Conserved domains of glycosyltransferases.

Glycosyltransferases catalyze the synthesis of glycoconjugates by transferring a properly activated sugar residue to an appropriate acceptor molecule or aglycone for chain initiation and elongation. The acceptor can be a lipid, a protein, a heterocyclic compound, or another carbohydrate residue. A catalytic reaction is believed to involve the recognition of both the donor and acceptor by suitable domains, as well as the catalytic site of the enzyme. To elucidate the structural requirements for substrate recognition and catalytic reactions of glycosyltransferases, we have searched the databases for homologous sequences, identified conserved amino acid residues, and proposed potential domain motifs for these enzymes. Depending on the configuration of the anomeric functional group of the glycosyl donor molecule and of the resulting glycoconjugate, all known glycosyltransferases can be divided into two major types: retaining glycosyltransferases, which transfer sugar residue with the retention of anomeric configuration, and inverting glycosyltransferases, which transfer sugar residue with the inversion of anomeric configuration. One conserved domain of the inverting glycosyltransferases identified in the database is responsible for the recognition of a pyrimidine nucleotide, which is either the UDP or the TDP portion of a donor sugar-nucleotide molecule. This domain is termed "Nucleotide Recognition Domain 1 beta," or NRD1 beta, since the type of nucleotide is the only common structure among the sugar donors and acceptors. NRD1 beta is present in 140 glycosyltransferases. The central portion of the NRD1 beta domain is very similar to the domain that is present in one family of retaining glycosyltransferases. This family is termed NRD1 alpha to designate the similarity and stereochemistry of sugar transfer, and it consists of 77 glycosyltransferases identified thus far. In the central portion there is a homologous region for these two families and this region probably has a catalytic function. A third conserved domain is found exclusively in membrane-bound glycosyltransferases and is termed NRD2; this domain is present in 98 glycosyltransferases. All three identified NRDs are present in archaebacterial, eubacterial, viral, and eukaryotic glycosyltransferases. The present article presents the alignment of conserved NRD domains and also presents a brief overview of the analyzed glycosyltransferases which comprise about 65% of all known sugar-nucleotide dependent (Leloir-type) and putative glycosyltransferases in different databases. A potential mechanism for the catalytic reaction is also proposed. This proposed mechanism should facilitate the design of experiments to elucidate the regulatory mechanisms of glycosylation reactions. Amino acid sequence information within the conserved domain may be utilized to design degenerate primers for identifying DNA encoding new glycosyltransferases.     

A sialyltransferase mutant with decreased donor hydrolysis and reduced sialidase activities for directly sialylating Lewis(x)

Glycosyltransferases are important catalysts for enzymatic and chemoenzymatic synthesis of complex carbohydrates and glycoconjugates. The glycosylation efficiencies of wild-type glycosyltransferases vary considerably when different acceptor substrates are used. Using a multifunctional Pasteurella multocida sialyltransferase 1 (PmST1) as an example, we show here that the sugar nucleotide donor hydrolysis activity of glycosyltransferases contributes significantly to the low yield of glycosylation when a poor acceptor substrate is used. With a protein crystal structure-based rational design, we generated a single mutant (PmST1 M144D) with decreased donor hydrolysis activity without significantly affecting its α2-3-sialylation activity when a poor fucose-containing acceptor substrate was used. The single mutant also has a drastically decreased α2-3-sialidase activity. X-ray and NMR structural studies revealed that unlike the wild-type PmST1, which changes to a closed conformation once a donor binds, the M144D mutant structure adopts an open conformation even in the presence of the donor substrate. The PmST1 M144D mutant with decreased donor hydrolysis and reduced sialidase activity has been used as a powerful catalyst for efficient chemoenzymatic synthesis of complex sialyl Lewis(x) antigens containing different sialic acid forms. This work sheds new light on the effect of donor hydrolysis activity of glycosyltransferases on glycosyltransferase-catalyzed reactions and provides a novel strategy to improve glycosyltransferase substrate promiscuity by decreasing its donor hydrolysis activity.     

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.     

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.     

Identification and characterization of large galactosyltransferase gene families: galactosyltransferases for all functions

Enzymatic glycosylation of proteins and lipids is an abundant and important biological process. A great diversity of oligosaccharide structures and types of glycoconjugates is found in nature, and these are synthesized by a large number of glycosyltransferases. Glycosyltransferases have high donor and acceptor substrate specificities and are in general limited to catalysis of one unique glycosidic linkage. Emerging evidence indicates that formation of many glycosidic linkages is covered by large homologous glycosyltransferase gene families, and that the existence of multiple enzyme isoforms provides a degree of redundancy as well as a higher level of regulation of the glycoforms synthesized. Here, we discuss recent cloning strategies enabling the identification of these large glycosyltransferase gene families and exemplify the implication this has for our understanding of regulation of glycosylation by discussing two galactosyltransferase gene families.     

Development of a highly sensitive,high-throughput assay for glycosyltransferases using enzyme-coupled fluorescence detection

Glycosyltransferases catalyze transfer of sugar moieties from activated donor molecules to specific acceptor molecules, forming glycosidic bonds. Identification of selective modulators of glycosyltransferases is important both to provide new tools for investigating pathophysiological roles of glycosylation reactions in cells and tissues, and as new leads in drug discovery. Here we describe a universal enzyme-coupled fluorescence assay for glycosyltransferases, based on quantification of nucleotides produced in the glycosyl transfer reaction. GDP, UDP, and CMP are phosphorylated with nucleotide kinase in the presence of excess ATP, generating ADP. Via coupled enzyme reactions involving ADP-hexokinase, glucose-6-phosphate dehydrogenase, and diaphorase, the ADP is utilized for conversion of resazurin to resorufin, which is determined by fluorescence measurement. The method was validated by comparison with an HPLC method, and employed to screen the LOPAC1280 library for inhibitors in a 384-well plate format. The assay performed well, with a Z′-factor of 0.80. We identified 12 hits for human galactosyltransferase B4GALT1 after elimination of false positives that inhibited the enzyme-coupled assay system. The assay components are all commercially available and the reagent cost is only 2 to 10 US cents per well. This method is suitable for low-cost, high-throughput assay of various glycosyltransferases and screening of glycosyltransferase modulators.     

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.     

糖基转移酶超家族

糖基转移酶在糖基化反应中发挥作用,能够催化活性糖基从糖基供体转移到糖基受体,并形成糖苷键。生物体中存在着数量庞大的糖基转移酶类,形成超基因家族。本文概述了目前糖基转移酶的鉴定方法、划分归类以及与该基因家族有关的系统进化问题,并对高等植物基因组中该家族的进化研究进行展望。     

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。     

Structural and functional features of glycosyltransferases

Most of the glycosylation reactions that generate the great diversity of oligosaccharide structures of eukaryotic cells occur in the Golgi apparatus. This review deals with the most recent data that provide insight into the functional organization of Golgi-resident glycosyltransferases. We also focus on the recent successes in X-ray crystal structure determination of glycosyltransferases. These new structures begin to shed light on the molecular bases accounting for donor and acceptor substrate specificities as well as catalysis.     

细胞代谢过程中的酶促糖基化及其功能

细胞代谢过程中多样的生化修饰反应能够精细调控细胞的活力与功能。其中,酶促糖基化是细胞代谢调控过程中普遍存在的一种分子修饰,对维持和调节细胞功能具有重要影响。糖基转移酶通过将糖基供体的糖基转移至相应的受体分子来实现糖基化修饰。受体分子经过糖基化修饰会改变其在细胞内的稳定性、溶解性和区域定位等特性,并在调节细胞周期、信号转导、蛋白质表达调控、应答反应和清除细胞异物等诸多生物过程中起着重要作用。简要介绍了细胞代谢过程中糖基转移酶超家族的分类、命名和催化机制。重点阐述细胞中蛋白质类生物大分子和小分子化合物的糖基化反应及其在细胞代谢过程中的功能。展望了细胞中糖基化反应及糖基转移酶在人类健康、医药产品、工业催化、食品和农业等领域的应用前景。     

Structural evidence of a passive base-flipping mechanism for AGT, an unusual GT-B glycosyltransferase

The Escherichia coli T4 bacteriophage uses two glycosyltransferases to glucosylate and thus protect its DNA: the retaining alpha-glucosyltransferase (AGT) and the inverting beta-glucosyltransferase (BGT). They glucosylate 5-hydroxymethyl cytosine (5-HMC) bases of duplex DNA using UDP-glucose as the sugar donor to form an alpha-glucosidic linkage and a beta-glucosidic linkage, respectively. Five structures of AGT have been determined: a binary complex with the UDP product and four ternary complexes with UDP or UDP-glucose and oligonucleotides containing an A:G, HMU:G (hydroxymethyl uracyl) or AP:G (apurinic/apyrimidinic) mismatch at the target base-pair. AGT adopts the GT-B fold, one of the two folds known for GTs. However, while the sugar donor binding mode is classical for a GT-B enzyme, the sugar acceptor binding mode is unexpected and breaks the established consensus: AGT is the first GT-B enzyme that predominantly binds both the sugar donor and acceptor to the C-terminal domain. Its active site pocket is highly similar to four retaining GT-B glycosyltransferases (trehalose-6-phosphate synthase, glycogen synthase, glycogen and maltodextrin phosphorylases) strongly suggesting a common evolutionary origin and catalytic mechanism for these enzymes. Structure-guided mutagenesis and kinetic analysis do not permit identification of a nucleophile residue responsible for a glycosyl-enzyme intermediate for the classical double displacement mechanism. Interestingly, the DNA structures reveal partially flipped-out bases. They provide evidence for a passive role of AGT in the base-flipping mechanism and for its specific recognition of the acceptor base.     

植物尿苷二磷酸糖基转移酶超家族晶体结构

糖基转移酶(Glycosyltransferases,GTs)催化的糖基化反应几乎是植物中最为重要的反应。GTs家族1中的植物UGTs(UDP-dependent glycosyltransferases)成员主要运用尿苷二磷酸活化的糖作为糖基供体,因其成员众多、生物功能多样,仅仅通过序列比较和进化分析不能够精确预测其复杂的底物专一性和特有的催化机制,需要后续生化实验的进一步验证。文中主要总结了目前在蛋白结构数据库(Protein Data Bank,PDB)中报道的5种植物UGTs的晶体三维结构和定点突变功能研究进展。详细介绍了植物UGTs整体结构的特点以及蛋白与底物相互作用的细节,为更有效地生化定性UGTs以便深入理解底物专一性提供了有力的工具,从而为植物UGTs在酶工程和基因工程中的应用奠定基础。     

Plant glycosyltransferases

Glycosyltransferases are involved in the biosyntheses of cell-wall polysaccharides, the addition of N-linked glycans to glycoproteins, and the attachment of sugar moieties to various small molecules such as hormones and flavonoids. In the past two years, substantial progress has been made in the identification and cloning of genes that encode glycosyltransferases. Moreover, analysis of the recently completed Arabidopsis genome sequence indicates the existence of several hundred additional genes encoding putative glycosyltransferases.     

An easy colorimetric assay for glycosyltransferases

Glycosyltransferases are involved in biosynthesis of both protein-bound and non-bound glycans that have multiple and important biological functions in all species. A variety of methods for assaying glycosyltransferase activity have been developed driven by the specific interests and type of information required by researchers. In this work, a novel colorimetric assay for the glycosyltransferase-catalyzed reaction was established. Compared with measuring the newly formed product, which might not exhibit visible absorption, the unreacted acceptor could be readily detected by measuring the visible absorption of the hydrolysis product. In the assay, 4-nitrophenyl-β-D-glycoside (glycosyl-β-pNP) is used as the glycosyl acceptor, which can be hydrolyzed by a special exoglycosidase to release the p-nitrophenol before glycosylation reactions. Absorbance change of the p-nitrophenolate corresponds to unreacted glycosyl acceptor that accompanied the glycosyl transfer. The assay is demonstrated to be useful in the initial characterization of recombinant glycosyltransferases for their kinetic parameters, optimal metal cofactor, and pH value. It provides a simple, sensitive, and quantitative method for assessing glycosyltransferase activity and is thus expected to have broad applications including automated high-throughput screening.     

Glycosyltransferases in plant natural product synthesis: characterization of a supergene family

Glycosyltransferases of plant secondary metabolism transfer nucleotide-diphosphate-activated sugars to low molecular weight substrates. Until recently, glycosyltransferases were thought to have only limited influence on the basic physiology of the plant. This view has changed. Glycosyltransferases might in fact have an important role in plant defense and stress tolerance. Recent results obtained with several recombinant enzymes indicate that many glycosyltransferases are regioselective or regiospecific rather than highly substrate specific. This might indicate how plants evolve novel secondary products, placing enzymes with broad substrate specificities downstream of the conserved, early, pivotal enzymes of plant secondary metabolism.     

Expedient synthesis of a series of N-acetyllactosamines.

A series of poly-N-acetyllactosamines representative of those found on complex N-glycans was synthesized for use in the kinetic characterization of recently cloned glycosyltransferases. The syntheses involved the iterative addition of a selectively protected N-acetyllactosaminyl donor to an octyl alpha-D-mannopyranosyl-1,6-beta-D-mannopyranoside acceptor, followed by deprotection. In addition, non-reducing galactosyl residues were removed with beta-galactosidase to furnish GlcNAc terminated compounds. In this manner tetra- to octasaccharides were efficiently produced.     

A fluorescence-based glycosyltransferase assay for high-throughput screening

Glycosyltransferases catalyze the transfer of a monosaccharide unit from a nucleotide or lipid sugar donor to polysaccharides, lipids, and proteins in a stereospecific manner. Considerable effort has been invested in engineering glycosyltransferases to diversify sugar-containing drugs. An important requirement for glycosyltransferase engineering is the availability of a glycosyltransferase assay system for high-throughput screening of glycosyltransferase mutants. In this study, a general glycosyltransferase assay system was developed based on an ATP sensor. This system showed submicromolar sensitivity and compatibility with both purified enzymes and crude cell extracts. The assay system will be useful for glycosyltransferase engineering based on high-throughput screening, as well as for general glycosyltransferase assays and kinetics.     

Geometric Attributes of Retaining Glycosyltransferase Enzymes Favor an Orthogonal Mechanism

Retaining glycosyltransferase enzymes retain the stereochemistry of the donor glycosidic linkage after transfer to an acceptor molecule. The mechanism these enzymes utilize to achieve retention of the anomeric stereochemistry has been a matter of much debate. Re-analysis of previously released structural data from retaining and inverting glycosyltransferases allows competing mechanistic proposals to be evaluated. The binding of metal-nucleotide-sugars between inverting and retaining enzymes is conformationally unique and requires the donor substrate to occupy two different orientations in the two types of glycosyltransferases. The available structures of retaining glycosyltransferases lack appropriately positioned enzymatic dipolar residues to initiate or stabilize the intermediates of a dissociative mechanism. Further, available structures show that the acceptor nucleophile and anomeric carbon of the donor sugar are in close proximity. Structural features support orthogonal (front-side) attack from a position lying ≤90° from the C1-O phosphate bond for retaining enzymes. These structural conclusions are consistent with the geometric conclusions of recent kinetic and computational studies.     

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.