Chemoenzymatic synthesis with distinct Pasteurella heparosan synthases: monodisperse polymers and unnatural structures

Heparosan (-GlcUA-beta1,4-GlcNAc-alpha1,4-)(n) is a member of the glycosaminoglycan polysaccharide family found in the capsule of certain pathogenic bacteria as well as the precursor for the vertebrate polymers, heparin and heparan sulfate. The two heparosan synthases from the Gram-negative bacteria Pasteurella multocida, PmHS1 and PmHS2, were efficiently expressed and purified using maltose-binding protein fusion constructs. These relatively homologous synthases displayed distinct catalytic characteristics. PmHS1, but not PmHS2, was able to produce large molecular mass (100-800 kDa) monodisperse polymers in synchronized, stoichiometrically controlled reactions in vitro. PmHS2, but not PmHS1, was able to utilize many unnatural UDP-sugar analogs (including substrates with acetamido-containing uronic acids or longer acyl chain hexosamine derivatives) in vitro. Overall these findings reveal potential differences in the active sites of these two Pasteurella enzymes. In the future, these catalysts should allow the creation of a variety of heparosan and heparinoids with utility for medical applications.     

Identification of a distinct, cryptic heparosan synthase from Pasteurella multocida types A, D, and F

The extracellular polysaccharide capsules of Pasteurella multocida types A, D, and F are composed of hyaluronan, N-acetylheparosan (heparosan or unsulfated, unepimerized heparin), and unsulfated chondroitin, respectively. Previously, a type D heparosan synthase, a glycosyltransferase that forms the repeating disaccharide heparosan backbone, was identified. Here, a approximately 73% identical gene product that is encoded outside of the capsule biosynthesis locus was also shown to be a functional heparosan synthase. Unlike PmHS1, the PmHS2 enzyme was not stimulated greatly by the addition of an exogenous polymer acceptor and yielded smaller- molecular-weight-product size distributions. Virtually identical hssB genes are found in most type A, D, and F isolates. The occurrence of multiple polysaccharide synthases in a single strain invokes the potential for capsular variation.     

Functional characterization of PmHS1, a Pasteurella multocida heparosan synthase

Heparosan synthase 1 (PmHS1) from Pasteurella multocida Type D is a dual action glycosyltransferase enzyme that transfers monosaccharide units from uridine diphospho (UDP) sugar precursors to form the polysaccharide heparosan (N-acetylheparosan), which is composed of alternating (-alpha4-GlcNAc-beta1,4-GlcUA-1-) repeats. We have used molecular genetic means to remove regions nonessential for catalytic activity from the amino- and the carboxyl-terminal regions as well as characterized the functional regions involved in GlcUA-transferase activity and in GlcNAc-transferase activity. Mutation of either one of the two regions containing aspartate-X-aspartate (DXD) residue-containing motifs resulted in complete or substantial loss of heparosan polymerizing activity. However, certain mutant proteins retained only GlcUA-transferase activity while some constructs possessed only GlcNAc-transferase activity. Therefore, it appears that the PmHS1 polypeptide is composed of two types of glycosyltransferases in a single polypeptide as was found for the Pasteurella multocida Type A PmHAS, the hyaluronan synthase that makes the alternating (-beta3-GlcNAc-beta1,4-GlcUA-1-) polymer. However, there is low amino acid similarity between the PmHAS and PmHS1 enzymes, and the relative placement of the GlcUA-transferase and GlcNAc-transferase domains within the two polypeptides is reversed. Even though the monosaccharide compositions of hyaluronan and heparosan are identical, such differences in the sequences of the catalysts are expected because the PmHAS employs only inverting sugar transfer mechanisms whereas PmHS1 requires both retaining and inverting mechanisms.     

Analysis of the polymerization initiation and activity of Pasteurella multocida heparosan synthase PmHS2, an enzyme with glycosyltransferase and UDP-sugar hydrolase activity

Heparosan synthase catalyzes the polymerization of heparosan (-4GlcUAβ1-4GlcNAcα1-)(n) by transferring alternatively the monosaccharide units from UDP-GlcUA and UDP-GlcNAc to an acceptor molecule. Details on the heparosan chain initiation by Pasteurella multocida heparosan synthase PmHS2 and its influence on the polymerization process have not been reported yet. By site-directed mutagenesis of PmHS2, the single action transferases PmHS2-GlcUA(+) and PmHS2-GlcNAc(+) were obtained. When incubated together in the standard polymerization conditions, the PmHS2-GlcUA(+)/PmHS2-GlcNAc(+) showed comparable polymerization properties as determined for PmHS2. We investigated the first step occurring in heparosan chain initiation by the use of the single action transferases and by studying the PmHS2 polymerization process in the presence of heparosan templates and various UDP-sugar concentrations. We observed that PmHS2 favored the initiation of the heparosan chains when incubated in the presence of an excess of UDP-GlcNAc. It resulted in a higher number of heparosan chains with a lower average molecular weight or in the synthesis of two distinct groups of heparosan chain length, in the absence or in the presence of heparosan templates, respectively. These data suggest that PmHS2 transfers GlcUA from UDP-GlcUA moiety to a UDP-GlcNAc acceptor molecule to initiate the heparosan polymerization; as a consequence, not only the UDP-sugar concentration but also the amount of each UDP-sugar is influencing the PmHS2 polymerization process. In addition, it was shown that PmHS2 hydrolyzes the UDP-sugars, UDP-GlcUA being more degraded than UDP-GlcNAc. However, PmHS2 incubated in the presence of both UDP-sugars favors the synthesis of heparosan polymers over the hydrolysis of UDP-sugars.     

In vitro synthesis of heparosan using recombinant Pasteurella multocida heparosan synthase PmHS2

In vertebrates and bacteria, heparosan the precursor of heparin is synthesized by glycosyltransferases via the stepwise addition of UDP-N-acetylglucosamine and UDP-glucuronic acid. As heparin-like molecules represent a great interest in the pharmaceutical area, the cryptic Pasteurella multocida heparosan synthase PmHS2 found to catalyze heparosan synthesis using substrate analogs has been studied. In this paper, we report an efficient way to purify PmHS2 and to maintain its activity stable during 6 months storage at −80 °C using His-tag purification and a desalting step. In the presence of 1 mM of each nucleotide sugar, purified PmHS2 synthesized polymers up to an average molecular weight of 130 kDa. With 5 mM of UDP-GlcUA and 5 mM of UDP-GlcNAc, an optimal specific activity, from 3 to 6 h of incubation, was found to be about 0.145 nmol/μg/min, and polymers up to an average of 102 kDa were synthesized in 24 h. In this study, we show that the chain length distribution of heparosan polymers can be controlled by change of the initial nucleotide sugar concentration. It was observed that low substrate concentration favors the formation of high molecular weight heparosan polymer with a low polydispersity while high substrate concentration did the opposite. Similarities in the polymerization mechanism between PmHS2, PmHS1, and PmHAS are discussed.     

Identification and molecular cloning of a heparosan synthase from Pasteurella multocida type D.

Pasteurella multocida Type D, a causative agent of atrophic rhinitis in swine and pasteurellosis in other domestic animals, produces an extracellular polysaccharide capsule that is a putative virulence factor. It was reported previously that the capsule was removed by treating microbes with heparin lyase III. We molecularly cloned a 617-residue enzyme, pmHS, which is a heparosan (nonsulfated, unepimerized heparin) synthase. Recombinant Escherichia coli-derived pmHS catalyzes the polymerization of the monosaccharides from UDP-GlcNAc and UDP-GlcUA. Other structurally related sugar nucleotides did not substitute. Synthase activity was stimulated about 7-25-fold by the addition of an exogenous polymer acceptor. Molecules composed of approximately 500-3,000 sugar residues were produced in vitro. The polysaccharide was sensitive to the action of heparin lyase III but resistant to hyaluronan lyase. The sequence of the pmHS enzyme is not very similar to the vertebrate heparin/heparan sulfate glycosyltransferases, EXT1 and 2, or to other Pasteurella glycosaminoglycan synthases that produce hyaluronan or chondroitin. The pmHS enzyme is the first microbial dual-action glycosyltransferase to be described that forms a polysaccharide composed of beta4GlcUA-alpha4GlcNAc disaccharide repeats. In contrast, heparosan biosynthesis in E. coli K5 requires at least two separate polypeptides, KfiA and KfiC, to catalyze the same polymerization reaction.     

Metabolic engineering of Bacillus subtilis for biosynthesis of heparosan using heparosan synthase from Pasteurella multocida,PmHS1

Heparosan, the capsular polysaccharide discovered in many pathogenic bacteria, is a promising material for heparin preparation. In this study, the Pasteurella multocida heparosan synthase 1 (PmHS1) module was used to synthesize heparosan with controlled molecular weight, while tuaD/gtaB module or gcaD module was responsible for UDP-precursors production in Bacillus subtilis 168. After metabolic pathway optimization, the yield of heparosan was as high as 237.6 mg/L in strain containing PmHS1 module and tuaD/gtaB module, which indicated that these two modules were key factors in heparosan production. The molecular weight of heparosan varied from 39 to 53 kDa, which indicated that heparosan molecular weight could be adjusted by the amount of PmHS1 and the ratio of two UDP precursors. The results showed that it would be possible to produce safe heparosan with appropriate molecular weight which is useful in heparin production.

     

Synthesis of heparosan oligosaccharides by Pasteurella multocida PmHS2 single-action transferases

Pasteurella multocida heparosan synthase PmHS2 is a dual action glycosyltransferase that catalyzes the polymerization of heparosan polymers in a non-processive manner. The two PmHS2 single-action transferases, obtained previously by site-directed mutagenesis, have been immobilized on Ni(II)-nitrilotriacetic acid agarose during the purification step. A detailed study of the polymerization process in the presence of non-equal amounts of PmHS2 single-action transferases revealed that the glucuronyl transferase (PmHS2-GlcUA(+)) is the limiting catalyst in the polymerization process. Using experimental design, it was determined that the N-acetylglucosaminyl transferase (PmHS2-GlcNAc(+)) plays an important role in the control of heparosan chain elongation depending on the number of heparosan chains and the UDP-sugar concentrations present in the reaction mixture. Furthermore, for the first time, the synthesis of heparosan oligosaccharides alternately using PmHS2-GlcUA(+) and PmHS2-GlcNAc(+) is reported. It was shown that the synthesis of heparosan oligosaccharides by PmHS2 single-action transferases do not require the presence of template molecules in the reaction mixture.     

Critical elements of oligosaccharide acceptor substrates for the Pasteurella multocida hyaluronan synthase

Three-dimensional structures are not available for polysaccharide synthases and only minimal information on the molecular basis for catalysis is known. The Pasteurella multocida hyaluronan synthase (PmHAS) catalyzes the polymerization of the alternating beta1,3-N-acetylglucosamine-beta1,4-glucuronic acid sugar chain by the sequential addition of single monosaccharides to the non-reducing terminus. Therefore, PmHAS possesses both GlcNAc-transferase and glucuronic acid (GlcUA)-transferase activities. The recombinant Escherichia coli-derived PmHAS enzyme will elongate exogenously supplied hyaluronan chains in vitro with either a single monosaccharide or a long chain depending on the UDP-sugar availability. Competition studies using pairs of acceptors with distinct termini (where one oligosaccharide is a substrate that may be elongated, whereas the other cannot) were performed here; the lack of competition suggests that PmHAS contains at least two distinct acceptor sites. We hypothesize that the size of the acceptor binding pockets of the enzyme corresponds to the size of the smallest high efficiency substrates; thus we tested the relative activity of a series of authentic hyaluronan oligosaccharides and related structural analogs. The GlcUA-transferase site readily elongates (GlcNAc-GlcUA)(2), whereas the GlcNAc-transferase elongates GlcUA-Glc-NAc-GlcUA. The minimally sized oligosaccharides, elongated with high efficiency, both contain a trisaccharide with two glucuronic acid residues that enabled the identification of a synthetic, artificial acceptor for the synthase. PmHAS behaves as a fusion of two complete glycosyltransferases, each containing a donor site and an acceptor site, in one polypeptide. Overall, this information advances the knowledge of glycosaminoglycan biosynthesis as well as assists the creation of various therapeutic sugars for medical applications in the future.     

Production of N-sulfated polysaccharides using yeast-expressed N-deacetylase/N-sulfotransferase-1 (NDST-1)

Heparan sulfate/heparin N-deacetylase/N-sulfotransferase-1 (NDST-1) is a critical enzyme involved in heparan sulfate/heparin biosynthesis. This dual-function enzyme modifies the GlcNAc-GlcA disaccharide repeating sugar backbone to make N-sulfated heparosan. N-sulfation is an absolute requirement for the subsequent epimerization and O-sulfation steps in heparan sulfate/heparin biosynthesis. We have expressed rat liver (r) NDST-1 in Saccharomyces cerevisiae as a soluble protein. The yeast-expressed enzyme has both N-deacetylase and N-sulfotransferase activities. N-acetyl heparosan, isolated from Escherichia coli K5 polysaccharide, de-N-sulfated heparin (DNSH) and completely desulfated N-acetylated heparan sulfate (CDSNAcHS) are all good substrates for the rNDST-1. However, N-desulfated, N-acetylated heparin (NDSNAcH) is a poor substrate. The rNDST-1 was partially purified on heparin Sepharose CL-6B. Purified rNDST-1 requires Mn(2+) for its enzymatic activity, can utilize PAPS regenerated in vitro by the PAPS cycle (PAP plus para-nitrophenylsulfate in the presence of arylsulfotransferase IV), and with the addition of exogenous PAPS is capable of producing 60-65% N-sulfated heparosan from E. coli K5 polysaccharide or Pasteurella multocida polysaccharide.     

Crystal structures of Pasteurella multocida sialyltransferase complexes with acceptor and donor analogues reveal substrate binding sites and catalytic mechanism

Sialyltransferases are key enzymes involved in the biosynthesis of biologically and pathologically important sialic acid-containing molecules in nature. Binary X-ray crystal structures of a multifunctional Pasteurella multocida sialyltransferase (Delta24PmST1) with a donor analogue CMP-3F(a)Neu5Ac or CMP-3F(e)Neu5Ac were determined at 2.0 and 1.9 A resolutions, respectively. Ternary X-ray structures of the protein in complex with CMP or a donor analogue CMP-3F(a)Neu5Ac and an acceptor lactose have been determined at 2.0 and 2.27 A resolutions, respectively. This represents the first sialyltransferase structure and the first GT-B-type glycosyltransferase structure that is bound to both a donor analogue and an acceptor simultaneously. The four structures presented here reveal that binding of the nucleotide-activated donor sugar causes a buried tryptophan to flip out of the protein core to interact with the donor sugar and helps define the acceptor sugar binding site. Additionally, key amino acid residues involved in the catalysis have been identified. Structural and kinetic data support a direct displacement mechanism involving an oxocarbenium ion-like transition state assisted with Asp141 serving as a general base to activate the acceptor hydroxyl group.     

Comamonas testosteronan synthase, a bifunctional glycosyltransferase that produces a unique heparosan polysaccharide analog

Glycosaminoglycans (GAGs) are linear hexosamine-containing polysaccharides. These polysaccharides are synthesized by some pathogenic bacteria to form an extracellular coating or capsule. This strategy forms the basis of molecular camouflage since vertebrates possess naturally occurring GAGs that are essential for life. A recent sequence database search identified a putative protein from the opportunistic pathogen Comamonas testosteroni that exhibits similarity with the Pasteurella multocida GAG synthase PmHS1, which is responsible for the synthesis of a heparosan polysaccharide capsule. Initial supportive evidence included glucuronic acid (GlcUA)-containing polysaccharides extracted from C. testosteroni KF-1. We describe here the cloning and analysis of a novel Comamonas GAG synthase, CtTS. The GAG produced by CtTS in vitro consists of the sugars d-GlcUA and N-acetyl-D-glucosamine, but is insensitive to digestion by GAG digesting enzymes, thus has distinct glycosidic linkages from vertebrate GAGs. The backbone structure of the polysaccharide product [-4-D-GlcUA-α1,4-D-GlcNAc-α1-](n) was confirmed by nuclear magnetic resonance. Therefore, this novel GAG, testosteronan, consists of the same sugars as the biomedically relevant GAGs heparosan (N-acetyl-heparosan) and hyaluronan but may have distinct properties useful for future medical applications.     

Evaluation of deoxygenated oligosaccharide acceptor analogs as specific inhibitors of glycosyltransferases

The glycosyltransferases controlling the biosynthesis of cell-surface complex carbohydrates transfer glycosyl residues from sugar nucleotides to specific hydroxyl groups of acceptor oligosaccharides. These enzymes represent prime targets for the design of glycosylation inhibitors with the potential to specifically alter the structures of cell-surface glycoconjugates. With the aim of producing such inhibitors, synthetic oligosaccharide substrates were prepared for eight different glycosyltransferases. The enzymes investigated were: A, alpha(1----2, porcine submaxillary gland); B, alpha(1----3/4, Lewis); C, alpha(1----4, mung bean); D, alpha(1----3, Lex)-fucosyltransferases; E, beta(1----4)-galactosyltransferase; F, beta(1----6)-N-acetylglucosaminyltransferase V; G, beta(1----6)-mucin-N-acetylglucosaminyltransferase ("core-2" transferase); and H, alpha(2----3)-sialyltransferase from rat liver. These enzymes all transfer sugar residues from their respective sugar nucleotides (GDP-Fuc, UDP-Gal, UDP-GlcNAc, and CMP-sialic acid) with inversion of configuration at their anomeric centers. The Km values for their synthetic oligosaccharide acceptors were in the range of 0.036-1.3 mM. For each of these eight enzymes, acceptor analogs were next prepared where the hydroxyl group undergoing glycosylation was chemically removed and replaced by hydrogen. The resulting deoxygenated acceptor analogs can no longer be substrates for the corresponding glycosyltransferases and, if still bound by the enzymes, should act as competitive inhibitors. In only four of the eight cases examined (enzymes A, C, F, and G) did the deoxygenated acceptor analogs inhibit their target enzymes, and their Ki values (all competitive) remained in the general range of the corresponding acceptor Km values. No inhibition was observed for the remaining four enzymes even at high concentrations of deoxygenated acceptor analog. For these latter enzymes it is suggested that the reactive acceptor hydroxyl groups are involved in a critical hydrogen bond donor interaction with a basic group on the enzyme which removes the developing proton during the glycosyl transfer reaction. Such groups are proposed to represent logical targets for irreversible covalent inactivation of this class of enzyme.     

Identification and molecular cloning of a chondroitin synthase from Pasteurella multocida type F

Pasteurella multocida Type F, the minor fowl cholera pathogen, produces an extracellular polysaccharide capsule that is a putative virulence factor. It was reported that the capsule was removed by treating microbes with chondroitin AC lyase. We found by acid hydrolysis that the polysaccharide contained galactosamine and glucuronic acid. We molecularly cloned a Type F polysaccharide synthase and characterized its enzymatic activity. The 965-residue enzyme, called P. multocida chondroitin synthase (pmCS), is 87% identical at the nucleotide and the amino acid level to the hyaluronan synthase, pmHAS, from P. multocida Type A. A recombinant Escherichia coli-derived truncated, soluble version of pmCS (residues 1-704) was shown to catalyze the repetitive addition of sugars from UDP-GalNAc and UDP-GlcUA to chondroitin oligosaccharide acceptors in vitro. Other structurally related sugar nucleotide precursors did not substitute in the elongation reaction. Polymer molecules composed of approximately 10(3) sugar residues were produced, as measured by gel filtration chromatography. The polysaccharide synthesized in vitro was sensitive to the action of chondroitin AC lyase but resistant to the action of hyaluronan lyase. This is the first report identifying a glycosyltransferase that forms a polysaccharide composed of chondroitin disaccharide repeats, [beta(1,4)GlcUA-beta(1,3)GalNAc](n). In analogy to known hyaluronan synthases, a single polypeptide species, pmCS, possesses both transferase activities.     

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.     

大肠杆菌K5产肝素前体heparosan的研究进展

肝素是一种被广泛临床应用的抗凝血药物多糖。Heparosan是某些细菌荚膜中的GAG成分,其二糖骨架结构与脊椎动物中的肝素类似,可以作为肝素和硫酸乙酰肝素的生物合成前体。本文综述了肝素及肝素前体heparosan的功能与应用,heparosan在大肠杆菌K5中合成转运相关酶的研究,以及发酵法生产heparosan的研究进展,并对其应用前景进行了展望。     

Glycosyltransferases involved in biosynthesis of the outer core region of Escherichia coli lipopolysaccharides exhibit broader substrate specificities than is predicted from lipopolysaccharide structures

The waaJ, waaT, and waaR genes encode alpha-1,2-glycosyltransferases involved in synthesis of the outer core region of the lipopolysaccharide of Escherichia coli. They belong to the glycosyltransferase CAZy family 8, characterized by the GT-A fold, DXD motifs, and by retention of configuration at the anomeric carbon of the donor sugar. Each enzyme adds a hexose residue at the same stage of core oligosaccharide backbone extension. However, they differ in the epimers for their donor nucleotide sugars, and in their acceptor residues. WaaJ is a UDP-glucose: (galactosyl) LPS alpha-1,2-glucosyltransferase, whereas WaaR and WaaT have UDP-glucose:(glucosyl) LPS alpha-1,2-glucosyltransferase and UDP-galactose:(glucosyl) LPS alpha-1,2-galactosyltransferase activities, respectively. The objective of this work was to examine their ability to utilize alternate donors and acceptors. When expressed in the heterologous host, each enzyme was able to extend the alternate LPS acceptor in vivo but they retained their natural donor specificity. In vitro assays were then performed to test the effect of substituting the epimeric donor sugar on incorporation efficiency with the natural LPS acceptor of the enzyme. Although each enzyme could utilize the alternate donor epimer, activity was compromised because of significant decreases in k(cat) and corresponding increases in K(m)(donor). Finally, in vitro assays were performed to probe acceptor preference in the absence of the cellular machinery. The results were enzyme-dependent: while an alternate acceptor had no significant effect on the kinetic behavior of His(6)-WaaT, His(6)-WaaJ showed a significantly decreased k(cat) and increased K(m)(acceptor). These results illustrate the differences in behavior between closely related glycosyltransferase enzymes involved in the synthesis of similar glycoconjugates and have implications for glycoengineering applications.     

Escherichia coli K5 heparosan fermentation and improvement by genetic engineering

N-acetyl heparosan is the precursor for the biosynthesis of the important anticoagulant drug heparin. The E. coli K5 capsular heparosan polysaccharide provides a promising precursor for in vitro chemoenzymatic production of bioengineered heparin. This article explores the improvements of heparosan production for bioengineered heparin by fermentation process engineering and genetic engineering.     

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.     

The N-acetyl-binding pocket of N-acetylglucosaminyltransferases also accommodates a sugar analog with a chemical handle at C2

In recent years, sugars with a unique chemical handle have been used to detect and elucidate the function of glycoconjugates. Such chemical handles have generally been part of an N-acetyl moiety of a sugar. We have previously developed several applications using the single mutant Y289L-β1,4-galactosyltransferase I (Y289L-β4Gal-T1) and the wild-type polypeptide-α-GalNAc-T enzymes with UDP-C2-keto-Gal. Here, we describe for the first time that the GlcNAc-transferring enzymes-R228K-Y289L-β4Gal-T1 mutant enzyme, the wild-type human β1,3-N-acetylglucosaminyltransferase-2 and human Maniac Fringe-can also transfer the GlcNAc analog C2-keto-Glc molecule from UDP-C2-keto-Glc to their respective acceptor substrates. Although the R228K-Y289L-β4Gal-T1 mutant enzyme transfers the donor sugar substrate GlcNAc or its analog C2-keto-Glc only to its natural acceptor substrate, GlcNAc, it does not transfer to its analog C2-keto-Glc. Thus, these observations suggest that the GlcNAc-transferring glycosyltransferases can generally accommodate a chemical handle in the N-acetyl-binding cavity of the donor sugar substrate, but not in the N-acetyl-binding cavity of the acceptor sugar.