Characterization of the ß-KDO Transferase KpsS, the Initiating Enzyme in the Biosynthesis of the Lipid Acceptor for Escherichia coli Polysialic Acid.

Antiphagocytic capsular polysaccharides are key components of effective vaccines against pathogenic bacteria. Neisseria meningitidis groups B and C, as well as Escherichia coli serogroups K1 and K92, are coated with polysialic acid capsules. Although the chemical structure of these polysaccharides and the organization of the associated gene clusters have been described for many years, only recently have the details of the biosynthetic pathways been discovered. The polysialic acid chains are synthesized by polysialyltransferases on a proposed phosphatidylglycerol lipid acceptor with a poly keto-deoxyoctulosonate (KDO) linker. Synthesis of this acceptor requires at least three enzymes in E. coli K1: KpsS, KpsC, and NeuE. In this report, we have characterized the ß-KDO glycosyltransferase KpsS, the first enzyme in the pathway for lipid acceptor synthesis. After purification of KpsS in a soluble active form, we investigated its function and substrate specificity and showed that KpsS can transfer a KDO residue to a fluorescently labeled phosphatidylglycerol lipid. The enzyme tolerated various lengths of fatty acid acyl chains on the phosphatidylglycerol, including fluorescent tags, but exhibited a preference for phosphatidylglycerol diacylated with longer fatty acid chains as indicated by the smaller Kd and Km values for substrates with chains with more than 14 members. Additional structural analysis of the KpsS product confirmed that KpsS transfers KDO from CMP-KDO to the 1-hydroxyl of phosphatidylglycerol to form a ß-KDO linkage.

Sequential One-Pot Multienzyme Chemoenzymatic Synthesis of Glycosphingolipid Glycans.

Glycosphingolipids are a diverse family of biologically important glycolipids. In addition to variations on the lipid component, more than 300 glycosphingolipid glycans have been characterized. These glycans are directly involved in various molecular recognition events. Several naturally occurring sialic acid forms have been found in sialic acid-containing glycosphingolipids, namely gangliosides. However, ganglioside glycans containing less common sialic acid forms are currently not available. Herein, highly effective one-pot multienzyme (OPME) systems are used in sequential for high-yield and cost-effective production of glycosphingolipid glycans, including those containing different sialic acid forms such as N-acetylneuraminic acid (Neu5Ac), N-glycolylneuraminic acid (Neu5Gc), 2-keto-3-deoxy-d-glycero-d-galacto-nononic acid (Kdn), and 8-O-methyl-N-acetylneuraminic acid (Neu5Ac8OMe). A library of 64 structurally distinct glycosphingolipid glycans belonging to ganglio-series, lacto-/neolacto-series, and globo-/isoglobo-series glycosphingolipid glycans is constructed. These glycans are essential standards and invaluable probes for bioassays and biomedical studies.

Effective one-pot multienzyme (OPME) synthesis of monotreme milk oligosaccharides and other sialosides containing 4-O-acetyl sialic acid.

A facile one-pot two-enzyme chemoenzymatic approach has been established for the gram (Neu4,5Ac2α3Lac, 1.33 g) and preparative scale (Neu4,5Ac2α3LNnT) synthesis of monotreme milk oligosaccharides. Other O-acetyl-5-N-acetylneuraminic acid (Neu4,5Ac2)- or 4-O-acetyl-5-N-glycolylneuraminic acid (Neu4Ac5Gc) -containing α2-3-sialosides have also been synthesized in the preparative scale. Used as an effective probe, Neu4,5Ac2α3GalßpNP was found to be a suitable substrate by human influenza A viruses but not bacterial sialidases.

Donor substrate promiscuity of bacterial ß1-3-N-acetylglucosaminyltransferases and acceptor substrate flexibility of ß1-4-galactosyltransferases.

ß1-3-N-Acetylglucosaminyltransferases (ß3GlcNAcTs) and ß1-4-galactosyltransferases (ß4GalTs) have been broadly used in enzymatic synthesis of N-acetyllactosamine (LacNAc)-containing oligosaccharides and glycoconjugates including poly-LacNAc, and lacto-N-neotetraose (LNnT) found in the milk of human and other mammals. In order to explore oligosaccharides and derivatives that can be synthesized by the combination of ß3GlcNAcTs and ß4GalTs, donor substrate specificity studies of two bacterial ß3GlcNAcTs from Helicobacter pylori (Hpß3GlcNAcT) and Neisseria meningitidis (NmLgtA), respectively, using a library of 39 sugar nucleotides were carried out. The two ß3GlcNAcTs have complementary donor substrate promiscuity and 13 different trisaccharides were produced. They were used to investigate the acceptor substrate specificities of three ß4GalTs from Neisseria meningitidis (NmLgtB), Helicobacter pylori (Hpß4GalT), and bovine (Bß4GalT), respectively. Ten of the 13 trisaccharides were shown to be tolerable acceptors for at least one of these ß4GalTs. The application of NmLgtA in one-pot multienzyme (OPME) synthesis of two trisaccharides including GalNAcß1-3Galß1-4GlcßProN3 and Galß1-3Galß1-4Glc was demonstrated. The study provides important information for using these glycosyltransferases as powerful catalysts in enzymatic and chemoenzymatic syntheses of oligosaccharides and derivatives which can be useful probes and reagents.

Facile chemoenzymatic synthesis of biotinylated heparosan hexasaccharide.

A biotinylated heparosan hexasaccharide was synthesized using a one-pot multi-enzyme strategy, in situ activation and transfer of N-trifluoroacetylglucosamine (GlcNTFA) to a heparin backbone significantly improved the synthetic efficiency. The biotinylated hexasaccharide could serve as a flexible core to diversify its conversion into heparan sulfate isoforms with potential biological applications and therapeutics.

Synthetic disialyl hexasaccharides protect neonatal rats from necrotizing enterocolitis.

Two novel synthetic α2-6-linked disialyl hexasaccharides, disialyllacto-N-neotetraose (DSLNnT) and α2-6-linked disialyllacto-N-tetraose (DS'LNT), were readily obtained by highly efficient one-pot multienzyme (OPME) reactions. The sequential OPME systems described herein allowed the use of an inexpensive disaccharide and simple monosaccharides to synthesize the desired complex oligosaccharides with high efficiency and selectivity. DSLNnT and DS'LNT were shown to protect neonatal rats from necrotizing enterocolitis (NEC) and are good therapeutic candidates for preclinical experiments and clinical application in treating NEC in preterm infants.

Donor substrate promiscuity of the N-acetylglucosaminyltransferase activities of Pasteurella multocida heparosan synthase 2 (PmHS2) and Escherichia coli K5 KfiA.

The biological activities of heparan sulfate (HS) and heparin (HP) are closely related to their molecular structures. Both Pasteurella multocida heparosan synthase 2 (PmHS2) and Escherichia coli K5 KfiA have been used for enzymatic and chemoenzymatic synthesis of HS and HP oligosaccharides and their derivatives. We show here that cloning using the pET15b vector and expressing PmHS2 as an N-His6-tagged fusion protein improve its expression level in E. coli. Investigation of the donor substrate specificity of the N-acetylglucosaminyltransferase activities of P. multocida heparosan synthase 2 (PmHS2) and E. coli K5 KfiA indicates the substrate promiscuities of PmHS2 and KfiA. Overall, both PmHS2 and KfiA can use uridine 5'-diphosphate-N-acetylglucosamine (UDP-GlcNAc) and some of its C2'- and C6'-derivatives as donor substrates for their α1-4-GlcNAcT activities. Nevertheless, PmHS2 has a broader tolerance towards substrate modifications. Other than the UDP-sugars that can be used by KfiA, additional C6'-derivatives of UDP-GlcNAc, UDP-glucose, and UDP-N-acetylgalactosamine (UDP-GalNAc) are tolerable substrates for the α1-4-GlcNAcT activity of PmHS2. The substrate promiscuities of PmHS2 and KfiA will allow efficient chemoenzymatic synthesis of diverse HS and HP oligosaccharide derivatives which may have improved or altered activities compared to their natural counterparts.

One-pot multi-enzyme (OPME) chemoenzymatic synthesis of sialyl-Tn-MUC1 and sialyl-T-MUC1 glycopeptides containing natural or non-natural sialic acid.

A series of STn-MUC1 and ST-MUC1 glycopeptides containing naturally occurring and non-natural sialic acids have been chemoenzymatically synthesized from Tn-MUC1 glycopeptide using one-pot multienzyme (OPME) approaches. In situ generation of the sialyltransferase donor cytidine 5'-monophosphate-sialic acid (CMP-Sia) using a CMP-sialic acid synthetase in the presence of an extra amount of cytidine 5'-triphosphate (CTP) and removal of CMP from the reaction mixture by flash C18 cartridge purification allow the complete consumption of Tn-MUC1 glycopeptide for quantitative synthesis of STn-MUC1. A Campylobacter jejuni ß1-3GalT (CjCgtBΔ30-His6) mutant has been found to catalyze the transfer of one or more galactose residues to Tn-MUC1 for the synthesis of T-MUC1 and galactosylated T-MUC1. Sialylation of T-MUC1 using Pasteurella multocida α2-3-sialyltransferase 3 (PmST3) with Neisseria meningitidis CMP-sialic acid synthetase (NmCSS) and Escherichia coli sialic acid aldolase in one pot produced ST-MUC1 efficiently. These glycopeptides are potential cancer vaccine candidates.

PmST3 from Pasteurella multocida encoded by Pm1174 gene is a monofunctional α2-3-sialyltransferase.

Pasteurella multocida (Pm) strain Pm70 has three putative sialyltransferase genes including Pm0188, Pm0508, and Pm1174. A Pm0188 gene homolog in Pm strain P-1059 encodes a multifunctional α2-3-sialyltransferase, PmST1, that prefers oligosaccharide acceptors. A Pm0508 gene homolog in the same strain encodes a monofunctional sialyltransferase PmST2 that prefers glycolipid acceptors. Here, we report that the third sialyltransferase from Pm (PmST3) encoded by gene Pm1174 in strain Pm70 is a monofunctional α2-3-sialyltransferase that can use both oligosaccharides and glycolipids as efficient acceptors. Despite the existence of both Pm0188 and Pm0508 gene homologs encoding PmST1 and PmST2, respectively, in Pm strain P-1059, a Pm1174 gene homolog appears to be absent from Pm strains P-1059 and P-934. PmST3 was successfully obtained by cloning and expression using a synthetic gene of Pm1174 with codons optimized for Escherichia coli expression system as the DNA template for polymer chain reactions. Truncation of 35 amino acid residues from the carboxyl terminus was shown to improve the expression of a soluble and active enzyme in E. coli as a C-His(6)-tagged fusion protein. This sialidase-free monofunctional α2-3-sialyltransferase is a useful tool for synthesizing sialylated oligosaccharides and glycolipids.

Defining function of lipopolysaccharide O-antigen ligase WaaL using chemoenzymatically synthesized substrates.

The WaaL-mediated ligation of O-antigen onto the core region of the lipid A-core block is an important step in the lipopolysaccharide (LPS) biosynthetic pathway. Although the LPS biosynthesis has been largely characterized, only a limited amount of in vitro biochemical evidence has been established for the ligation reaction. Such limitations have primarily resulted from the barriers in purifying WaaL homologues and obtaining chemically defined substrates. Accordingly, we describe herein a chemical biology approach that enabled the reconstitution of this ligation reaction. The O-antigen repeating unit (O-unit) of Escherichia coli O86 was first enzymatically assembled via sequential enzymatic glycosylation of a chemically synthesized GalNAc-pyrophosphate-undecaprenyl precursor. Subsequent expression of WaaL through use of a chaperone co-expression system then enabled the demonstration of the in vitro ligation between the synthesized donor (O-unit-pyrophosphate-undecaprenyl) and the isolated lipid A-core acceptor. The previously reported ATP and divalent metal cation dependence were not observed using this system. Further analyses of other donor substrates revealed that WaaL possesses a highly relaxed specificity toward both the lipid moiety and the glycan moiety of the donor. Lastly, three conserved amino acid residues identified by sequence alignment were found essential for the WaaL activity. Taken together, the present work represents an in vitro systematic investigation of the WaaL function using a chemical biology approach, providing a system that could facilitate the elucidation of the mechanism of WaaL-catalyzed ligation reaction.

One-pot three-enzyme synthesis of UDP-GlcNAc derivatives.

A Pasteurella multocida N-acetylglucosamine 1-phosphate uridylyltransferase (PmGlmU) was cloned and used efficiently with an N-acetylhexosamine 1-kinase (NahK_ATCC55813) and an inorganic pyrophosphatase (PmPpA) for one-pot three-enzyme synthesis of UDP-GlcNAc derivatives with or without further chemical diversification.

Substrate promiscuity of N-acetylhexosamine 1-kinases.

N-Acetylhexosamine 1-kinase (NahK) catalyzes the direct addition of a phosphate from adenosine 5'-triphosphate (ATP) to the anomeric position of N-acetylhexosamine and shows similar activity towards N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc). Herein we report the cloning, characterization, and substrate specificity studies of two NahKs from Bifidobacterium infantis ATCC15697 and Bifidobacterium longum ATCC55813, respectively. A new capillary electrophoresis assay method has been developed for enzyme activity assays. Both enzymes have a good expression level in E. coli (180-185 mg/L culture) and can tolerate diverse modifications at C2 of GlcNAc and GalNAc. Various GlcNAc derivatives with C6, both C2 and C6, as well as both C2 and C3 modifications are tolerable substrates for the newly cloned NahKs. Quite interestingly, despite of their low activities toward glucose and galactose, the activities of both NahKs are much higher for mannose and some of its C2, C4, and C6 derivatives. These NahKs are excellent catalysts for enzymatic and chemoenzymatic synthesis of carbohydrates.

PmST2: a novel Pasteurella multocida glycolipid α2-3-sialyltransferase.

Pasteurella multocida (Pm) is a multi-species pathogen that causes diseases in animals and humans. Sialyltransferase activity has been detected in multiple Pm strains and sialylation has been shown to be important for the pathogenesis of Pm. Three putative sialyltransferase genes have been identified in Pm genomic strain Pm70. We have reported previously that a Pm0188 gene homolog in Pm strain P-1059 (ATCC 15742) encodes a multifunctional sialyltransferase (PmST1). We demonstrate here that while PmST1 prefers to use oligosaccharides as acceptors, PmST2 encoded by the Pm0508 gene homolog in the same Pm strain is a novel glycolipid α2-3-sialyltransferase that prefers to use lactosyl lipids as acceptor substrates. PmST2 and PmST1 thus complement each other for an efficient synthesis of α2-3-linked sialosides with or without lipid portion. In addition, ß1-4-linked galactosyl lipids are better PmST2 substrates than ß1-3-linked galactosyl lipids. PmST2 has been used successfully in the preparative scale synthesis of sialyllactosyl sphingosine (lyso-GM3), which is an important glycolipid and an intermediate for synthesizing more complex glycolipids such as gangliosides.

Sequential two-step multienzyme synthesis of tumor-associated sialyl T-antigens and derivatives.

A series of α2-3-sialylated ß1-3-linked galactosides, including sialyl T-antigens, 3'-sialyl galacto-N-biose, 3'-sialyl lacto-N-biose, and their derivatives containing natural and non-natural sialic acid forms have been synthesized from simple monosaccharides using an efficient sequential two-step multienzyme approach.

Identifying selective inhibitors against the human cytosolic sialidase NEU2 by substrate specificity studies.

Aberrant expression of human sialidases has been shown to associate with various pathological conditions. Despite the effort in the sialidase inhibitor design, less attention has been paid to designing specific inhibitors against human sialidases and characterizing the substrate specificity of different sialidases regarding diverse terminal sialic acid forms and sialyl linkages. This is mainly due to the lack of sialoside probes and efficient screening methods, as well as limited access to human sialidases. A low cellular expression level of the human sialidase NEU2 hampers its functional and inhibitory studies. Here we report the successful cloning and expression of the human sialidase NEU2 in E. coli. About 11 mg of soluble active NEU2 was routinely obtained from 1 L of E. coli cell culture. Substrate specificity studies of the recombinant human NEU2 using twenty p-nitrophenol (pNP)-tagged α2-3- or α2-6-linked sialyl galactosides containing different terminal sialic acid forms including common N-acetylneuraminic acid (Neu5Ac), non-human N-glycolylneuraminic acid (Neu5Gc), 2-keto-3-deoxy-D-glycero-D-galacto-nonulosonic acid (Kdn), or their C5-derivatives in a microtiter plate-based high-throughput colorimetric assay identified a unique structural feature specifically recognized by the human NEU2 but not two bacterial sialidases. The results obtained from substrate specificity studies were used to guide the design of a sialidase inhibitor that was selective against human NEU2. The selectivity of the inhibitor was revealed by the comparison of sialidase crystal structures and inhibitor docking studies.

Cloning and characterization of a viral α2-3-sialyltransferase (vST3Gal-I) for the synthesis of sialyl Lewisx.

Sialyl Lewis(x) (SLe(x), Siaα2-3Galß1-4(Fucα1-3)GlcNAcßOR) is an important sialic acid-containing carbohydrate epitope involved in many biological processes such as inflammation and cancer metastasis. In the biosynthetic process of SLe(x), α2-3-sialyltransferase-catalyzed sialylation generally proceeds prior to α1-3-fucosyltransferase-catalyzed fucosylation. For the chemoenzymatic synthesis of SLe(x) containing different sialic acid forms, however, it would be more efficient if diverse sialic acid forms are transferred in the last step to the fucosylated substrate Lewis(x) (Le(x)). An α2-3-sialyltransferase obtained from myxoma virus-infected European rabbit kidney RK13 cells (viral α2-3-sialyltransferase (vST3Gal-I)) was reported to be able to tolerate fucosylated substrate Le(x). Nevertheless, the substrate specificity of the enzyme was only determined using partially purified protein from extracts of cells infected with myxoma virus. Herein we demonstrate that a previously reported multifunctional bacterial enzyme Pasteurella multocida sialyltransferase 1 (PmST1) can also use Le(x) as an acceptor substrate, although at a much lower efficiency compared to nonfucosylated acceptor. In addition, N-terminal 30-amino-acid truncated vST3Gal-I has been successfully cloned and expressed in Escherichia coli Origami™ B(DE3) cells as a fusion protein with an N-terminal maltose binding protein (MBP) and a C-terminal His(6)-tag (MBP-Δ30vST3Gal-I-His(6)). The viral protein has been purified to homogeneity and characterized biochemically. The enzyme is active in a broad pH range varying from 5.0 to 9.0. It does not require a divalent metal for its α2-3-sialyltransferase activity. It has been used in one-pot multienzyme sialylation of Le(x) for the synthesis of SLe(x) containing different sialic acid forms with good yields.

Highly efficient chemoenzymatic synthesis of ß1-3-linked galactosides.

A novel D-galactosyl-ß1-3-N-acetyl-D-hexosamine phosphorylase cloned from Bifidobacterium infantis (BiGalHexNAcP) was used with a recombinant E. coli K-12 galactokinase (GalK) for efficient one-pot two-enzyme synthesis of T-antigens, galacto-N-biose (Galß1-3GalNAc), lacto-N-biose (Galß1-3GlcNAc), and their derivatives.

Highly efficient chemoenzymatic synthesis of beta1-4-linked galactosides with promiscuous bacterial beta1-4-galactosyltransferases.

Two bacterial beta1-4-galactosyltransferases, NmLgtB and Hp1-4GalT, exhibit promiscuous and complementary acceptor substrate specificity. They have been used in an efficient one-pot multienzyme system to synthesize LacNAc, lactose, and their derivatives including those containing negatively charged 6-O-sulfated GlcNAc and C2-substituted GlcNAc or Glc, from monosaccharide derivatives and inexpensive Glc-1-P.

Helicobacter hepaticus Hh0072 gene encodes a novel alpha1-3-fucosyltransferase belonging to CAZy GT11 family.

Lewis x (Le(x)) and sialyl Lewis x (SLe(x))-containing glycans play important roles in numerous physiological and pathological processes. The key enzyme for the final step formation of these Lewis antigens is alpha1-3-fucosyltransferase. Here we report molecular cloning and functional expression of a novel Helicobacter hepaticus alpha1-3-fucosyltransferase (HhFT1) which shows activity towards both non-sialylated and sialylated Type II oligosaccharide acceptor substrates. It is a promising catalyst for enzymatic and chemoenzymatic synthesis of Le(x), sialyl Le(x) and their derivatives. Unlike all other alpha1-3/4-fucosyltransferases characterized so far which belong to Carbohydrate Active Enzyme (CAZy, http://www.cazy.org/) glycosyltransferase family GT10, the HhFT1 shares protein sequence homology with alpha1-2-fucosyltransferases and belongs to CAZy glycosyltransferase family GT11. The HhFT1 is thus the first alpha1-3-fucosyltransferase identified in the GT11 family.