Glycosyltransferases:key players involved in the modification of plant secondary metabolites

Glycosyltransferases are members of the multigene superfamily in plants that can transfer single or multiple activated sugars to a range of plant molecules,resulting in the glycosylation of plant compounds.Although the activities of many glycosyltransferases and their products have been recognized for a long time,only in recent years were some glycosyltransferase genes identified and a few functionally characterized in detail.Glycosylation is thought to be one of the most important modification reactions towards plant secondary metabolites,and plays a key role in maintaining cell homeostasis,thus likely participating in the regulation of plant growth,development and in defense responses to stress environments.With advances in plant genome projects and the development of novel technologies in analyzing gene function,significant progress could be made in gaining new insights into the properties and precise biological roles of plant secondary product glycosyltransferases,and the new knowledge will have extensive application prospects in the catalytic synthesis of glycoconjugates and metabolic engineering of crops.In this review,we summarize the current research,highlighting the possible biological roles,of plant secondary metabolite glycosyltransferases and discuss their potential applications as well as aspects to be further studied in the near future.     

Glycosyltransferases: key players involved in the modification of plant secondary metabolites

Glycosyltransferases are members of the multigene superfamily in plants that can transfer single or multiple activated sugars to a range of plant molecules, resulting in the glycosylation of plant compounds. Although the activities of many glycosyltransferases and their products have been recognized for a long time, only in recent years were some glycosyltransferase genes identified and a few functionally characterized in detail. Glycosylation is thought to be one of the most important modification reactions towards plant secondary metabolites, and plays a key role in maintaining cell homeostasis, thus likely participating in the regulation of plant growth, development and in defense responses to stress environments. With advances in plant genome projects and the development of novel technologies in analyzing gene function, significant progress could be made in gaining new insights into the properties and precise biological roles of plant secondary product glycosyltransferases, and the new knowledge will have extensive application prospects in the catalytic synthesis of glycoconjugates and metabolic engineering of crops. In this review, we summarize the current research, highlighting the possible biological roles, of plant secondary metabolite glycosyltransferases and discuss their potential applications as well as aspects to be further studied in the near future.     

Discovery of glycosyltransferases using carbohydrate arrays and mass spectrometry

Glycosyltransferases catalyze the reaction between an activated sugar donor and an acceptor to form a new glycosidic linkage. Glycosyltransferases are responsible for the assembly of oligosaccharides in vivo and are also important for the in vitro synthesis of these biomolecules. However, the functional identification and characterization of new glycosyltransferases is difficult and tedious. This paper describes an approach that combines arrays of reactions on an immobilized array of acceptors with an analysis by mass spectrometry to screen putative glycosyltransferases. A total of 14,280 combinations of a glycosyltransferase, an acceptor and a donor in four buffer conditions were screened, leading to the identification and characterization of four new glycosyltransferases. This work is notable because it provides a label-free method for the rapid functional annotation of putative enzymes.     

Glycosyltransferases and their products: cryptococcal variations on fungal themes

Glycosyltransferases are specific enzymes that catalyse the transfer of monosaccharide moieties to biological substrates, including proteins, lipids and carbohydrates. These enzymes are present from prokaryotes to humans, and their glycoconjugate products are often vital for survival of the organism. Many glycosyltransferases found in fungal pathogens such as Cryptococcus neoformans do not exist in mammalian systems, making them attractive potential targets for selectively toxic agents. In this article, we present the features of this diverse class of enzymes, and review the fungal glycosyltransferases that are involved in synthesis of the cell wall, the cryptococcal capsule, glycoproteins and glycolipids. We specifically focus on enzymes that have been identified or studied in C. neoformans, and we consider future directions for research on glycosyltransferases in the context of this opportunistic pathogen.     

A class of plant glycosyltransferases involved in cellular homeostasis

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

An evolutionary view of functional diversity in family 1 glycosyltransferases

Glycosyltransferases (GTs) (EC 2.4.x.y) catalyze the transfer of sugar moieties to a wide range of acceptor molecules, such as sugars, lipids, proteins, nucleic acids, antibiotics and other small molecules, including plant secondary metabolites. These enzymes can be classified into at least 92 families, of which family 1 glycosyltransferases (GT1), often referred to as UDP glycosyltransferases (UGTs), is the largest in the plant kingdom. To understand how UGTs expanded in both number and function during evolution of land plants, we screened genome sequences from six plants (Physcomitrella patens, Selaginella moellendorffii, Populus trichocarpa, Oryza sativa, Arabidopsis thaliana and Arabidopsis lyrata) for the presence of a conserved UGT protein domain. Phylogenetic analyses of the UGT genes revealed a significant expansion of UGTs, with lineage specificity and a higher duplication rate in vascular plants after the divergence of Physcomitrella. The UGTs from the six species fell into 24 orthologous groups that contained genes derived from the common ancestor of these six species. Some orthologous groups contained multiple UGT families with known functions, suggesting that UGTs discriminate compounds as substrates in a lineage-specific manner. Orthologous groups containing only a single UGT family tend to play a crucial role in plants, suggesting that such UGT families may have not expanded because of evolutionary constraints.     

Comparative aspects of glycosyltransferases

Glycosyltransferases, the enzymes that build oligosaccharides and glycoconjugates, have received much interest in recent years owing to their biological functions and their potential uses in biotechnology. Despite the fact that many glycosyltransferases recognize similar donor or acceptor substrates, there is surprisingly limited sequence identity between different classes. On the one hand, the glycosyltransferases are found in a large number of families, by sequence-based classification. On the other hand, only two structural folds have been identified among the fewer than one dozen glycosyltransferases that have been crystallized at present. Detection of conserved motifs that have a direct role in the functional aspects of glycosyltransferases is one approach for identifying remote similarity. With the availability of more crystal structures, the use of the fold-recognition approach is also very promising.     

Molecular dynamics simulations of solvated UDP-glucose in interaction with Mg2+ cations.

Glycosyltransferases are key enzymes involved in biosynthesis of oligosaccharides. Nucleotide-sugars, the glycosyltransferase substrates, serve as activated donors of sugar residues during the enzymatic reaction Although very little is known about the catalytic mechanism of these enzymes, it appears that the catalytic activity in most glycosyltransferases is dependent upon the presence of a divalent cation, for example Mn2+ or Mg2+. It is not known whether the ion is bound to the enzyme before its interaction with the substrate, or if it binds the substrate before the enzymatic reaction to modify its conformation to fit better the active site of the enzyme. We have inspected the latter possibility by running four 2-ns molecular dynamics trajectories on fully solvated UDP-glucose in the presence of Mg2+ ions. Our results indicate that the divalent cation interacts strongly with the nucleotide-sugar in solution, and that it can alter its conformational behavior. It is also shown that a conformation of the pyrophosphate moiety that results in an eclipsed or almost eclipsed orientation of two of the oxygen atoms, and which is found in protein interacting with a nucleotide di- or tri-phosphate X-ray data, is energetically favored. The results are also discussed in light of existing NMR data, and are found to be in a good agreement with them.     

A bacterial glycosyltransferase gene toolbox: Generation and applications

The bioactivity of many natural products produced by microorganisms can be attributed to their sugar substituents. These substituents are transferred as nucleotide-activated sugars to an aglycon by glycosyltransferases. Engineering these enzymes can broaden their substrate specificity and can therefore have an impact on the bioactivity of the secondary metabolites.In this review we present the generation of a glycosyltransferase gene toolbox which contains more than 70 bacterial glycosyltransferases to date. Investigations of the function, specificity and structure of these glycosyltransferases help to understand the great potential of these enzymes for natural product biosynthesis.     

Genetics and biochemistry of secondary metabolites in plants: an evolutionary perspective

The evolution of new genes to make novel secondary compounds in plants is an ongoing process and might account for most of the differences in gene function among plant genomes. Although there are many substrates and products in plant secondary metabolism, there are only a few types of reactions. Repeated evolution is a special form of convergent evolution in which new enzymes with the same function evolve independently in separate plant lineages from a shared pool of related enzymes with similar but not identical functions. This appears to be common in secondary metabolism and might confound the assignment of gene function based on sequence information alone.     

Crystal structure of vancosaminyltransferase GtfD from the vancomycin biosynthetic pathway: interactions with acceptor and nucleotide ligands

The TDP-vancosaminyltransferase GtfD catalyzes the attachment of L-vancosamine to a monoglucosylated heptapeptide intermediate during the final stage of vancomycin biosynthesis. Glycosyltransferases from this and similar antibiotic pathways are potential tools for the design of new compounds that are effective against vancomycin resistant bacterial strains. We have determined the X-ray crystal structure of GtfD as a complex with TDP and the natural glycopeptide substrate at 2.0 A resolution. GtfD, a member of the bidomain GT-B glycosyltransferase superfamily, binds TDP in the interdomain cleft, while the aglycone acceptor binds in a deep crevice in the N-terminal domain. However, the two domains are more interdependent in terms of substrate binding and overall structure than was evident in the structures of closely related glycosyltransferases GtfA and GtfB. Structural and kinetic analyses support the identification of Asp13 as a catalytic general base, with a possible secondary role for Thr10. Several residues have also been identified as being involved in donor sugar binding and recognition.     

常见致病菌糖基转移酶的结构与功能

糖基转移酶(glycosyltransferases,GTs)将糖基从活化的供体转移到糖、脂、蛋白质和核酸等受体,其参与的蛋白质糖基化是最重要的翻译后修饰(post-translational modifications,PTMs)之一。近年来越来越多的研究证明,糖基转移酶与致病菌毒力密切相关,在致病菌的黏附、免疫逃逸和定殖等生物学过程中发挥关键作用。目前,已鉴定的糖基转移酶根据其蛋白质三维结构特征分为3种类型GT-A、GT-B和GT-C,其中常见的是GT-A和GT-B型。在致病菌中发挥黏附功能的糖基转移酶,在结构上属于GT-B或GT-C型,对致病菌表面蛋白质(黏附蛋白、自转运蛋白等)进行糖基化修饰,在致病菌黏附、生物被膜的形成和毒力机制发挥具有重要作用。糖基转移酶不仅参与致病菌黏附这一感染初始过程,其中属于GT-A型的一类致病菌糖基转移酶会进入宿主细胞,通过糖基化宿主蛋白质影响宿主信号传导、蛋白翻译和免疫应答等生物学功能。本文就常见致病菌糖基转移酶的结构及其糖基化在致病机制中的作用进行综述,着重介绍了特异性糖基化高分子量(high-molecular-weight,HMW)黏附蛋白的糖基转移酶、针对富丝氨酸重复蛋白(serine-rich repeat proteins,SRRP)糖基化修饰的糖基转移酶、细菌自转运蛋白庚糖基转移酶(bacterial autotransporter heptosyltransferase,BAHT)家族、N-糖基化蛋白质系统和进入宿主细胞发挥毒力作用的大型梭菌细胞毒素、军团菌(Legionella)葡萄糖基转移酶以及肠杆菌科的效应子NleB。为揭示致病菌中糖基转移酶致病机制的系统性研究提供参考,为未来致病菌的诊断、药物设计研发以及疫苗开发等提供科学依据和思路。     

The yeast expression system for recombinant glycosyltransferases

Glycosyltransferases are increasingly being used for in vitro synthesis of oligosaccharides. Since these enzymes are difficult to purify from natural sources, expression systems for soluble forms of the recombinant enzymes have been developed. This review focuses on the current state of development of yeast expression systems. Two yeast species have mainly been used, i.e. Saccharomyces cerevisiae and Pichia pastoris. Safety and ease of fermentation are well recognized for S. cerevisiae as a biotechnological expression system; however, even soluble forms of recombinant glycosyltransferases are not secreted. In some cases, hyperglycosylation may occur. P. pastoris, by contrast, secrete soluble orthoglycosylated forms to the supernatant where they can be recovered in a highly purified form. The review also covers some basic features of yeast fermentation and describes in some detail those glycosyltransferases that have successfully been expressed in yeasts. These include beta1,4galactosyltransferase, alpha2,6sialyltransferase, alpha2,3sialyltransferase, alpha1,3fucosyltransferase III and VI and alpha1,2mannosyltransferase. Current efforts in introducing glycosylation systems of higher eukaryotes into yeasts are briefly addressed.     

Nucleotide sugars and glycosyltransferases for synthesis of cell wall matrix polysaccharides

The current interest in cell wall biosynthesis is expanding because of the increasing evidence that the properties of the cell wall mediate cellular interactions during growth, development and differentiation. Much effort has been put forward to the identification of glycosyltransferases because of their obvious importance in polysaccharide synthesis. Enzymes involved in nucleotide sugar production and transport are also important because of the potential to manipulate the composition of cell walls through substrate level control. Molecular genetics have begun to uncover genes for important enzymes in polysaccharide biosynthesis including glycosyltransferases and enzymes of nucleotide sugar metabolism; but at this time, much is inferred from comparisons to bacteria, yeast and animal cells. This review examines the production and transport of nucleotide sugars, the protein structure of glycosyltransferases, and implications for the cellular mechanisms of cell wall biosynthesis.     

On the origin of family 1 plant glycosyltransferases

The phylogeny of highly divergent multigene families is often difficult to validate but can be substantiated by inclusion of data outside of the phylogeny, such as signature motifs, intron splice site conservation, unique substitutions of conserved residues, similar gene functions, and out groups. The Family 1 Glycosyltransferases (UGTs) comprises such a highly divergent, polyphyletic multigene family. Phylogenetic comparisons of UGTs from plants, animals, fungi, bacteria, and viruses reveal that plant UGTs represent three distinct clades. The majority of the plant sequences appears to be monophyletic and have diverged after the bifurcation of the animal/fungi/plant kingdoms. The two minor clades contain the sterol and lipid glycosyltransferases and each show more homology to non-plant sequences. The lipid glycosyltransferase clade is homologous to bacterial lipid glycosyltransferases and reflects the bacterial origin of chloroplasts. The fully sequenced Arabidopsis thaliana genome contains 120 UGTs including 8 apparent pseudogenes. The phylogeny of plant glycosyltransferases is substantiated with complete phylogenetic analysis of the A. thaliana UGT multigene family, including intron-exon organization and chromosomal localization.     

Targeting of active sialyltransferase to the plant Golgi apparatus.

Glycosyltransferases in the Golgi apparatus synthesize cell wall polysaccharides and elaborate the complex glycans of glycoproteins. To investigate the targeting of this type of enzyme to plant Golgi compartments, we generated transgenic Arabidopsis plants expressing alpha-2,6-sialyltransferase, a glycosyltransferase of the mammalian trans-Golgi cisternae and the trans-Golgi network. Biochemical analysis as well as immunolight and immunoelectron microscopy of these plants indicate that the protein is targeted specifically to the Golgi apparatus. Moreover, the protein is predominantly localized to the cisternae and membranes of the trans side of the organelle. When supplied with the appropriate substrates, the enzyme has significant alpha-2,6-sialyltransferase activity. These results indicate a conservation of glycosyltransferase targeting mechanisms between plant and mammalian cells and also demonstrate that glycosyltransferases can be subcompartmentalized to specific cisternae of the plant Golgi apparatus.     

An evolving hierarchical family classification for glycosyltransferases

Glycosyltransferases are a ubiquitous group of enzymes that catalyse the transfer of a sugar moiety from an activated sugar donor onto saccharide or non-saccharide acceptors. Although many glycosyltransferases catalyse chemically similar reactions, presumably through transition states with substantial oxocarbenium ion character, they display remarkable diversity in their donor, acceptor and product specificity and thereby generate a potentially infinite number of glycoconjugates, oligo- and polysaccharides. We have performed a comprehensive survey of glycosyltransferase-related sequences (over 7200 to date) and present here a classification of these enzymes akin to that proposed previously for glycoside hydrolases, into a hierarchical system of families, clans, and folds. This evolving classification rationalises structural and mechanistic investigation, harnesses information from a wide variety of related enzymes to inform cell biology and overcomes recurrent problems in the functional prediction of glycosyltransferase-related open-reading frames.     

Glycosyltransferases as biocatalysts

Glycosyltransferases are useful synthetic tools for the preparation of natural oligosaccharides, glycoconjugates and their analogues. High expression levels of recombinant enzymes have allowed their use in multi-step reactions, on mg to multi-gram scales. Since glycosyltransferases are tolerant with respect to utilizing modified donors and acceptor substrates they can be used to prepare oligosaccharide analogues and for diversification of natural products. New sources of enzymes are continually discovered as genomes are sequenced and they are annotated in the Carbohydrate Active Enzyme (CAZy) glycosyltransferase database. Glycosyltransferase mutagenesis, domain swapping and metabolic pathway engineering to change reaction specificity and product diversification are increasingly successful due to advances in structure-function studies and high throughput screening methods.