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

Antiphagocytic capsular polysaccharides are key components of effective vaccines against pathogenic bacteria. groups B and C, as well as 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.

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.

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.

Donor substrate promiscuity of bacterial β1-3-N-acetylglucosaminyltransferases and acceptor substrate flexibility of β1-4-galactosyltransferases.

β1-3-N-Acetylglucosaminyltransferases (β3GlcNAcTs) and β1-4-galactosyltransferases (β4GalTs) 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 β3GlcNAcTs and β4GalTs, donor substrate specificity studies of two bacterial β3GlcNAcTs from Helicobacter pylori (Hpβ3GlcNAcT) and Neisseria meningitidis (NmLgtA), respectively, using a library of 39 sugar nucleotides were carried out. The two β3GlcNAcTs have complementary donor substrate promiscuity and 13 different trisaccharides were produced.

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.

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 β1-3GalT (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.

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.

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.

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.

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.

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

A series of α2-3-sialylated β1-3-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.

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

Sialyl Lewis(x) (SLe(x), Siaα2-3Galβ1-4(Fucα1-3)GlcNAcβOR) 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)).

Highly efficient chemoenzymatic synthesis of β1-3-linked galactosides.

A novel D-galactosyl-β1-3-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 (Galβ1-3GalNAc), lacto-N-biose (Galβ1-3GlcNAc), 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.

Combinatorial chemoenzymatic synthesis and high-throughput screening of sialosides.

Although the vital roles of structures containing sialic acid in biomolecular recognition are well documented, limited information is available on how sialic acid structural modifications, sialyl linkages, and the underlying glycan structures affect the binding or the activity of sialic acid-recognizing proteins and related downstream biological processes. A novel combinatorial chemoenzymatic method has been developed for the highly efficient synthesis of biotinylated sialosides containing different sialic acid structures and different underlying glycans in 96-well plates from biotinylated sialyltransferase acceptors and sialic acid precursors. By transferring the reaction mixtures to NeutrAvidin-coated plates and assaying for the yields of enzymatic reactions using lectins recognizing sialyltransferase acceptors but not the sialylated products, the biotinylated sialoside products can be directly used, without purification, for high-throughput screening to quickly identify the ligand specificity of sialic acid-binding proteins.

The Hd0053 gene of Haemophilus ducreyi encodes an alpha2,3-sialyltransferase.

Haemophilus ducreyi is a Gram-negative bacterium that causes chancroid, a sexually transmitted genital ulcer disease. Different lipooligosaccharide (LOS) structures have been identified from H. ducreyi strain 35000, including those sialylated glycoforms.

NeuA sialic acid O-acetylesterase activity modulates O-acetylation of capsular polysaccharide in group B Streptococcus.

Group B Streptococcus (GBS) is a common cause of neonatal sepsis and meningitis. A major GBS virulence determinant is its sialic acid (Sia)-capped capsular polysaccharide. Recently, we discovered the presence and genetic basis of capsular Sia O-acetylation in GBS.