes, as: article this cite Please Catharanthus roseus NA 7-Deoxyloganetin 7-deoxyloganin 0.088 μM 0.202 μM 1.99 μM KF411463 –– – – WsGT from FJ560880 8.488 μM 37.79 Flavonoid-7-ols Diadzein 7-O-glucoside Singh et al. (2013) Withania somnifera 9.335 μM 39.79 Diadzein Naringenin 7-O-glucoside 11.61 μM 9.89 Naringenin Genistein 7-O-glucoside 12.79 μM 9.885 Genistein Luteolin 7-O-glucoside 13.01 μM 6.961 Luteolin Apigenin 7-O-glucoside Apigenin NSGT1 from KC696865 –– Phenylpropanoid volatiles-V diglycosides PhP-V triglycosides Tikunov et al. (2013) Solanum lycopersicum Eugenol Eugenol-2-O-b-D-glucopyranosyl-(1 → Guaiacol 2)-[O-b-D-xylopyranosyl-(1 → 6)]-O-b-D-glucopyranoside (GXG) http://dx.doi.org/10.1016/j.biotechadv.2016.03.006 Methyl salicylate Guaiacol-GXG Methyl Salicylate-GXG UGT94F2 and UGT86C4 from Picrorhiza JQ996408 JQ996409 –– – Picrosides Bhat et al. (2013)) kurrooa UGT73C11 and UGT73C13 from JQ291614 UGT73C11 Aglycone sapogenins Augustin et al. (2012) − Barbarea vulgaris JQ291616 9.7 μM 817 nmol min 1 Oleanolic acid 3-O-β-D-Glc oleanolic acid − 3.3 μM mg 1 Hederagenin 3-O-β-D-Glc hederagenin

UGT73C13 390 Oleanolic acid xxx (2016) xxx Advances Biotechnology / al. et Tiwari P. 12.5 μM 231 Hederagenin 22.9 μM 131 UGT75L6 and UGT94E5 from AB555731 UGT94E5 – Crocetin Crocin-5, crocetin-mono-(b-glucosyl)-ester Nagatoshi et al. (2012) Gardenia jasminoides AB555739 0.072 mM Crocin-3, crocetin-di-(b-glucosyl)-ester 0.023 mM UGT71A15 from DQ103712 82 μM 8 μmol/s kg Phloretin Phloridzin (phloretin 2′-O-glucoside) Gosch et al. (2012) Malus x domestica 188 μM 7-O-glucoside of kaempferol 243 μM 54 Kaempferol 3-O-glucoside of kaempferol 244 μM 49 Quercetin 7-O-glucoside of quercetin 354 μM 79 3-O-glucoside of quercetin 85 UGTSr from –––Stevioside Rebaudioside A Madhav et al. (2012) Stevia rebaudiana UGT79B1 and UGT84A2 from AB018115 –– Cyanidin 3-O-glucoside Cyanidin 3-O-xylosyl(1 → 2)glucoside Yonekura-Sakakibara Arabidopsis thaliana AB019232 Sinapic acid 1-O-sinapoylglucose et al. (2012) UGT707B1 from HE793682 –– Kaempferol Kaempferol-3-O-b-D-Glucopyranosyl-(1–2)-β-D-Glucopyranoside Trapero et al. (2012) Crocus sativus Quercetin Quercetin-3-O-rhamnosyl(1 → 2)-glucoside-7-O-rhamnoside sgtl3.1, sgtl3.2 and sgtl3.3 from EU342379 –– Sterols Sterol glucoside Chaturvedi et al.

Withania somnifera EU342374 (2012) – EU342375 xxx –––– – − UGT72B14 and UGT74R1 from EU567325 UGT72B14 57.8 pkat mg 1 Tyrosol Salidroside (tyrosol 8-O-b-D-glucoside) Yu et al. (2011) Rhodiola sachalinensis A. Bor. EF508689 4.7 μM 293.1 UGT74R1 172.4 μM UGT85A24 from AB555732 8.8 mM – Genipin Geniposide Nagatoshi et al. (2011) Gardenia jasminoides 0.61 mM 7-deoxyloganetin Gardenoside Loganetin 7-deoxyloganetin 1-O-glucoside Loganetin 1-O-glucoside UGT85K4 and UGT85K5 from Manihot JF727883 –– Acetone cyanohydrin Cyanogenic glucosides Linamarin Kannangara et al. esculenta JF727884 2-hydroxy-2-methylbutyronitrile Lotaustralin (2011) − SlUGT5 from HM209439 – 22.1 nkat/mg 1 Methyl salicylate Methyl salicylate glucoside Louveau et al., 2011 Solanum lycopersicum protein Guaiacol glucoside 19.8 Guaiacol Eugenol glucoside 7.62 Eugenol Benzyl alcohol glucoside 4.43 Benzyl alcohol Not Detected – Phenyl ethanol Hydroquinone glucoside 121.3 Hydroquinone β-isosalicin 77.5 Salicyl alcohol TaGTa-TaGTd from Triticum aestivum AB547237 –– Benzoxazinones Sue et al. (2011))

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GT/plant system NCBI/Genbank/DDBJ/Uniprot Km Vmax Substrate Products Reference accession number

ScGT from Secale cereale AB547238 2,4-dihydroxy-1,4-benzoxazin-3-one DIBOA glucose AB547239 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one AB547240 DIMBOA glucose F3GT1 and F3GGT1 from Actinidia GU079683 –– Anthocyanidin – Monteﬁori et al. chinensis FG404013 Cyanidin (2011) THC2’GT from Carnation, Cyclamen and –––Tetrahydroxychalcone 4,2′,4′,6′-Tetrahydroxychalcone (THC) 2′-glucoside Togami et al. (2011) Catharanthus roseus UGT76B1 from AC008153 –– Isoleucic acid Isoleucic acid glucoside Paul et al. (2011) Arabidopsis thaliana PlUGT1 from EU889119 –– Daidzein Diadzin Zhou et al. (2011) http://dx.doi.org/10.1016/j.biotechadv.2016.03.006 Pueraria lobata VvgGT1, VvgGT2 and VvgGT3 from JN164679 –– Hydroxybenzoic acids (C6C1) p-Hydroxybenzoyl-D-glucose Khater et al. (2011) Vitis vinifera JN164680 JN164681 4-hydroxybenzoic acid Protocatechoyl-D-glucose Protocatechuic acid Galloyl-D- glucose Gallic acid Syringoyl-D- glucose Syringic acid p-Coumaroyl-D-glucose Hydroxycinnamic acids (C6C3) Caffeoyl-D- glucose

p-coumaric acid Sinapoyl-D- glucose xxx (2016) xxx Advances Biotechnology / al. et Tiwari P. Caffeic acid Sinapic acid NA Stilbene Trans-resveratrol NA Flavonoids NA Quercetin NA Cyanidin Catechin − UGT78K1 from GU434274 174 μM 24.8 pKat μg 1 Anthocyanidins and ﬂavonol aglycones Kaempferol 3-O-glucoside Kovinich et al. Glycine max (L.) Merr. 16 μM – Kaempferol Cyanidin 3-O-glucoside (2010) Cyanidin ScUGT1-ScUGT5 from Sinningia AB537178 Flavonoids Nakatsuka and − cardinalis AB537179 145.8 μM 60.2 nmole mg 1 Apigeninidin Apigeninidin 5-O-glucoside Nishihara (2010) − AB537180 min 1 AB537181 633.2 μM 635.3 Lutcolinidin Luteolinidin 5-O-glucoside AB537182 UGT88D8 from AB465708 10.72 μM – Apigenin – Ono et al. (2010b) Veronica persica

CsUGT1, CsUGT2 and CsUGT3 from GQ221686 –– Terpenoids Terpenoid glycosides Fan et al. (2010) – xxx Citrus sinensis L.Osbeck GQ221687 GQ221688 SbUGT from GU339042 –– Flavonol Chiou et al. (2010) Scutellaria barbata Kaempferol Kaempferol 7-O-glucoside Flavanone Kaempferol 3-O-glucoside Naringenin Naringenin 7-O-glucoside Flavone Apigenin Apigenin 7-O-glucoside Isoflavones Daidzein Diadzein 7-O-glucoside GmSGT2 and GmSGT3 from Glycine max AB473730 –– – Soyasapogenol B Shibuya et al. (2010) AB473731 UDP-GT from Catharanthus roseus – 0.112 mM – Scopoletin Scopoletin 7-O-b-monoglucoside Piovan et al. (2010) 0.077 mM 5,7-dihydroxyflavone Dihydroxyflavone 5-O-b-monoglucoside 0.064 mM 5,7-dihydroxyflavanone Dihydroxyflavanone 7-O-b-monoglucoside 1.0 mM – Umbelliferone 7-O-b-monoglucoside – – Isoscopoletin 6-O-b-monoglucoside UDPG Esculetin 6-O-b-monoglucoside Umbelliferone

Isoscopoletin

Esculetin laect hsatcea:Twr,P,e l,Patscnaymtbls ikdgyoytaseae:A paeo xadn nweg and knowledge expanding on update An glycosyltransferases: linked metabolism secondary Plant al., et (2016), P., Adv Biotechnol Tiwari, scopes, as: article this cite Please CaUGT3, CaUGT4 and CaUGT5 from AB443870 CaUGT3 F Quercetin 3- O -gentiobioside Masada et al. (2009) Catharanthus roseus AB443871 0.38 μM lavonol glycosides Quercetin 3-O-gentiotrioside AB443872 0.36 μM Quercetin 3-O-glucoside Quercetin 3-O-gentiotetroside 0.69 μM Kaempferol 3-O-glucoside 2.00 μM Myricetin 3-O-glucoside 0.14 μM Quercetin 3-O-gentiobioside 0.18 μM Flavone glycosides 1.11 μM Apigenin 7-O-glucoside 0.32 μM Luteolin 7-O-glucoside N2.50 μM Flavanone glucoside 0.41 μM Liquiritigenin 4′-O-glucoside 0.67 μM Isoflavone glucoside 0.58 μM Genistein 7-O-glucoside N2.50 μM Cyanidin 3-O-glucoside http://dx.doi.org/10.1016/j.biotechadv.2016.03.006 N2.50 μM Curcumin glycosides 1.95 μM Curcumin monoglucoside N2.50 μM Curcumin diglucoside N2.50 μM Curcumin gentiobioside 0.72 μM Coumarin Esculin Phenolics Arbutin .Twr ta./BoehooyAvne x 21)xxx (2016) xxx Advances Biotechnology / al. et Tiwari P. p–Nitrophenylglucoside Purnasin Salicin UDP-glucose PaGT 1, PaGT2 and PaGT 3 from AB458516 PaGT3 – – Noguchi et al. (2009) Phytolacca americana L. AB458515 145 μM Capsaicin AB458517 20 μM Quercetin 66 μM Apigenin 20 μM Genistein UGT90A7, UGT95A1 and UGT72B11 EU561019 UGT90A7 4.7 pkat/μg Luteolin Witte et al. (2009) from EU561020 9.5 μM 4.0 Eriodictyol Luteolin 3′-O-glucoside Hieracium pilosella L. EU561016 11.8 μM Eriodictyol 3′-O-glucoside UGT95A1 27.2 Luteolin 46.8 μM Luteolin 3′-O-glucoside UGT72B11 7.6 Kaempferol 20.2 μM Kaempferol 3-O-glucoside Kaempferol 7-O-glucoside − UGT85H2 from DQ875463 35.5 μM 4.1 μM min 1 mg Flavonol Kaempferol 3-O-glucoside Modolo et al. (2009) Medicago truncatula 118.9 μM prot Kaempferol – 4.0 Isoflavone Biochanin A 7-O-glucoside xxx Biochanin A CsGT45 from FJ194947 15.6 μM 366 pkat/mg Kaempferol 7-OH Kaempferol 7-O-glucoside Moraga et al. (2009) Crocus sativus 86.95 μM 186 Quercetin 4′-OH Quercetin 7-O-glucoside Quercetin 4′-O-glucoside 30.3 μM 22.9 Quercetin 3′-OH Quercetin 3′-O-glucoside 21.50 μM 104 Quercetin 7-OH OsCGT from FM179712 16.5 μM – 2,5,7-trihydroxyflavanone 2,5,7-hydroxyflavanone-C-glucoside Brazier et al. (2009) Oryza sativa ssp. Indica – 2-Hydroxyflavanone 2-hydroxyflavanone-C-glucosides – 2,5-Dihydroxyflavanone 2-hydroxyflavanone-O-glucosides – 2,7-Dihydroxyflavanone 2,5-Dihydroxyflavanone C-glucosides 2.5 μM 2-Hydroxynaringenin 2,7-Dihydroxyflavanone C-glucosides NA 2-Hydroxyeriodictyol 2-Hydroxynaringenin C-glucosides Flavones 2-Hydroxyeriodictyol C-glucosides – Chrysin Chrysin 6-C-glucosides – Apigenin Chrysin 8-C-glucosides – Luteolin Apigenin C-glucosides – Naringenin Luteolin C-glucosides Naringenin chalcone Naringenin C-glucosides 8.3 μM 2′,4′,6′-Trihydroxydihydrochalcone Naringenin chalcone C-glucosides Phoretin 2′,4′,6′-Trihydroxydihydrochalcone 4.78 μM 2,4,6-Trihydroxybenzophenone Phoretin C-glucosides Maclurin Maclurin C-glucosides

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GT/plant system NCBI/Genbank/DDBJ/Uniprot Km Vmax Substrate Products Reference accession number

8.0 μM 1,3,5-Trihydroxybenzoic acid 1,3,5-Trihydroxybenzoic acid C-glucosides – 1,3,5-Trihydroxyacetophenone 1,3,5-Trihydroxyacetophenone C-glucosides – Benzyl 2,4,6-trihydroxybenzoate Benzyl 2,4,6-trihydroxybenzoate C-glucosides – UGT73F2 from DQ278439 28.33 μM – Daidzein Daidzin Dhaubhadel et al. Glycine max 6.3 μM Glycitein Glycitin (2008) 164.37 μM Genistein Genistin UDP-glucose: sterol GT from Solanum –– Steroid alkaloids Steryl glucosides Potocka and melongena a) Spirosolane type Zimowski (2008) Tomatidine http://dx.doi.org/10.1016/j.biotechadv.2016.03.006 Solasodine b) Solanidane type Solanidine Demissidine Steroid sapogenin Nuatigenin Isonuatigenin

Hecogenin xxx (2016) xxx Advances Biotechnology / al. et Tiwari P. Diosgenin Tigogenin Sarsasapogenin Yamogenin Sterols and their derivatives Sitosterol Stigmasterol Stigmasta-5,24(28)-dien-3β-ol Stigmastan-3β-ol Cholesterol 25-Hydroxycholesterol Cholest-5-en-3β,20α-diol Cholest-5-en-3β,19-diol Cholest-5-en-3β-ol-7-on 22-Oxycholesterol 5α-Cholest-7-en-3β-ol Thiocholesterol 5α-Cholestan-3β-ol

5β-Cholestan-3β-ol – xxx 5α-Cholestan-3α-ol 5β-Cholestan-3α-ol Androstane or pregnane derivatives Androstenolon Pregnenolon Triterpenic alcohols Lanosterol β-Amyrin FaGT1 from AAU09442 30 μM 21 nkat/mg Anthocyanidins Pelargonidin 3-O-glucoside Griesser et al. Fragaria x ananassa Pelargonidin Cyanidin 3-O-glucoside (2008a, 2008b) Cyanidin Peonidin 3-O-glucoside Peonidin Kaempferol 3-O-glucoside Kaempferol Quercetin 3-O-glucoside Quercetin Delphinidin Petunidin Malvidin Flavonol Galangin Fisetin Isorhamnetin Myricetin UGT73B4 and UGT73C1 from AC006248 UGT73B4 Gandia-Herrero et al. − laect hsatcea:Twr,P,e l,Patscnaymtbls ikdgyoytaseae:A paeo xadn nweg and knowledge expanding on update An glycosyltransferases: linked metabolism secondary Plant al., et (2016), P., Adv Biotechnol Tiwari, scopes, as: article this cite Please Arabidopsis thaliana AC006282 0.95 mM 4.36 nkat mg 1 2-hydroxylaminodinitrotoluene 2-HADNT-O-monoglucoside (2008) 0.44 3.32 4-hydroxylaminodinitrotoluene 4-HADNT-O-monoglucoside – – 0.15 0.008 2-aminodinitrotoluene 0.29 0.021 4-aminodinitrotoluene − Gt5GT7 from AB363839 29.5 mM 1.49 nmol min 1 Delphinidin Delphinidin 3’O glucoside Nakatsuka et al. − Gentiana triﬂora 20.9 mM mg protein 1 Cyanidin Cyanidin 3’O glucoside (2008) 12.2 mM 0.98 Pelargonidin Pelargonidin 3’O glucoside 3.4 0.68 Malvidin Malvidin 3’O glucoside NA 0.52 Kaempferol NA Apigenin NA Naringenin UGT706C1, UGT706D1, UGT707A3 and AP003560 –– Apigenin Apigenin-7-O-glucoside Ko et al. (2008) UGT709A4 from AP003560 Daidzein Diadzein 7-O-glucoside http://dx.doi.org/10.1016/j.biotechadv.2016.03.006 Oryza sativa AP005186 Genistein Genistein 7-O-glucoside AP005190 Kaempferol Kaempferol 3-O-glucoside Luteolin Luteolin 3′-O-glucoside Luteolin 4′-O-glucoside Luteolin 7-O-glucoside Naringenin Naringenin 7-O-glucoside Quercetin Quercetin 3-O-glucoside GmIF7GT from AB292164 3.6 μM – Genistein Genistein 7-O-β-D-glucopyranoside Noguchi et al. (2007) .Twr ta./BoehooyAvne x 21)xxx (2016) xxx Advances Biotechnology / al. et Tiwari P. Glycine max (genistin) Daidzein Daidzein 7-O-glucoside Formononetin Formononetin 7-O-glucoside Quercetin Quercetin 7-O-glucoside Kaempferol Kaempferol 7-O-glucoside 4,2′-tetrahydoxychalcone 4,2′-tetrahydoxychalcone 7-O-glucoside 4′,6′-tetrahydoxychalcone 7-O-glucoside 4′,6′-tetrahydoxychalcone Apigenin 7-O-glucoside Apigenin Aureusidin 6-O-glucoside Aureusidin Esculetin 7-O-glucoside Esculetin Naringenin 7-O-glucoside Naringenin − SGTL1, SGTL2 and SGTL3 from DQ356887 40 μM 8 pmol mg 1 Dehydroepiandrosterone Sterol glucosdes Sharma et al. (2007) − Withania somnifera DQ356888 min 1 Deacetyl 16-DPA DQ356889 10 7.2 Transandrosterone 25 2.5 3-b-hydroxy 16,17- α 1.8 epoxypregnenolene Pregnenolene 10 Stigmasterol

0.6 – 6 0.8 β-sitosterol xxx 27 0.6 Ergosterol 40 0.8 Brassicasterol 4 0.2 Solasodine 4.5 0.2 5-α-chol estan-3-β-ol 7 0.5 Cholesterol ND ND 27β-hydroxy GT from Withania – 0.17 mM 0.08 pmol/min 17α-OH withaferin A – Madina et al. (2007) somnifera 0.11 0.05 27β-OH withanone 0.11 0.04 Withanolide A 0.01 0.02 5α,6β,17α,27β- Tetrahydroxywithanolide 0.05 0.03 Withanolide U 0.17 1.05 Testosterone 0.04 0.15 Estradiol 0.07 0.06 21β-OH progesterone − UGT85H2 from DQ875463 2.9 μM 0.1073 μM min 1 Kaempferol Kaempferol 3-O-glucosde Li et al. (2007) Medicago truncatula 2.8 μM 0.0184 Quercetin Quercetin 3-O-glucosde 4.8 μM 0.0257 Biochanin A Biochanin A 7-O-glucoside 1918 μM 0.0124 Genistein Genistein 7-O-glucoside 9.9 μM 0.0742 Isoliquiritigenin – UGT74M1 from DQ915168 170 μM – Gypsogenic acid Gypsogenin 28-glucoside Meesapyodsuk et al.

(continued on next page) 13 14 laect hsatcea:Twr,P,e l,Patscnaymtbls ikdgyoytaseae:A paeo xadn nweg and knowledge expanding on update An glycosyltransferases: linked metabolism secondary Plant al., et (2016), P., Adv Biotechnol Tiwari, scopes, as: article this cite Please http://dx.doi.org/10.1016/j.biotechadv.2016.03.006

Table 1 (continued) .Twr ta./BoehooyAvne x 21)xxx (2016) xxx Advances Biotechnology / al. et Tiwari P.

GT/plant system NCBI/Genbank/DDBJ/Uniprot Km Vmax Substrate Products Reference accession number

Saponaria vaccaria 51 μM 16-OH gypsogenic acid (2007) 42 μM Gypsogenin 37 μM Quillaic acid UGT84A10 and UGT84A11 from AM231594 –– Hydroxycinnamates β-acetal esters Mittasch et al. Brassica napus AM231595 Ferulate (2007) Sinapate 4-coumarate Cinnamates Cinnamate Caffeate PsUGT1 from –––Kaempferol Kaempferol-C-glucuronide Woo et al. (2007) Pisum sativum Quercetin Quercetin-C-glucuronide Apigenin Apigenin-C-glucuronide Taxifolin Taxifolin-C-glucuronide Naringenin NA Indole Acetic Acid NA – Cytokinin NA xxx RUGT-10 from AP006584 –– Flavanone Naringenin 7-O-glucoside Hong et al. (2007)) Oryza sativa Naringenin Apigenin 7-O-glucoside Apigenin 4′-O-glucoside Flavone Kaempferol 3-O-glucoside Kamepferol 7-O-glucoside Apigenin Kamepferol 4′-O-glucoside Flavonol Kaempferol − FaGT2 from AY663785 356.9 μM 2.34 nkat mg 1 Cinnamic acid Cinnamoyl –D-glucose Lunkenbein et al. Fragaria ananassa 603.5 μM 2.69 p-Coumaric acid p-coumaroyl-D- glucose (2006) 707.7 μM 2.59 Caffeic acid Caffeoyl –D-glucose 358.5 μM 1.65 Ferulic acid Feruloyl-D-glucose 315.7 μM 2.24 5-Hydroxyferulic acid 5-Hydroxyferuloyl-Dglucose 300.2 μM 1.21 Sinapic acid Sinapoyl-D-glucose 502.6 μM 1.08 Benzoic acid Benzoyl-D-glucose 464.4 μM 1.05 3-Hydroxybenzoic acid 3-Hydroxybenzoyl-D-glucose 642.4 μM 0.77 para-Hydroxybenzoic acid p-Hydroxybenzoyl-Dglucose 515.3 μM 1.69 Vanillic acid Vanillin-D-glucose 108.0 μM 0.68 3,4-Dimethoxycinnamic acid Phenylpropionyl-D-glucose 411.2 μM 0.17 Phenylpropionic acid Phenylbutyoyl-D-glucose 431 μM 0.22 Phenylbutyric acid 3-Aminobenzoyl-D-glucose 488.2 μM 0.75 3-Aminobenzoic acid 4-Aminobenzoyl-D-glucose 437.1 μM 1.73 4-Aminobenzoic acid RuGT5 from Oryza sativa – 239.5 μM 1666.7 pkat/mg Kaempferol – Ko et al. (2006) 327 μM 2000 Apigenin laect hsatcea:Twr,P,e l,Patscnaymtbls ikdgyoytaseae:A paeo xadn nweg and knowledge expanding on update An glycosyltransferases: linked metabolism secondary Plant al., et (2016), P., Adv Biotechnol Tiwari, scopes, as: article this cite Please 120.7 μM 733.3 Genistein − UGT73A4 and UGT71F1from AY526080 UGT73A4 16.4 pkat μg 1 Quercetin – Isayenkova et al. Beta vulgaris AY526081 11.0 μM 12.8 Apigenin (2006) 19.2 μM 0.2 Betanidin 333 μM UGT71F1 0.7 Quercetin 2.4 μM 0.1 Apigenin 26.8 μM 0.03 Betanidin 29.0 μM UGT75L4 and UGT88A4 from DQ985179 UGT75L4 – Dihydrokaempferol – Tian et al. (2006) Maclura pomifera DQ985176 20.51 μM Dihydroquercetin 23.05 μM Kaempferol 2.99 μM Genistein 93.69 μM Isoliquiritigenin http://dx.doi.org/10.1016/j.biotechadv.2016.03.006 59.33 μM AtGT-1 from AY14269 18.0 μM 333.3 pkat/mg Eriodictyol Eriodictyol 7-O-glucoside Kim et al. (2006a, b) Arabidopsis thaliana 26.3 μM 333.3 Kaempferol Kaempferol 3-O-glucoside 35.3 μM 250.0 Quercetin Quercetin 3-O-glucoside – – Naringenin Naringenin 7-O-glucoside – – Apigenin Apigenin 7-O-glucoside – – Luteolin Luteolin 7-O-glucoside Sgt1 and Sgt2 from Solanum tuberosum DQ218276 –– Solanaceous aglycones α-chaconine McCue et al. (2006) .Twr ta./BoehooyAvne x 21)xxx (2016) xxx Advances Biotechnology / al. et Tiwari P. Solanidine α-solanine UGT73K1 and UGT71G1 from AY747626 GT029H – Achnine et al. (2005) Medicago truncatula AY747627 25 μM Quercetin Quercetin glycosides 32 μM Genistein Genistein 7-O-glucoside 45 μM Biochanin A Biochanin A 7-O-glucoside 166 μM Hederagenin Hederagenin 3- or 28-O-glucoside GT049F Soyasapogenol B 3-, 22-, 165 μM Soyasapogenol B or 23-O-glucoside Hederagenin 3- or 28-O-glucoside 235 μM Hederagenin UGT85B1 from Sorghum bicolor AF199453 –– p-hydroxymandelonitrile Cyanogenic glucoside Thorsoe et al. (2005) dhurrin Ih3GT from Iris hollandica AB161175 –– Anthocyanidins Delphinidin 3 glucoside Yoshihara et al. Delphinidin Malvidin 3 glucoside (2005) Malvidin Cyanidin 3 glucoside Cyanidin Peonidin 3 glucoside Peonidin /Pelargonidin Pelargonidin 3 glucoside

ND—Not detected. –

NA—No activity. xxx NS—Not submitted. 15 16 P. Tiwari et al. / Biotechnology Advances xxx (2016) xxx–xxx

GTs had been classified into two structural superfamilies, GT-A and its function and substrate specificities. Substrate docking, kinetics and GT-B, initially reported in SpsA and β-glucosyltransferase (BGT) struc- mutational studies decoded the complex structure of the enzyme and tures, respectively (Charnock and Davies, 1999; Vrielink et al., 1994). its regiospecificity in terms of preference for different isoflavonoids. In 2008, a third family of GTs was reported. The crystal structure of Heterologous expression in E.coli and kinetic characterization showed GT-C enzyme: STT3 from the archaea Pyrococcus furiosus was that UGT85H2 glucosylated diverse flavonoids namely the isoflavones established (Igura et al., 2008). The structure consists of a C-terminal genistein and biochanin A, flavonols kaempferol and quercetin, and soluble domain and central core domain including WWDYG motif the chalcone isoliquiritigenin utilizing UDP-glucose as sugar donor (Li responsible for its catalytic activity. The GT folds comprise of α/β/α et al., 2007). 7-O-positions in biochanin A and genistein are preferred sandwich pattern similar to the Rossmann type fold (six-stranded par- for regiospecific glucosylation as well as for kaempferol, producing allel β-sheet with 321,456 topology) present in many nucleotide bind- their respective 3-O-glucosides. ing proteins (Lesk, 1995). Till date, four crystal structures of plant GTs As compared to other plant GTs (VvGT1 and UGT71G1), linker re- are available namely AtUGT72B1, MtUGT71G1 and MtUGT85H2 and gion in UGT85H2 is longer and includes several other insertions and de- VvGT1. While, the first three belongs to family 72, 71 and 85, VvGT1 letions. A long insertion with seven or six residues more is seen between has not been nomenclature according to the classification (Mackenzie Nα5andNα5a in the N-terminal region. In the C-terminal domain five et al., 1997). The four crystallized structures of GTs show 20–35% and four residue insertions are present between Cβ2andCα2, and also, amino acid identity and remarkable conservation in their secondary a deletion between Cβ6andCα6 is present. Further, Nβ2 is followed by and tertiary structures (Osmani et al., 2009). Nα2 directly with the absence of a loop linker region similar to VvGT1 but different from UGT71G1. Several disordered regions were also 7.1. GT-A family found. These flexible regions differ in their length and conformations and are present around cleft formed by the N and C-terminal domains The GT-A structure consists of α/β/α sandwich, similar to Rossmann of the enzymes. This likely explains the substrate binding and specificity fold and consists of a seven stranded β-sheet (with 3,214,657 topology and the flexibility might be essential for recognition of different/diverse in which strand 6 is antiparallel to the rest). The β-sheet in the center is acceptors. The conformational changes occurring during substrate bind- flanked by a smaller one and both are associated to form an active site ing and glycosylation due to flexible regions in enzyme's active site offer (Breton et al., 2006). The presence of DxD motif and a divalent cation a possible explanation. The study showed that the presence of histidine is a conserved feature of members in GT-A family, essential component at 21st position and aspartic acid at 125th position is crucial and impor- for enzymatic function (Breton et al., 1998; Breton and Imberty, 1999). tant for the enzymatic activity of UGT85H2 enzyme (Li et al., 2007).

7.2. GT-B family 8.2. Flavonoid specific glycosyltransferase (WsFGT) from W. somnifera The GT-B structure comprises of two separate Rossmann domains 3D protein modeling and ligand docking studies have been reported with a connecting linker region and a catalytic site present between for a flavonoid glycosyltransferase from W. somnifera by Jadhav et al. the domains. The structure of the GT-B family had shown remarkable (2012). A protein model was generated for WsFGT through homology conservation mainly in the C-terminal domain which is the nucleotide modeling approach using MODELLER 9v9 and the model was subjected binding domain of the enzyme. Structural variations have been found to loop refinement and energy minimization through various bioinfor- in the N-terminal domains, in the helices and loops and active site matic tools. Further, the protein model showed a significant homology which evolved to accommodate different acceptors (Breton et al., to M. truncatula UDP-glucuronosyl/UDPglucosyltransferase (2PQ6: 31% 2006). A peptide motif, a glutamate residue and glycine-rich loops identity) and the docking studies were performed with various flavo- which interacts with the ribose and phosphate moieties of nucleotide noid acceptors such as luteolin, diadzein, apigenin, naringenin, genistein donor, respectively is present in members of GT-B family (Wrabl and and kaempferol and UDP-glucose as sugar donor. The binding pocket of Grishin, 2001). the model consists of 13 amino acid residues and these interacted with substrates with hydrogen bond formation. The sugar donor, UDP- 8. 3D structure of plant glycosyltransferases glucose was completely buried in the C-terminal domain of the enzyme while the residues involved in the interaction of enzyme with UDP- The structural information is an important tool to understand the glucose, were present in PSPG motif (Jadhav et al., 2012). evolutionary trends and catalytic mechanism of the proteins. Despite great progress in isolation and characterization of plant GTs, only a few crystal structures of plant GTs are available. Shao et al. (2005) and 8.3. Flavonoid glucosyltransferase (CaUGT3) from C. roseus He et al. (2006) presented the crystal structures of a flavonoid/ triterpene glycosyltransferase (UGT) from M. truncatula. It consists of A unique glucosyltransferase catalyzing the 1,6-glucosylation of two Rossmann folds and acceptors bind the residues in the N-terminal flavonol and flavone glucosides was isolated from suspension culture portion of the protein whereas the activated donor sugars bind to of C. roseus. The functional properties of the enzyme were investigated amino acid residues in the C-terminal region. The present trends in GT through homology modeling and site-directed mutagenesis. A research highlight the importance of X-ray crystallographic studies in homology model of CaUGT3 docked with UDP-glucose and quercetin elucidation of 3D structure of plant GTs with an aim to unravel the struc- 3-O-glucoside was generated. Vitis vinifera flavonoid 3-O- tural complexity and utilization of bioinformatic strategies to decode glucosyltransferase, VvGT1, crystallized with an acceptor substrate the enzyme functionality through protein modeling and active site kaempferol, was used as template. CaUGT3 model has GT-B fold confor- docking studies. Recent studies on protein modeling and active site ma- mation and includes N and C terminal domains which form a deep cleft nipulations of GTs are described in Table 2. which site for binding of the sugar donor and sugar acceptor substrates. The docking energy of the model was determined to be 8.1. Flavonoid/triterpene GT from M. truncatula −32.84 kcal mol−1. Further, it was assumed that replacement of Phe121and Phe200 in VvGT1 to His125 and Asn206 in CaUGT3 is UGT85H2 from M. truncatula, involved in glycosylation of responsible for broad acceptability for substrates. The point mutation isoflavonoid class of secondary metabolites was cloned by Li et al. in His125Phe and Asn206Phe showed that His125 plays a more important (2007). Further, crystal structure of UGT85H2 was deduced at 2.1 Å res- role as compared to Asn206 in binding with the acceptor substrates olution which revealed new structural insights about the enzyme and (Masada et al., 2009).

9. Biotechnological and biomedical applications of GTs 9.2. Synthesis of valuable glycoconjugates

Considerable work has been carried out on GTs and the availability Plant glycosyltransferases are involved in in vitro biosynthesis of of genomic and biochemical data has made important contributions in glycoconjugates having diverse properties. The addition of sugar moie- biological studies of plant glycosyltransferases. Techniques involving ties to aglycones or to glycones in chains leads to generation of broad functional genomics, gene knock-outs/silencing, overexpression of spectrum of secondary/specialized metabolites with unique properties. genes, bioinformatic approaches including protein docking and site- For instance, quercetin, a flavonol occurs in 300 different glycosidic directed mutagenesis provide a good insight into the physiological forms in plants. Glycosylation is usually the terminal modification step roles of GTs in plants. This indicates that glycosyltransferases might in the biosynthesis of secondary metabolites, for example in case of play an important role in plant growth, development, defense responses W. somnifera, sterol glucosyltransferases were found to catalyze the and interaction with the environment. glucosylation of sterol, probably leading to its stabilization and storage The major classes of secondary metabolites such as the (Sharma et al., 2007; Madina et al., 2007). Such enzymate catalyzed phenylpropanoids, flavonoids and benzoates in plants and hormones synthesis of natural product glycosides are often preferred over the syn- namely auxins, gibberellins, cytokinins and abscisic acid occur as glyco- thetic processes using chemical methods as synthesis of stereospecific sides (Kleczkowski and Schell, 1995; Ostrowski and Jakubowska, 2014). glycosides does not involve the use of chemicals as blocking and Plant UGTs play a significant role in defense responses to stress condi- deblocking reagents, with low cost and fewer synthetic steps and the tions (Lim and Bowles, 2004; Kanoh et al., 2014), detoxification of pesti- microbial whole-cell systems could be used for production in fermenta- cides and herbicides (Wetzel and Sandermann, 1994), enhanced tion reaction at a much larger levels. Therefore, the application of GTs in bioavailability (Thorson et al., 2001), detoxification of xenobiotics (Bra- synthesis of glycoconjugates has biotechnological relevance (Wang and zier-Hicks et al., 2007; Messner et al., 2003) biosynthesis (Bhat et al., Hou, 2009). Recent research by Bhat et al. (2013) identified and charac- 2013), storage and transport of secondary metabolites (Gachon et al., terized UGT94F4 and UGT86C4, involved in the biosynthesis of 2005), hormone homeostasis and synthesis of bioactive natural prod- picroside, an iridoid glycoside possessing pharmacological properties ucts (Lim and Bowles, 2004; Paquette et al., 2003; Asada et al., 2013; (Bhat et al., 2013). The biochemical characterization of GTs in near fu- Ito et al., 2014). Glycosylation is an important regulatory mechanism ture and establishment of their catalytic properties would facilitate which plays a key role in maintaining cellular homeostasis. A wide their application as biocatalysts. Similarly, a C-glucoside specificGT range of sugar moieties is added either independently (monoglycosides) was cloned from Fagopyrum esculentum, demonstrating catalytic effi- or in chains (di- or tri-glycosides), resulting in a broad category of glyco- ciency for flavonoids and THAP-like compounds in E.coli cultures sug- sides possessing glycodiversity among them (Jones et al., 1999). Studies gesting the possibilities of efficient biotransformation yielding have reported that more than 6000 different glycoconjugates of flavo- glucosylated products of therapeutic value (Ito et al., 2014). Other im- noids occur in plants (Anderson and Markham, 2006). portant applications of GTs include the use of purified enzymes in fer- mentors and enzyme immobilization to recycle enzymes ((Dulik and Fenselau, 1988), in plants employing whole cell systems by internal 9.1. Role in defense mechanism GTs and supplementation of exogenous aglycones. The main advantage is that the activated sugar donor for the reaction is provided by the liv- The addition of a carbohydrate moiety to a toxic substance converts ing cells particularly UDP-rhamnose, which is not available commercial- it to non-reactive and stable form which can be stored within the cell. ly. Various types of living cells from seedlings to plant cell suspension Further, the attachment of a sugar residue would limit the interaction cultures have been employed as biocatalysts (Koen and Thiem, 1997). with other cellular components thereby reducing the chances of electron transfer from the aglycone to other components resulting 9.3. GTs involved in hormonal regulation in lower reactivity and thus, better stability of the molecule. Several examples show the importance of secondary metabolism specific The regulation of hormone level in plants is critical for plant growth glycosylation subjecting to defense responses. For example, the to- and adaptation to environmental changes. Glycosylation defines a key bacco glycosyltransferases, TOGTsglycosylateshydroxycoumarin, mechanism in regulation of phytohormones in glycosidic form, with lit- scopoletin, and hydroxycinnamic acids. Furthermore, decline in erature suggesting the presence of hormone glycosides except ethylene scopoletin glucoside levels and impaired resistance to Tobacco Mo- (Fujioka and Yokota, 2003; Woodward and Bartel, 2005; Ostrowski and saic Virus (TMV) were observed with downregulation of TOGTs in Jakubowska, 2014). The UDP-glycosyltransferases (UDP1 class) play an the transgenic tobacco (Chong et al., 2002). These findings suggested important role in the biosynthesis of ether-type and ester-type of phy- that glycosylation of scopoletin by TOGTs increases plant resistance tohormone conjugates in plants. Widely known in plant kingdom, the to pathogens. glycosylation of plant hormones defines an important regulatory mech- Glycosylation of saponins is another remarkable example of defense anism for assessing the phytohormone levels in plants during growth mechanism. Saponins are triterpene glycosides in which the oligosac- and developmental stages (Table 3). The phytohormones execute di- charide chain consists of glucose, galactose, arabinopyranose, GlcUA, xy- verse functions like the storage and transport of hormones and their lose or rhamnose attached to the C-3 of saponins which may be crucial degradation in metabolic pathway contrary to acting as signaling mole- for resistance against fungal pathogens. Removal or alteration in the po- cules (Ostrowski and Jakubowska, 2014). The glycosylation of plant hor- sition of sugar residues leads to loss of bioactivity. It is interesting to mones plays a critical role in influencing hormone homeostasis by note that fungal pathogen produces hydrolases which attack the C3 temporary inactivation and further by hydrolysis and conjugation with- chain to detoxify saponins. For instance, Gaeumannomyces graminis, out de novo synthesis. The benefit of such system is to minimized energy the oat root infecting pathogen synthesize avenacinase, (a β- loss due to recycling of intact molecules and rapid responses (Jones and glucosidase) for detoxification of avenicin, (Wang and Hou, 2009). In Vogt, 2001). another study, Kanoh et al. (2014) reported the isolation and biochem- The mechanism of hormone glycosylation is attributed to the chang- ical characterization of PNgt1 and PNgt2, GTs from the hairy roots of es in substrate recognition by hormone or changes in their properties Pharbitis nil. The biochemical characterization of the recombinant pro- (Kleczkowski and Schell, 1995). Studies involving endogenous glycosyl- tein showed significant glycosylation for coumarins and benzaldehyde ation of hormones have been reviewed, an example includes a recent derivatives, suggesting a correlation with skimmin biosynthesis, report that suggested the effect of O-glucosylation of cytokinin in highlighting the significance of plant GTs in defense responses and maize. Similar studies on glucosylation of zeatin, a naturally occurring phytoalexin production respectively (Kanoh et al., 2014). cytokinin identified a cis-zeatin O-glucosyltransferase (cisZOG1) from

Table 2 Protein modeling and docking studies of plant glycosyltransferases.

Plant source Classiﬁcation Template Resolution Substrates Catalytic Software used/type of Reference residues protein modeling

WsGTL1 and GT-B type Chimeric – β-sitosterol Asp535 GENO3D Pandey et WsGTL4 from glucosyltransferase from Brassicasterol Phe506 al. (2015) Withania Actinoplanes Deactyl-16-DPA Pro55 somnifera teichomyceticus, Dehydro-epiandrosteron 3H4T Epoxypregnenolone Ergosterol Pregnenolone Transandrosterone Solasodine Stigmasterol 24-methylene cholesterol Withaferin A Withanolide A Withanolide B Withanolide D Flavonoid GT from GT-B type Medicago truncatula 2.1 Å–3.1 Diadzein His18 Modeler 9v9, Jadhav Withania UDP-glucuronosyl/ Å Apigenin Asp110 homology modeling et al. somnifera UDPglucosyltransferase, Luteolin Trp352 Asn Autodock vina 1.1.2. (2012) 2PQ6 Naringenin 353 (31% identity) Genistein Kaempferol SlUGT5 from UDP-glycosyltransferase Arabidopsis UGT72B1 – Methyl salicylate His17 Glu Modeler 9.7, homology Louveau Solanum 72 family (60.5% Guaiacol 81 Phe 311 modeling, et al. lycopersicum identity) Benzyl alcohol AutoDock 4.2 tool (2011) Phenyl ethanol Hydroquinone Eugenol VvGT5 and VvGT6 GT1 family VvGT1 – Kaempferol Pro 19 Insight II modeling Ono et al. from Vitis UDP-glucose Arg 140 homology modeling (2010a, vinifera Gln 373 2010b) CaUGT3 from GT-B type VvGT1+ kaempferol – Quercetin 3-O-glucoside His125 Asn Molecular operating Masada Catharanthus complex, 206 environment software, et al. roseus (18% identity) ASEDock (2009) BpUGT94B1 from GT-B type MtUGT71G1(26%identity) – Cyanidin 3ʹglucoside Arg (R25) Sybyl protein modeling, Osmani Bellis perennis VvGT1(20% identity) 6″-O-malonylglucoside Procheck, et al. Delphinidin 3′ O homology modeling (2008) glucoside UGT71G1 from GT-B type A. orientalis GtfD (10% 2.6 Å Quercetin His22 Multi-wavelength anomalous Shao et al. Medicago identity) Genistein Asp121 dispersion (MAD) method, (2005) truncatula RESOLVE, PROCHECK UGT85B1 from GT1 family GtfA and GtfB – Mandelonitrile His23 Homology modeling program Thorsoe Sorghum bicolor (15% identity each) Ser391 COMPOSER, Sybyl software et al. R201 (SYBYL) (2005) UGT73A5 from GT-B type UDP-glucosyltransferase – UDP-glucose Glu378 Homology modeling tool in Hans et al. Dorotheanthus (Gtfb) Betanidin His22 molecular operating (2004) bellidiformis from A. orientalis, 1IIR environment (14% identity) (MOE) AMBER PROCHECK PROSA II

Z. mays and suggested that cis-zeatin and derivatives act as regulatory Ostrowski and Jakubowska (2014), an IAA specific glucosyltransferase elements in cytokinin homeostasis in plants (Martin et al., 2001a, was isolated from immature pea seeds and biochemically characterized. 2001b). Another example includes the overexpression of zeatin O- The study revealed the possible role of the enzyme in the glycoprotein glucosyltransferase from Phaseolus lunatus L. in the roots and leaves of modification in Pisum sativum. Interestingly, the glycosides of plant hor- the maize transgenic plant that led to higher fold expression and accu- mones have been reported for almost all the classes of phytohormones mulation of zeatin-O-glucoside and phenotype leading to growth retar- in plants. An abscisic acid glucosyltransferase was cloned and character- dation and tassel seed formation characteristic of cytokinin-deficient ized from Phaseolus vulgaris L. (Palaniyandi et al., 2015). The identified plants. These studies suggest that the regulation of cytokinin levels in gene was able to glucosylate the abscisic acid into an inactive form plant is influenced by O-glucosylation of cytokinins (Rodo et al., 2008). ABA-glucose ester, thereby playing a regulatory role in ABA homeostasis Auxins constitute an important class of phytohormones, indole during stress response and development in bean plant. acetic acid being a key example and exist in conjugated form. Glycosyl- Further, several studies have demonstrated the role of glycosyltrans- transferases catalyze the synthesis of ester conjugates through covalent ferase in hormone glycosylation, like N-glycosylation of cytokinins bond formation between the carboxyl group of the hormone and the (Veach et al., 2003; Wang et al., 2011; Hou et al., 2014), auxins hydroxyl group present at the C1 position in β-D-glucopyranose hemi- (Szerszen et al., 1994; Jackson et al., 2001; Tognetti et al., 2010), salicylic acetal form. In an attempt to address the function of IAA glycosides in acid (Lee and Raskin, 1999; Song, 2006), brassinosteroids plants, several studies investigated the glucosylation of auxins and (Poppenberger et al., 2005; Husar et al., 2011), and abscisic acid their effect on the phytohormone metabolism. In a recent study by (Priest et al., 2005; Palaniyandi et al., 2015; Liu et al., 2015). An

Table 3 Glycosyltransferases involved in hormonal regulation in plants.

Glycosyltransferase Phytohormone Glycosylation product Reference

UGT71C5 from Arabidopsis thaliana Abscisic acid Abscisic acid-glucose ester Liu et al. (2015) ABAGT from Phaseolus vulgaris L. Abscisic acid Abscisic acid-glucose ester Palaniyandi et al. (2015) UGT73C14 from Gossypium hirsutum L. Abscisic acid ABA-glucoside Gilbert et al. (2013) ABA GT from Abscisic acid Abscisic acid-glucose ester Xu et al. (2002) Vigna angularis 2-trans-(+)-Abscisic acid Trans-cinnamic acid UGT71B6 from Arabidopsis thaliana Abscisic acid and structural analogs Abscisic acid glucose ester Priest et al. (2005) PBI-413 PBI-410 PBI-82 IAA GT from Pisum sativum Auxin 1-O-IAA-glucose Ostrowski et al. (2015) Indole-acetic-acid UGT84B1 from Arabidopsis thaliana Auxin 1-O-indole acetyl glucose ester Jackson et al., 2001 Indole-3-acetic acid UGT74D1 from Arabidopsis thaliana Auxin Glucose esters Jin et al. (2013) Indole-3-acetic acid Indole-3-propionic acid Indole-3-butyric acid naphthalene acetic acid 2,4-dichlorophenoxyacetic acid Indole-3-carboxylic acid UGT73C6 from Arabidopsis thaliana Brassinosteroids Brassinolide-23-O-glucoside Husar et al. (2011) Brassinolide (BR) BR malonyl glucosides Castasterone Castasterone -23-O-glucoside UGT73C5 from Arabidopsis thaliana Brassinosteroids Brassinolide-23-O-glucoside Poppenberger et al. (2005) Brassinolide Castasterone -23-O-glucoside Castasterone UGT85A1 from Arabidopsis thaliana Cytokinin trans-Zeatin O-glucosides Jin et al. (2013) trans-zeatin UGT76C1 from Arabidopsis thaliana Cytokinin Cytokinin N-glucosides Wang et al. (2013) Trans-zeatin N6-isopentenyladenine UGT76C2 from Arabidopsis thaliana Cytokinin Cytokinin N-glucosides Wang et al. (2011) UGT84B1 from Arabidopsis thaliana Indole-3-acetic acid Indole acetyl glutamate Jackson et al. (2002) UGT74E2 from Arabidopsis thaliana Indole butryic acid IBA glucose Tognetti et al. (2010) OsSGT from Tuberonic acid Tuberonic acid glucoside Seto et al. (2009) Solanum tuberosum L. Salicylic acid Salicylic acid glucoside ZOG1 from Cytokinin Zeatin-O-glucoside Rodo et al. (2008) Phaseolus lunatus L. Zeatin UGT74F1 and UGT74F2 from Salicylic acid SA 2-O-beta-D-glucose Dean and Delaney (2008) Arabidopsis thaliana SA glucose ester InGTase1 from 2-trans-abscisic acid Glucose esters Suzuki et al. (2007) Ipomoea nil Indole-3-acetic acid Salicylic acid (±)-Jasmonic acid AtJGT1 from Arabidopsis thaliana Jasmonic acid Glucose esters Song et al. (2005) Dihydro jasmonic acid Indole-3-acetic acid Indole-3-propionic acid Indole-3-butyric acid ZOG1 from Cytokinin O-glucoside of cis-zeatin Mok et al. (2005) Phaseolus lunatus Cis-zeatin O-glucoside of trans-zeatin cisZOG1 from Zea mays Trans-zeatin O-glucoside of m-topolin Hydroxylated derivatives of benzyladenine (topolins) UGT76C2 from Arabidopsis thaliana Cytokinin N6-benzyladenine-7-N-glucoside Hou et al. (2004) N6-benzyladenine N6-benzyladenine-9-N glucoside Trans-zeatin Trans-zeatin-7-N-glucoside Cis-zeatin Trans-zeatin-9-N-glucoside Dihydrozeatin Dihydrozeatin-7-N-glucoside N6-(+2-isopentenyl)-adenine Dihydrozeatin-9-N-glucoside Kinetin N6-isopentenyladenine-7-N-glucoside N6-isopentenyladenine-9-N-glucoside Kinetin-3-N-glucoside cisZOG2 from Cytokinin Cis-zeatin riboside Veach et al. (2003) Zea mays Cis-zeatin Cis-zeatin-O-glucoside cis-zeatin-O-GT from Zea mays Cytokinin Cis-zeatin Martin et al. (2001b) Zeatin SA GTase from Nicotiana tabacum Salicylic acid SA 2-O-β-D-glucoside Lee and Raskin (1999) Glucosyl salicylate AtSAGT1 from Arabidopsis thaliana Salicylic acid Glucosyl salicylic acid Song et al. (2009) AtSGT1 from Arabidopsis thaliana Salicylic acid SA 2-O-β-D-glucoside Song (2006) Salicylic acid glucose ester OsSGT1 from Salicylic acid Salicylic acid O-beta-glucoside Umemura et al. (2009) Oryza sativa

Please cite this article as: Tiwari, P., et al., Plant secondary metabolism linked glycosyltransferases: An update on expanding knowledge and scopes, Biotechnol Adv (2016), http://dx.doi.org/10.1016/j.biotechadv.2016.03.006 20 P. Tiwari et al. / Biotechnology Advances xxx (2016) xxx–xxx interesting example includes the isolation of a glucosyltransferase from the vacuolar region. Functional genomic approaches identified three sa- I. nil and its ability to glucosylate different phytohormones (Suzuki et al., lient GTs shown to be involved in the synthesis of the major sweet 2007). The enzyme exhibited a broad substrate preference ranging from phyto-glucosides of S. rebaudiana. The heterologous expression and char- indole-3-acetic acid to salicylic acid and from 2-trans-abscisic acid to (± acterization of the GTs revealed their regioselectivity towards steviol )-jasmonic acid forming the respective glucose esters. Such studies glucosylating activities (Richman et al., 2005). would shed light on the physiological roles of phytohormones GTs and their regulatory mechanism in plants (Table 3). Moreover, it is assumed 9.6. Stabilization of secondary metabolites that studies involving transgenics in sense and antisense orientation would shed some light on the actual role of glycosylation mechanism Glycosylation plays a key role in stabilization of phytomolecules. For in regulation of hormone metabolism in plants. example, the anthocyanins which are the water soluble pigments present in the vacuole constitute the secondary metabolites, widely taken 9.4. GTs involved in modification of xenobiotics and detoxification of up for the studies related with glycoside formation versus stabilization. pollutants Glycosylation together with the acylation of the sugars takes place in the cytosol and then the conjugated anthocyanins are made transport- The glycosylation mechanism in plant secondary metabolism is an able to get accumulated in the vacuoles. It is interesting to note the efficient way to neutralize the toxic effect of foreign pathogens, pollut- mechanism of glycosylation may not participate in the color formation ants, toxic substances and xenobiotics. For example, the detoxification of the anthocyanin structure but plays a critical role in the maintenance of DON (trichothecene deoxynivalenol), a toxic substance produced by of the structural integrity and stability of the flavylium cation. Further- Fusarium, a common pathogen of cereals such as wheat, maize and bar- more, together with acylation process, complex anthocyanin structures ley, is toxic to plant growth and development. A putative glycosyltrans- are formed (Strack and Wray, 1989). Furthermore, mutational studies in ferase from Arabidopsis, UGT73C5, catalyzes the conversion of DON to grapevine suggested the absence of anthocyanidin glucosyltransferase nontoxic DON 3-O-glucoside. Further, the overexpression of UGT73C5 leading to anthocyanidin-less berries. Glycosylation at the C-3, C-5 or in transgenic tobacco increased the resistance to DON (Poppenberger C-7 position of anthocyanidins inhibits conversion into non-colored et al., 2003) showing the detoxification role of GTs in plant. forms, prevents its breakdown and improves stability of the molecule. Additionally, the activity of the enzymes against exogenous com- Therefore, the improved stability of glycosides limits their interaction pounds like insecticides, xenobiotics, pollutants and herbicides has with the catabolic enzymes (Wajant et al., 1994). The cyanohydrins been reported (Lim et al., 2002; Loutre et al., 2003). Several Arabidopsis (Moller and Seigler, 1998) and thihydroxymates (Halkier and Du, UGTs are involved in the detoxification of 2, 4, 5-trichlorophenol (a xe- 1997) breakdown into smaller constituents and glycosylation stabilizes nobiotic) (Messner et al., 2003) or the pollutant 3, 4-dichloroaniline these molecules and protects the structure thereby facilitating their (DCA) (Loutre et al., 2003). Other examples include the detoxification accumulation in plant. of 2, 4-dihydroxy-1,4-benzoxazin-3-one (DIBOA) and 2,4-dihydroxy- Another example of stabilization of molecule by glycosylation 7-methoxy-1,4-benzoxazin-3-one (DIMBOA) by overexpression of GT process highlights the 2-O-glucosides of L-ascorbic acid, generally BX8 or GT BX 9 in transgenic Arabidopsis plants (von-Rad et al., 2001). more resistant to enzymatic and chemical oxidation compared to 6-O- Ferreyra et al. (2013) identified and cloned a bifunctional C and O glucosides or aglycones (Yamamoto et al., 1990). The addition of sugar glycosyltransferase involved in biosynthesis of insecticidal C-glycosyl moieties at specific position is essential in improving chemical stability flavones, maysin and flavanone O-glycosides. of the plant metabolites. The role of glycosylation mechanism in stabilization of secondary metabolites is significant in case of cyanogen- 9.5. GTs involved in secondary metabolite biosynthesis ic glycosides and glucosinolates. The addition of a sugar moiety to the molecule prevents its degradation to aldehydes, cyanide or isothiocya- The role of glycosyltransferases and glycosylation mechanism in the nates and is essential for the maintenance of stability (Kahn et al., 1997). biosynthesis, storage and transport of secondary metabolites has been demonstrated in several studies. In lignin biosynthesis, lignin mono- 9.7. Plant-microbe interactions mers (coumaryl, coniferyl and sinapyl alcohols) are required to be translocated for polymerization to lignin. UGT72E2 and UGT72E3, the It is assumed that glycosylation mechanism might play role in inter- recombinant glycosyltransferases of Arabidopsis, display 4-O- specific or intraspecific signaling in plant-microbe interactions. Bacteria glucosylation of phenylpropanoids (Lim et al., 2005b), suggesting that implicated in the plant to microbe interactions detect the presence of these enzymes might play a role in the biosynthesis of lignin. The plant secondary metabolites such as phenolics (Peters and Verma, exact mechanism of association between monolignol glycosylation 1990). It is believed that the glycosylation status of the phenolics may and lignin synthesis is not clear but it has been assumed that the down- be involved in influencing the signaling and perception response. In regulation of these GTs led the lowered glucoside levels of the mono- Prunus avium, secondary metabolite phenolic glycosides enhance mers of lignins in transformed Arabidopsis plants (Lanot et al., 2006). phytotoxin synthesis in Pseudomonas, while the phenolic aglycones do Secondary metabolites such as monoterpenoids, hydroxybenzoic not exhibit inductive response indicating the involvement of glycosyla- acids and flavonols accumulate in plants both as aglycones and glycones. tion (Mo et al., 1995). Similar example shows that in symbiotic bacteria, Jones et al. (2003) isolated 3 GTs, UGT75C1, UGT78D2 and UGT79B1 Rhizobium meliloti,luteolinactsasanod-gene inducer (Peters et al., from Arabidopsis involved in flavonol glycosides biosynthesis. Glycosyla- 1986) while luteolin 7-O-glucoside is a weak inducer and proceed via tion is the terminal step in flavonol biosynthesis exhibiting a require- free aglycone hydrolysis for signaling mechanism (Hartwig and ment of stability and translocation of molecule. Another example is the Phillips, 1991). biosynthesis of steviol glycosides in Stevia rebaudiana.Theplantextract Several other studies indicate the role of glucosyltransferases in comprises of a mixture of as many as eight different glycosides of response to pathogen infection. A study by Jimenez et al. (2005) showed diterpenoi steviol (the most intense sweet compound, 300 times sweeter that infection of Beta vulgaris with Pseudomonas syringae or than sugar). The glycosylation mechanism begins with steviol and lead- Agrobacterium tumefacians and mechanical wounding induced ing to formation of mono-, di-, tri- and tetraglycosides. S. rebaudiana glucosyltransferase (BvGT) expression in the plant. Furthermore, the leaves comprise of stevioside and the tetraglycoside rebaudioside as correlation between the BvGT antisense construct transient expression the major steviol glycosides (Wang and Hou, 2009). The first step and decrease in BvGT transcript accumulation suggested the role of GT (glucosylation of the C-4 carboxyl position of steviolbioside) occurs in gene in betacyanin glucosylation and the reactive oxygen species plastids and the glycosides are transported into the leaf cells occupying (ROS) produced by membrane associated NADPH oxidase may induce

BvGT expression. In a similar study, a UDP-glucosyltransferase induced Recently, a rice glycosyltransferase phylogenomic database was cre- in response to P. syringae infection was cloned and characterized from ated by Cao et al. (2008),whoidentified rice-diverged glycosyltransfer- the bean plant. The cDNA clone was designated as Hra25 (for hypersen- ases. It would provide promising lead in studying GTs in rice and other sitive reaction associated) and its activation in response to non-avr crop plants. Strategies involving manipulation of active site residues (general signals) or avr-derived signals (specific) was studied in re- through docking studies and generation of mutants through knockouts sponse to P. syringae infection and wounding (Sullivan et al., 2001). An- and gene silencing are the emerging research trends in genetic engi- other study by Langlois-Meurinne et al. (2005) reported the function of neering of crops and would establish the biological role of GTs in plants. plant secondary metabolism glucosyltransferases in plant-pathogen interactions. UGT73B3 and UGT73B5 from A. thaliana exhibited distinct 9.9. Pharmacological studies using UGTs expression profiles on P. syringae infection, defense-signaling mutants showing salicylic acid induced and methyljasmonate independent Many of the phytomolecules are known to exist as glycosides namely expression. The study highlighted the contribution of UDP-GTs in flavonoids, hormones, sweeteners, antibiotics, and alkaloids (Blanchard plant-microbe interactions and their role in hypersensitivity response. and Thorson, 2006).Thepresenceofcarbohydratemoietyisacriticalre- The role of a glucosyltransferase gene during superoxide-dependent quirement for pharmacological parameters of drug and its activity (Kren cell death in A. thaliana was studied by Mazel and Levine in 2002.The and Martínkova, 2001). Techniques in molecular biology and biochemis- gene, UGT73B5 was induced by the superoxides, in the presence of try have made significant contributions in biosynthetic studies of glyco- salicylic acid or cycloheximide and mechanical crushing and infection sylated molecules both at genomics and metabolomics level. It has with Botrytis cinerea. It was presumed that UGT73B5 may act in been reported that the attachment of a sugar moiety to a molecule en- transport of cellular components from the tissues undergoing cell hances its solubility and hence, bioavailability (Thorson et al., 2001). death, acting in coordination of metabolites during defense mechanisms Studies have reported that glycosylation of bioactive compounds at spe- (Mazel and Levine, 2002). Furthermore, several studies have made cific positions modify their pharmaceutical properties (Kren and major contributions in deciphering the function of glucosyltransferase Martínkova, 2001; Mijatovic et al., 2007). However, synthesis of specific class of enzymes in plant-microbe interactions namely conversion of glycoconjugates employing organic chemistry is difficult and enzymatic Deoxynivalenol (DON) produced by Fusarium graminearum to DON-3- glycosylation is a better alternative strategy for biosynthesis of desired glucoside (Schweiger et al., 2013; Xin et al., 2014; Michlmayr et al., glycoconjugates. These parameters are important aspects to be consid- 2015), induction of tobacco genes (TOGT) in response to fungal elicitors ered in rational drug designing. The availability of plethora of bioactive and synthesis of conjugated aromatic metabolites (Fraissinet-Tachet natural products with medicinal properties and their glycosylation et al., 1998) and benzoxazinones synthesis in T. aestivum and Secale mechanism provides a key platform for identification of drug targets cereale against microbial or herbivore attack (Sue et al., 2011). Role of and pharmacophore development. Studies involving active site muta- glucosyltransferase in Mi-mediated (nematode root-knot nematode tions would probably confer new property/function to the existing en- Meloidogyne species) nematode resistance was demonstrated (Schaff zyme with an aim of refinement in certain specifictraits. et al., 2007). In the study, the root transcriptome of tomato nematode The use of GT enzymes in pharmaceutical industry holds good pros- resistant (Mi+) and susceptible (Mi−) cultivars of the plant, Motelle pects but the unavailability of nucleotide activated sugars has posed a and Moneymaker was studied following a time-course infection with limitation on further applications of these enzymes (Luzhetskyy et al., the pathogen. Furthermore, virus induced gene silencing of 2007). Another aspect in drug engineering is the availability of informa- glucosyltransferase reintroduced susceptibility to M. incognita in tion of a vast set of GT genes which may catalyze side chains formation Motelle plant highlighting the functional role of the gene in resistance and glycosylate molecules of diverse properties. With the progress in to the nematode (Schaff et al., 2007). whole-genome sequencing projects, novel GTs are being identified At the intraspecies levels, glycosylation process may also influence and their pharmacological prospects are explored immensely. plant-to-plant signaling related responses. An example is flavonol aglycones, kaempferol and quercetin in maize and petunia, which are 10. Future prospects in glycosyltransferase research involved in the germination of pollen while the corresponding glycosides are shown to be non-responsive (Mo et al., 1992). The future prospects of plant secondary metabolic glycosyltransferases in commercial and economic applications look promising. With 9.8. Metabolic engineering of crops the range of activities catalyzed by GTs from glycoconjugate synthesis to detoxification of pollutants and xenobiotics to drug targeting and The genetic manipulation of economically viable crops is an impor- crop engineering, the benefits of glycosylation are immense. The rising tant biotechnological application for improving food (nutritional con- trends in world population are creating more consumption demands tent) or the quality of crop. By employing the technique of enzyme and genetic engineering of economically viable crops for quality im- immobilization, a recombinantly cloned GT from pummelo converted provement is a step ahead. Mutational studies in plants including the bitter limonoids of lemon juice into tasteless glycosides (Karim sense and antisense approaches would demonstrate the biological role and Hashinaga, 2002). The techniques involving isolation and establish- of GTs in plants. For instance, GTs involved in homeostasis of plant hor- ment of enzymatic activities and biological roles of GTs in plants would mones such as cytokinins, brassicasteroids and auxins hold good pros- serve as a platform for crop improvement. GTs play diverse functional pects in creating crops with desired phenotypic traits by manipulating roles in plant secondary metabolism from defense responses to detoxi- the hormone levels which would further influence the growth and de- fication and regulation of hormone levels to increase in stability of mol- velopment of the plants. Other desired features of crop engineering in- ecules and would be an ideal candidate for creating transgenics with clude insertion of GTs involved in detoxification of pesticides and improved traits. For instance, GTs involved in homeostasis of plant hor- xenobiotics to enhance food quality and safety, to create plants with in- mones such as auxins, cytokinins and brassicasteroids offers new pros- creased resistance to abiotic and biotic stress and increased levels of gly- pects in creating crops with desired phenotypic traits by manipulating cosides in food with antioxidant and anticancer properties. the hormone levels which would further influence the growth and Further, it has been demonstrated that addition of a carbohydrate development of the plants. Other desired features of crop engineering moiety to a metabolite leads to enhanced solubility and reduced toxicity include insertion of GTs involved in detoxification of pesticides and thereby improvement in bioavailability. These properties are important xenobiotics to enhance food quality and safety, to create plants with criteria for drug designing. The glycosylated molecules serve as an ideal increased resistance to abiotic and biotic stress and increased levels of target in pharmacophore development. Certain GTs with broad glycosides in food with antioxidant and anticancer properties. spectrum activity are permissible candidates for enzyme manipulations

Please cite this article as: Tiwari, P., et al., Plant secondary metabolism linked glycosyltransferases: An update on expanding knowledge and scopes, Biotechnol Adv (2016), http://dx.doi.org/10.1016/j.biotechadv.2016.03.006 22 P. Tiwari et al. / Biotechnology Advances xxx (2016) xxx–xxx with some others with stringent specificity which are disadvantageous Plants (CSIR-CIMAP) for the constant encouragement and support. PT and a hindrance in glycoengineering. However, the active site mutation- is thankful to CSIR, New Delhi for the award of Senior Research Fellow- al studies have been a remarkable achievement in creating novel ship. RSS and NSS are responsible for the study concept, design, analysis “chimeras” with the desired traits and properties. and interpretation of the article. PT contributed in literature collection The biotechnological applications of GTs make them superior candi- and writing of the manuscript. RSS and NSS have critically read the man- dates in several important areas of research. However, certain aspects uscript. NSS holds the decision to submit the review in Biotech Advances. on GT studies still need to be explored in detail. The availability of crys- Finally, we would like to extend our sincere thanks to everyone whose tal structures of very few plant GTs (triterpene GT from M. truncatula, support directly or indirectly, have made this manuscript possible. flavonoid GT from Avena sativa and few more) is a major limitation in GT research. The correlation between three dimensional structure and References functional properties of the enzyme (acceptor binding specificity) is awaited. The elucidation of GT crystal structure would shed light on Achnine, L., Huhman, D.V., Farag, M.A., Sumner, L.W., Blount, J.W., Dixon, R.A., 2005. Genomics-based selection and functional characterization of triterpene glycosyl- the functional role of GTs and the biosynthesis of phytoconstituents – fi transferases from the model legume Medicago truncatula. Plant J. 41, 875 887. with pharmacological signi cance in important medicinal plants. Akhtar, N., Gupta, P., Sangwan, N.S., Sangwan, R.S., Trivedi, P.K., 2012. Cloning and func- The evolution of catalytic preferences for substrate highlights tional characterization of 3-hydroxy-3-methylglutaryl coenzyme A reductase gene regioselectivity of GTs for O glycosylation as compared to relatively from Withania somnifera: an important medicinal plant. Protoplasma 250, 613–622. http://dx.doi.org/10.1007/s00709-012-0450-2. very few C, N and S glycosylation respectively. Studies by Lim et al. Alseekh, S., Tohge, T., Wendenberg, R., Scossa, F., Omranian, N., Li, J., et al., 2015. Identifica- (2002) and Loutre et al. (2003) demonstrated that a bifunctional GT tion and mode of inheritance of quantitative trait loci for secondary metabolite abun- from Arabidopsis GT72B1 catalyzes an O-glycosidic linkage with 3, dance in tomato. Plant Cell (www.plantcell.org/cgi/doi/10.1105/tpc.114.132266). Anderson, O.M., Markham, K.R., 2006. Flavonoids: Chemistry, Biochemistry, and Applica- 4-dihydroxybenzoic acid and an N-glucosidic bond with 3, 4- tions. CRC Press, Boca Raton. dichloroaniline (a pollutant). GTs display enzymatic catalysis for Asada, K., Salim, V., Masada-Atsumi, S., Edmunds, E., Nagatoshi, M., Terasaka, K., et al., oxygen, carbon, nitrogen and sulfur and mostly involved in heteroatom 2013. A 7-deoxyloganetic acid glucosyltransferase contributes a key step in secologanin biosynthesis in Madagascar periwinkle. Plant Cell 25, 4123–4134. glycosylation (O-glycosylation, N-glycosylation and S-glycosylation) Augustin, J.M., Drok, S., Shinoda, T., Sanmiya, K., Nielsen, J.K., Khakimov, B., et al., 2012. except C-glycosylation GTs (Chang et al., 2011). Recently, a bifunctional UDP-glycosyltransferases from the UGT73C subfamily in Barbarea vulgaris catalyze maize GT was identified displaying C-glycosylation for natural insecti- sapogenin 3-O-glucosylation in saponin-mediated insect resistance. Plant Physiol. – cide, maysin as well as O-glycosylation for flavones as substrates. The 160, 1881 1895. Bhat, W.W., Dhar, N., Razdan, S., Rana, S., Mehra, R., et al., 2013. Molecular characteriza- evolution of dual catalytic mechanism in the maize GT highlights the tion of UGT94F2 and UGT86C4, two glycosyltransferases from Picrorhiza kurrooa: multiple functional roles, one in defense mechanism against insects comparative structural insight and evaluation of substrate recognition. 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