Glycosyltransferases as biocatalysts

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Glycosyltransferases as biocatalysts Monica M Palcic 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.

together these applications have resulted in great advancements in both chemical and enzymatic methods for oligosaccharide production.

Address Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Valby, Copenhagen, Denmark

Glycosyltransferases

Corresponding author: Palcic, Monica M ([email protected])

Current Opinion in Chemical Biology 2011, 15:226–233 This review comes from a themed issue on Biocatalysis and Biotransformation Edited by Sabine Flitsch and Vicente Gotor Available online 19th February 2011 1367-5931/$ – see front matter # 2010 Elsevier Ltd. All rights reserved. DOI 10.1016/j.cbpa.2010.11.022

Introduction Cell surface oligosaccharides have numerous biological roles in cell recognition, signalling, infection, adhesion, inflammation, and normal and abnormal cellular developments. Thus there is great interest in access to defined oligosaccharides to determine the exact roles of a given saccharide in these processes. Additionally there is therapeutic potential for using saccharides or their analogues for intervention when their biological roles are deleterious. There is interest in producing oligosaccharide prebiotics and in generating uniform, defined glycosylated protein biopharmaceuticals. Sugars are also found conjugated to clinically important antibacterial and anti-cancer natural products where they can determine or alter bioactivities. These pharmaceutical properties can be tailored by altering the saccharides and new lead compounds can be discovered by glycodiversification. Taken Current Opinion in Chemical Biology 2011, 15:226–233

The chemical synthesis of oligosaccharides is feasible though it can be laborious since multiple protection, deprotection and purification steps are required [1,2]. Glycosyltransferases (GTs) are the class of enzymes that synthesizes glycosidic linkages and thus are ideal choices for the synthesis of natural oligosaccharides. Several recent reviews cover GTs in oligosaccharide [1–5] and natural product sugar syntheses [6–8]. This review will provide examples of the use and the engineering of glycosyltransferases for biocatalytic applications in natural and oligosaccharide analogue syntheses.

Glycosyltransferases catalyze the formation of glycosidic linkages by the transfer of a saccharide, typically a monosaccharide from a donor substrate to an acceptor substrate [9,10]. The majority of donors are nucleotide sugars, however, sugar phosphates, sugar lipids and saccharides are also donors. Acceptors can be other saccharides, proteins, lipids, nucleic acids, natural products and unnatural products thus GTs exhibit the greatest chemical diversity with respect to substrates and products of any enzyme class. Glycosyltransferase reactions are, with rare exceptions, stereospecific and regiospecific and transfer can occur with either retention or inversion of configuration at the anomeric carbon of the transferred sugar. Enzyme classification is based on the donor utilized by the enzyme, the position and the stereochemistry of transfer. The most widely used and studied GT for biocatalysis is bovine milk b1,4-galactosyltransferase (b1,4-GalT) since it has been commercially available for over thirty years. This enzyme catalyzes the transfer of galactose (Gal) from UDP-Gal to the 4-OH group of terminal N-acetylglucosamine (GlcNAc) acceptors giving Galb1-4GlcNAc-R (N-acetyllactosamine, LacNAc) structures found on mammalian cell surfaces (Figure 1). As genomes are sequenced increasing numbers of microbial enzymes that synthesize mammalian glycans like LacNAc are being discovered and employed in syntheses due to the ease of their cloning into high level bacterial expression systems relative to mammalian expression systems. Open reading frames that encode for glycosyltransferases are continually added to the Carbohydrate Active Enzyme (CAZy) glycosyltransferase database. Their functional characterization remains an enormous challenge given that the www.sciencedirect.com

Glycosyltransferases as biocatalysts Palcic 227

Figure 1

α2,3-SiaT α1,2-FucT 8.7g 1.9g UDP-Glc

OH HO HO

OH OH

UDP-Gal

O

HO

O N3

NHAc

O

β1,4-GalT UDP-Gal Epimerasefusion

O

O HO

NHAc

OH

HO

UDP-GlcNAc β1,3-GlcNAcT

OR

LacNAc 10g

OH

α2,6-SiaT α1,3-FucT 7.5g 2.3g

OH O

HO

OH O

NHAc

β1,4-GalT β UDP-Gal Epimerasefusion

UDP-Glc

OH

UDP-Gal O

HO

O HO

O

α1,2-FucT

HO

Difucosylated Le y:

HO O HO

OH

OH O HO

OH

O HO

O

O

NHAc HO

NHAc

O HO

OH

Sialyl Lewis X α1,3-FucT

O OR NHAc

OH

β1,3-GlcNAcT β1,4-GalT OH

OH O

O HO

O HO

OH

OH

OH O

OH

α1,3-FucT

HO

O HO

O

NHAc HO

α 2,3-SiaT

O

O

OH

4g

OH

O

O HO

OR NHAc

OH

HO

O

O HO

O

NHAc

β1,3-GlcNAcT β1,4-GalT

OH

OH

OH

OH O

OH

OR NHAc

OH

HO

OH

O

O HO

O

HO

3.2g OH

OH

O O

O

NHAc HO

OH

O O HO

OH

OH O O

NHAc HO

OH

O HO

O OR NHAc

100mg Current Opinion in Chemical Biology

Large-scale enzymatic synthesis of poly-N-acetyllactosamine (LacNAc) sequences using b1,4-galactosyltransferase and b1,3-Nacetylglucosaminytransferase. These were elaborated to numerous blood group and tumor-associated antigens with fucosyltransferases and sialyltransferases [11].

donor and acceptor substrates of less than 5% of the entries are known [9].

Glycosyltransferases for chemoenzymatic synthesis of mammalian oligosaccharides Mammalian cell surface oligosaccharides have roles in diverse biological recognition events and are the most frequent synthetic targets. Only nine nucleotide donor sugars are used by approx. 300 different mammalian GTs; UDP-Gal, UDP-GlcNAc, UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-glucose (UDP-Glc), UDP-xylose (UDP-Xyl), UDP-glucuronic acid (UDP-GlcA), GDPfucose (GDP-Fuc), GDP-mannose (GDP-Man) and CMP-N-acetylneuraminic acid (CMP-NeuAc, CMP-sialic acid). The state-of-the-art for GTs as biocatalysts is exemplified by the production of 24 different mammalian blood group www.sciencedirect.com

and tumor associated antigens based on poly-N-acetyllactosamine in gram-scale using six different GT enzymes and four nucleotide donors (Figure 1) [11]. The starting GlcNAc was chemically synthesized with an azidoethyl spacer that allows for its further modification including incorporation into glycan microarrays. The monosaccharide was elaborated to LacNAc disaccharide with a recombinant Neisseria meningitidis b1,4-GalT fused with a UDPGal epimerase enzyme. This allowed for the use of less expensive UDP-Glc as a donor. The product LacNAc was converted to poly-N-acetyllactosamine repeating units by the action of b1,3-GlcNAcT and b1,4-GalT (Figure 1). All LacNAc-terminating compounds were converted to either a2,3-sialylated, a2,6-sialylated, a1,2-fucosylated or a1,3fucosylated (Lewis X) structures (Figure 1). The difucosylated Lewis Y and a2,3-sialyl Lewis X structures were also prepared on all terminal LacNAcs. The quantities of isolated product ranged from 100 mg to 10 gram (Figure 1). Current Opinion in Chemical Biology 2011, 15:226–233

228 Biocatalysis and Biotransformation

A recent review summarizes chemical and enzymatic approaches for the preparation of sialyl Lewis X which has long been a synthetic target due to its location as a terminal structure on cell surfaces and potential importance in inflammation [12]. Poly-N-acetyllactosamines have also been chemoenzymatically prepared with tBoc protected amino linkers as aglycones. These allowed in situ deprotection and saccharide coupling onto amino reactive microtiter plates for studying galectin binding [13]. The use of recombinant human His6-propeptide b1,4-GalT-1 enzyme was required for this synthesis since the b1,4-GalTs from N. meningitidis and Helicobacter pylori were not sufficiently active with this aglycone. The use of bacterial GTs to synthesize mammalian structures is exemplified in a three-step synthesis to produce 14 mg of Forssman antigen pentasaccharide from lactose-pNP using novel a1,4-GalT and b1,3-GalNAcT from Campylobacteri jejuni and a novel a1,3-GalNAcT from Pasteurella multocida (Figure 2) [14]. In this synthesis UDP-GalNAc epimerase was employed to allow the use of less costly UDP-GlcNAc donor. The corresponding

Forssman pentasaccharide and precursors were also prepared starting with LacNAc-pNP [14]. Heparosan, comprising linear repeating units of GlcAb1,4GlcNAca- is a precursor of both heparan sulfates, a highly sulfated class of polysaccharides, and the anticoagulant heparin. Heparosan polysaccharides have been prepared using b1,4-GlcAT and a1,4-GlcNAcT enzymes [15,16]. A recent review summarizes progress in the production and use of specific N-sulfotransferase and O-sulfotransferase and heparin sulfate C5-epimerase to convert heparosan to heparan sulfates and heparin [5]. The heparosan synthesizing GTs and the saccharide modifying enzymes have been employed in the elegant production of libraries of heparin sulfates, ranging from hepta-saccharide to dodecasaccharide starting with a disaccharide prepared by nitrous-acid degradation of heparosan [17]. A representative structure from the library is shown in Figure 3a [17]. Biocatalytic approaches are thus increasingly promising alternatives to the multi-step chemical synthesis currently employed for an antithrombin pentasaccharide (Arixtra) [18] and for closely related compounds [19].

Figure 2

HO OH

HO

OH O

OH O

HO OH

HO

O O HO OH

OH O

UDP-Gal

O NO2

OH

OH O

C. jejuni α1,4-GalT

HO OH

Lactose-pNP

O

O HO

O OH

C. jejuni β1,3-GalNAcT

UDP-GlcNAc

Epimerase

OH

HO

OH

HO

UDP-GalNAc

O

O O

HO

OH

NHAc

O

OH

OH O

HO

HO

OH O HO

HO

OH

OH

OH

O

AcHN O AcHN

OH O

O HO

P. multocida α1,3-GalNAcT

O

O

HO

NO2

O O OH

NO2

UDP-GlcNAc OH

Epimerase

OH O

HO OH

O HO

UDP-GalNAc

O O OH

NO2

Forssman Antigen Current Opinion in Chemical Biology

Synthesis of the Forssman antigen with three novel bacterial glycosyltransferases [14]. Current Opinion in Chemical Biology 2011, 15:226–233

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Glycosyltransferases as biocatalysts Palcic 229

Figure 3

(a) CF3CO NH

HO HO

O O

O

OH

OH

HO O

O

O HO

O

O

OSO3

NH O SO3

O

OH

(b)

OH HO

HO

O OH

OH Lacto-N-biose phosphorylase

NHAc OH

HO O

O HO

OR

NH

HO

OH

HO

SO3

O

O

OH O

HO

HO

O

NHAc

OH

OH

O

+

βGal(1→3)GlcNAc

Pi Recycled

1.7 Kg

HO

OH

OPO32-

O

(c)

O

(d) O

OleD mutant

OH

15 different sugars

N HO

O

N O

O

HO O

O

O

O

O

O

OH

O

O

O

Current Opinion in Chemical Biology

(a) A representative member of a library of heparan sulfates. The pentasaccharide was built up on a disaccharide scaffold (R) by the repetitive action of a1,4-GlcNAcT and b1,4-GlcAT followed by modification using specific sulfotransferases and an epimerase [17]. (b) Lacto-N-biose prepared with a pyrophosphorylase using Gal-1phosphate donor which was regenerated from the released phosphate via a three-enzyme recycling system [27]. (c) A triple mutant of OleD identified in a directed evolution screen capable of transferring fifteen different C-2, C-3, C-4 or C-6 modified sugars from their corresponding UDP-sugar donors while wild-type enzyme utilized only three of the sugars [42]. The mutant also had increased activity for several other natural products and was capable of forming O-glycoside, N-glycoside and S-glycoside [43]. (d) Product of the DesVII reaction, a TDP-D-desosamine glycosyltransferase that requires an auxiliary protein DesVIII for activity [49].

GTs can be used to remodel glycoproteins to give a single uniform glycoform. The N-linked glycans on human serum immunoglobulin G antibodies were fully galactosylated with recombinant bovine b1,4-GalT on a 1 kg scale [20]. 13C-Sialic acid has been incorporated into the N-linked glycans of a2,6-sialyltransferase for NMR studies by auto-sialylation with CMP-13C-NeuAc [21]. Glycan arrays composed of saccharides immobilized on solid supports are widely used to examine carbohydrate recognition by receptors, enzymes, antibodies, pathogens, toxins, lectins, etc. [22]. The diversity of the structures on arrays can be expanded by chemoenzymatic synthesis www.sciencedirect.com

with GTs as exemplified in Figure 1. This topic has been well covered in recent reviews including the use of arrays to examine GT activities by monitoring saccharide transfer to the immobilized glycans [1,23,24]. Specific glycan arrays for biomarker screening have been constructed by high-throughput chemoenzymatic synthesis of 160 mucin GalNAc-peptides further diversified with b1,3-GalT and a2,6-SialylT [25]. Another innovative scaffold to probe sialic acid binding proteins is GlcNAc which has been conjugated to virus particles. The GlcNAc was converted to LacNAc with b1,4-GalT then elaborated with a2,6SialylT (as in Figure 1) providing a polyvalent presentation of glycans [26]. Current Opinion in Chemical Biology 2011, 15:226–233

230 Biocatalysis and Biotransformation

In addition to GTs with nucleotide sugar donors, pyrophosphorylases that utilize sugar-phosphates as donors can also be employed. Figure 3b shows the preparation of Galb1-3GlcNAc (Lacto-N-biose I), believed to be a bifidus factor in human milk oligosaccharides responsible for the intestinal colonization of beneficial bifidobacteria bacteria in infants. The lacto-N-biose pyrophorylase enzyme utilizes galactose 1-phosphate donor and GlcNAc acceptor giving Galb1-3GlcNAc disaccharide as a product [27]. The phosphate produced in the reaction is recycled via three enzymes to regenerate galactose 1-phosphate. The preparation of lacto-N-biose was achieved on an impressive 1.5 kg scale [27] while 45 g of the Galb13GalNAc analogue was produced by the use of GalNAc as an acceptor rather than GlcNAc [28].

Glycosyltransferases for mammalian oligosaccharide analogue synthesis Enormous product diversity can be generated by the use of modified substrates. The nine donors of mammalian metabolism can be used by hundreds of different mammalian and bacterial GTs. Analogues of the sugars of the nine nucleotide sugar donors prepared by chemical [29] and enzymatic methods [6] have been evaluated with many glycosyltransferases [30]. The synthesis of the heptasaccharide in Figure 3a required the use of UDPGlcNTFA as a donor for a1,4-GlcNAcT. This was deprotected under mild basic conditions to give GlcNH2 which was sulfated with N-sulfotransferase [17]. Other examples of the use of modified donors include GDPC-5 substituted-Fuc analogues prepared enzymatically then transferred to LacNAc to make panels of Lewis X derivatives [31] and CPM-9-azido-NeuAc donors used with a2,3 or a2,6-SialylTs, transferred to LacNAc then acylation with a library of acylchlorides to give a sialoside analogue library in a glycan array [32]. GD3 disialylgangliosides have been prepared using an a2,8-sialyltransferase and CMP-Neu with various substitutions on C-5 or C-9 of NeuAc [33]. The latter donor analogues were produced with a one-pot-three enzyme strategy starting with ManNAc or derivatives and pyruvate [33]. Acceptor analogues have been used to generate unique mannose sialosides with marine bacterial a2,3-sialyltransferase [34]. A series of 6-sulfated and C2-substituted GlcNAc and Glc acceptors were converted to the corresponding LacNAc analogues with bacterial b1,4-GalTs of complementary specificities [35].

Engineering glycosyltransferases to change reaction specificity Two structural folds or their variants have been identified for glycosyltransferases that utilize nucleotide sugar donors, the GT-A and GT-B folds [9,10]. The GT-A fold consists of two closely associated b/a/b domains that resemble a Rossmann-fold with a contiguous central bsheet. The GT-B fold is characterized by two separate b/ Current Opinion in Chemical Biology 2011, 15:226–233

a/b Rossmann type domains with a connecting linker. The active site is located in a deep cleft between the domains. Changes in the reaction specificity for members of both fold families have been achieved by point mutagenesis. Some recent examples are the conversion of a human b1,4-GalT to a b1,4-GalNAcT with a single Y284L mutation [36], enhancement of the UDP-Glc transfer activity of a Lamiales UDP-GlcA transferase with a R350W mutation [37] and conversion of an a1,3-GalT to an a1,3-GalNAc-T that could also transfer UDP-GalNAz and UDP-2-keto-Gal with a triple mutation [38]. The Y289L mutant of b1,4Gal-T1 has been employed to label therapeutic antibodies by transferring 2-keto-Gal from UDP-2-keto-Gal followed by linking of a fluorescent dye or biotin [39]. A 37-fold increase in catalytic efficiency was observed with a single Ile305Thr mutation in a UDP-Glc isoflovanoid transferase from Medicago [40]. The regiospecific production of only the 40 -O-glucoside of the flavonoid quercetin was achieved by a single N142Y mutation of an Arabidopsis thaliana GlcT after domainswapping of two closely related GlcTs [41]. A triple mutant of Streptomyces antibioticus oleandomycin glucosyltransferase (OleD) identified in a directed evolution screen with a high throughput fluorescence screen utilized 15 of 22 donor sugar analogues that were modified at C-2, C-3, C-4 or C-6 of the sugar while the wild-type enzyme used only three of the analogues (Figure 3c) [42]. The acceptor specificity of this mutant was expanded as well with enhanced transfer to 71 diverse drug-like compounds and the ability to synthesize O-glycoside, S-glycoside and N-glycoside — a powerful example of enzymatic glycodiversification of natural products [43]. The GT-A fold enzymes are not as amenable to domain swapping while chimeric enzymes of the GT-B fold type can be constructed by swapping the two separate domains around the linker. In one example, a mutant was produced comprising the N-terminal domain acceptor binding domain of a glycopeptide antibiotic glycosyltransferase, GtfA a TDP-epi-vancosamine transferase that transfers to desvancosaminyl vancomycin (DVV) and the C-terminal donor binding domain of Orf1 a UDP-GlcNAc transferase that transfers to teichoplanin glucosaminyl-pseudoaglycone. These original enzymes are both very specific for their donors and acceptors and had different stereochemical courses, with the production of a-linkage and b-linkage, respectively. The hybrid enzyme GtfAH1 had considerably expanded acceptor and donor specificity. It transferred not only to DVV but also to the teichoplanin acceptor, vancomycin aglycone (AGV) and vancomycin (Figure 4). GtfAH1 utilized UDP-Glc as a donor in addition to UDP-GlcNAc of the original wild-type Orf1 enzyme [44]. In other examples, hybrid enzymes with new donor specificities were obtained by domain swapping between a kanamycin GT and a vancomycin GT using a high throughput pH indicator assay to screen the chimeric library [45]. Domain swaps have also been reported for www.sciencedirect.com

Glycosyltransferases as biocatalysts Palcic 231

Figure 4 OR1

HO O HN

O

Cl O

O

O

H N

N H H

OR1

Cl

O

O

OH O

H N

N H

O O

N H

OH HO HO

H N

O R

Cl O OH

Cl

O 2

GtfAH1

H2N

HO2C

HO

UDP-Glc or UDP-GlcNAc

OH OH

R1

Vancomycin aglycone

H OH HO HO

HO HO

O

H

OH NHAc

GlcNAc-Vancomycin aglycone

O

OH

O-vancosamine

O

HO HO

OH NHAc

NHAc OH

Vancomycin

OH NHAc

O

HO HO OH

NHAc OH

R2

OH

Desvancosaminyl vancomycin

OH OH

HO HO

O

R1

O

HO HO

OH NHAc

O-vancosamine Current Opinion in Chemical Biology

A domain swapping mutant GtfAH1 comprised the N-terminal acceptor binding domain of a TDP-epi-vancosamine transferase that only transfers to desvancosaminyl vancomycin (DVV) acceptor and the C-terminal donor binding domain of a highly specific UDP-GlcNAc transferase that transfers to teichoplanin glucosaminyl-pseudoaglycone (AGT) [44]. GtfAH1 had expanded acceptor recognition and transferred to vancomycin, vancomycin aglycone (AGV), GlcNAc-AGV and AGT in addition to DVV of the wild-type parent (Figure 4). GtfAH1 also utilized UDP-Glc donor in addition to UDPGlcNAc of the wild-type parent.

landomycin glycosyltransferases [46], plant flavanoid glucosyltransferases [41] and closely and distantly related CAZy family 1 plant glucosyltransferases from A. thaliana and Stevia rebaudiana to obtain novel specificities [47].

Glycosyltransferases for natural product analogue synthesis While mammalian oligosaccharide structures are generated from only nine nucleotide sugar donors, prokaryotic polysaccharides and natural products contain hundreds of unusual sugars in addition to the nine found in mammals. These include deoxy-, amino-, methyl, amino deoxy-, amino dideoxy-, and trideoxy-sugars which are transferred from their corresponding nucleotides by bacterial GTs [6–8]. Glycosylated natural products include therapeutically compounds such as vancomycin, novobiocin, megalomicin, streptomycin and erythromycin. The sugars are frequently essential for the pharmacological properties and/or biological activity of the compounds. Since their biological activity can be altered by adding or changing sugars, numerous approaches have been developed for natural product diversification based on chemical and chemoenzymatic glycorandomization or glycodiverwww.sciencedirect.com

sification [6–8]. In vitro enzymatic approaches require access to libraries of rare and unusual nucleotide sugar donors which can be produced by promiscuous nucleotide donor sugar biosynthetic enzymes [42] or by taking advantage of the reversibility of some bacterial GT reactions [48]. Other examples of in vitro glycodiversification besides OleD as discussed above have been reported for methymycin, vancomycin derivatives and calicheamicins [6–8]. Auxiliary proteins have also been discovered that are required for the activation of bacterial GTs including DesVII a TDP-D-desosamine (a 4,6-dideoxy-3-N,Ndimethyl sugar) GT in the pathway for methymicin biosynthesis (Figure 3d) [49].

Whole-cell approaches Considerable progress has been made in the large-scale biocatalytic production of oligosaccharides by metabolic engineering of microorganisms especially for the production of novel glycosylated natural products. Whole cells are advantageous since there is no need to isolate GTs and entire donor biosynthetic pathways can be coexpressed in the host. Representative examples of whole cell engineering are the production of a2,6-sialyllactose at Current Opinion in Chemical Biology 2011, 15:226–233

232 Biocatalysis and Biotransformation

34 g/L in Escherichia coli [50] and the production of perosaminyl-amphoteronolide [51]. E. coli strains that can be used for in vivo glycorandomization have also been developed [52]. These strains contain promiscuous nucleotide donor synthases [42] and promiscuous GTs similar to the OleD triple mutant (described above). The cells are permeable such that diverse acceptors and monosaccharide analogues can be taken up and converted to their respective nucleotide sugar donors and then transferred to the acceptors.

Future perspectives There has been tremendous progress in the use of glycosyltransferases for the production of oligosaccharides and their analogues. This has been driven by advancements in the discovery, production and characterization of GTs, high throughput screening for enzyme evolution and metabolic pathway engineering. The synthesis of oligosaccharide libraries and diversification of natural products has been achieved. Breakthroughs continue, such as the development of an artificial Golgi apparatus that allows for fully automated glycan synthesis using GTs with saccharide assembly on protein-like dendrimers as polymer supports [53]. For the future, the functional characterization of the 95% of GTs with unconfirmed specificities remains an ongoing challenge.

Acknowledgements The author thanks Professor Ole Hindsgaul for helpful discussion and the Danish Council for Independent Research/Natural Sciences (FNU) for support.

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Chemically synthesized O-GlcNAc-mucin peptides were elaborated with galactosyltransferases and sialyltransferases to give a 160 compound library for biomarker discovery.

42. Williams GJ, Zhang C, Thorson JS: Expanding the promiscuity of a natural-product glycosyltransferase by directed evolution. Nat Chem Biol 2007, 3:657-662.

26. Kaltgrad E, O’Reilly MK, Liao L, Han S, Paulson JC, Finn MG: Onvirus construction of polyvalent glycan ligands for cell-surface receptors. J Am Chem Soc 2008, 130:4578-4579.

43. Gantt RW, Goff RD, Williams GJ, Thorson JS: Probing the  aglycon promiscuity of an engineered glycosyltransferase. Angew Chem Int Ed 2008, 47:8889-8892. Further characterization of a natural-product glycosyltransferase discovered in a directed evolution screen in [42] reveals unexpectedly broad tolerance for natural product acceptors and the unusual ability to produce O-glycoside, N-glycoside and S-glycoside.

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44. Truman AW, Dias MVB, Wu S, Blundell TL, Huang F, Spencer JB:  Chimeric glycosyltransferases for the generation of hybrid glycopeptides. Chem Biol 2009, 16:676-685. A chimeric enzyme GtfAH1 was produced by swapping the N-terminal acceptor binding domain and C-terminal donor binding domain of two highly specific natural product glycosyltransferases. The GtfAH1 mutant enzyme was very promiscuous, exhibiting relaxation in donor specificity and much broader acceptor tolerances. X-ray structural studies provided a possible rationale for the increased acceptor promiscuity. 45. Park SH, Park HY, Sohng JK, Lee HC, Liou K, Yoon YJ, Kim BG: Expanding substrate specificity of GT-B fold glycosyltransferases via domain swapping and highthroughput screening. Biotechnol Bioeng 2009, 102:988-994. 46. Krauth C, Fedoryshyn M, Schleberger C, Luzhetskyy A, Bechthold A: Engineering a function into a glycosyltransferase. Chem Biol 2009, 16:28-35. 47. Hansen EH, Osmani SA, Kristensen C, Møller BL, Hansen J: Substrate specificities of family 1 UGTs gained by domain swapping. Phytochemistry 2009, 70:473-482. 48. Zhang C, Griffith BR, Fu Q, Albermann C, Fu X, Lee IK, Li L, Thorson JS: Exploiting the reversibility of natural product glycosyltransferase-catalyzed reactions. Science 2006, 313:1291-1294.

35. Lau K, Thon V, Yu H, Ding Y, Chen Y, Muthana MM, Wong D, Huang R, Chen X: Highly efficient chemoenzymatic synthesis of b1-4-linked galactosides with promiscuous bacterial b1-4-galactosyltransferases. Chem Commun 2010, 46:6066-6068.

49. Borisova SA, Liu HW: Characterization of glycosyltransferase  DesVII and its auxiliary partner protein DesVIII in the methymycin/pikromycin biosynthetic pathway. Biochemistry 2010, 49:8071-8084. DesVII, a natural product glycosyltransferase has an unusual requirement for an additional protein partner DesVIII for full activity in vitro. This study shows that DesVIII assists the folding of DesVII and that it remains tightly bound during catalysis which will be important when designing combinatorial biosynthetic schemes for glycodiversification.

36. Bojarova´ P, Krˇenek K, Wetjen K, Adamiak K, Pelantova H, Brezousˇka K, Elling L, Krˇen V: Synthesis of LacdiNActerminated glycoconjugates by mutant galactosyltransferase — a way to new glycodrugs and materials. Glycobiology 2009, 19:509-517.

50. Drouillard S, Mine T, Kajiwara H, Yamamoto T, Samain E: Efficient synthesis of 60 -sialylactose, 6,60 -disialyllactose, and 60 -KDOlactose by metabolically engineered E. coli expressing a multifunctional sialyltransferase from Photobacterium sp. JTISH-224. Carbohydr Res 2010, 345:1394-1399.

37. Noguchi A, Horikawa M, Fukui Y, Fukuchi-Mizutani M, IuchiOkada A, Ishiguro M, Kiso Y, Nakayama T, Ono E: Local differences of sugar donor specificity of flavonoid glycosyltransferase in Lamiales. Plant Cell 2009, 21:1556-1572.

51. Hutchinson E, Murphy B, Dunne T, Breen C, Rawlings B, Caffrey P: Redesign of polyene macrolide glycosylation: engineered biosynthesis of 19-(O)-perosaminyl-amphoteronolide B. Chem Biol 2010, 17:174-182.

38. Pasek M, Ramakrishnan B, Boeggeman E, Manzoni M, Waybright TJ, Qasba PK: Bioconjugation and detection of lactosamine moiety using a1,3-galactosyltransferase mutants that transfer C2-modified galactose with a chemical handle. Bioconj Chem 2009, 20:608-618. 39. Boeggeman E, Ranakrishnan B, Pasek M, Manzoni M, Puri A, Loomis KH, Waybright TJ, Qasba PK: Site specific conjugation of fluoroprobes to the remodeled Fc N-glycans of monoclonal antibodies using mutant glycosyltransferases: application for cell surface antigen detection. Bioconj Chem 2009, 20:1228-1236. 40. Modolo LV, Escamilla-Trevino LL, Dixon RA, Wang X: Single amino acid mutations of Medicago glycosyltransferase UGT85H2 enhance activity and impart reversibility. FEBS Lett 2009, 583:2131-2135. 41. Cartwright AM, Lim EK, Kleanthous C, Bowles DJ: A kinetic analysis of regiospecific glucosylation by two glycosyltransferases of Arabidopsis thaliana: domain swapping to introduce new activities. J Biol Chem 2008, 283:15724-15731.

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52. Williams GJ, Yang J, Zhang C, Thorson JS: Recombinant E. coli  prototype strains for in vivo glycorandomization. ACS Chem Biol, in press, October 1, 2010. doi:10.1021/cb100267k The first example of engineering strains of E. coli for glycorandomization of natural products. The strains contain promiscuous donor synthesizing enzymes (anomeric kinase, sugar 1-phosphate nucleotidyltransferase) and glycosyltransferases such that monosaccharides or their analogues are added to the culture medium along with a small molecule acceptor. These are taken up by the cells, the monosaccharides or their analogues are converted to nucleotide donor sugars then transferred to the acceptors by the glycosyltransferase. 53. Matsushita T, Nagashima I, Fumoto M, Ohta T, Yamada K,  Shimizu H, Hinou H, Naruchi K, Ito T, Kondo H, Nishimura S-I: Artificial Golgi apparatus: globular protein-like dendrimer facilitates fully automated enzymatic glycan synthesis. J Am Chem Soc 2010, 132:16651-16656. An automated glycan synthesizer has been developed. The artificial Golgi was utilized to synthesize sialyl Lewis X from GlcNAc-linked with a tetrapeptide linker to a high molecular weight aminooxy-functionalized G7 poly(amidoamine) dendrimer. All steps are automated, including the glycosyltransferase reactions, exchange of buffers, recovery of elaborated dendrimer, protease cleavage and product collection.

Current Opinion in Chemical Biology 2011, 15:226–233