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. They were used to investigate the acceptor substrate specificities of three β4GalTs from Neisseria meningitidis (NmLgtB), Helicobacter pylori (Hpβ4GalT), and bovine (Bβ4GalT), respectively. Ten of the 13 trisaccharides were shown to be tolerable acceptors for at least one of these β4GalTs. The application of NmLgtA in one-pot multienzyme (OPME) synthesis of two trisaccharides including GalNAcβ1-3Galβ1-4GlcβProN3 and Galβ1-3Galβ1-4Glc 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.

[1]  P. Wang,et al.  Improved one-pot multienzyme (OPME) systems for synthesizing UDP-uronic acids and glucuronides. , 2015, Chemical communications.

[2]  L. Bode,et al.  Synthetic disialyl hexasaccharides protect neonatal rats from necrotizing enterocolitis. , 2014, Angewandte Chemie.

[3]  L. Berliner,et al.  The lactose synthase acceptor site: a structural map derived from acceptor studies , 1984, Molecular and Cellular Biochemistry.

[4]  O. Blixt,et al.  High-level expression of the Neisseria meningitidis lgtA gene in Escherichia coli and characterization of the encoded N-acetylglucosaminyltransferase as a useful catalyst in the synthesis of GlcNAc beta 1-->3Gal and GalNAc beta 1-->3Gal linkages. , 1999, Glycobiology.

[5]  Lipids of abducted Antarctic pteropods, Spongiobranchaea australis, and their hyperiid amphipod host , 1999 .

[6]  B. Priem,et al.  A new fermentation process allows large-scale production of human milk oligosaccharides by metabolically engineered bacteria. , 2002, Glycobiology.

[7]  C. Dumon,et al.  Production of Lewis x Tetrasaccharides by Metabolically Engineered Escherichia coli , 2006, Chembiochem : a European journal of chemical biology.

[8]  T. Saito,et al.  Chemical characterization of the oligosaccharides in Bactrian camel (Camelus bactrianus) milk and colostrum. , 2010, Journal of dairy science.

[9]  Hai Yu,et al.  Characterizing non-hydrolyzing Neisseria meningitidis serogroup A UDP-N-acetylglucosamine (UDP-GlcNAc) 2-epimerase using UDP-N-acetylmannosamine (UDP-ManNAc) and derivatives. , 2016, Carbohydrate research.

[10]  S. Do,et al.  Expression and Characterization of β-1,4-Galactosyltransferase from Neisseria meningitidis and Neisseria gonorrhoeae , 2002 .

[11]  Ryan McBride,et al.  Recognition of sialylated poly-N-acetyllactosamine chains on N- and O-linked glycans by human and avian influenza A virus hemagglutinins. , 2012, Angewandte Chemie.

[12]  P. Wang,et al.  Substrate Promiscuity of N-Acetylhexosamine 1-Kinases , 2011, Molecules.

[13]  C Kunz,et al.  Lactose-derived oligosaccharides in the milk of elephants: comparison with human milk , 1999, British Journal of Nutrition.

[14]  J. Feeney,et al.  Separation of human milk oligosaccharides by recycling chromatography. First isolation of lacto-N-neo-difucohexaose II and 3'-Galactosyllactose from this source. , 1988, Carbohydrate research.

[15]  M. Palcic.  Glycosyltransferases as biocatalysts. , 2011, Current opinion in chemical biology.

[16]  M. Mrksich,et al.  Combining carbochips and mass spectrometry to study the donor specificity for the Neisseria meningitidis β1,3-N-acetylglucosaminyltransferase LgtA. , 2011, Bioorganic & Medicinal Chemistry Letters.

[17]  H. Gruppen,et al.  Comparison of milk oligosaccharides pattern in colostrum of different horse breeds. , 2015, Journal of agricultural and food chemistry.

[18]  K. Fukuda,et al.  Chemical characterization of milk oligosaccharides of the koala (Phascolarctos cinereus) , 2013, Glycoconjugate Journal.

[19]  W. Wakarchuk,et al.  Functional genomics of Helicobacter pylori: identification of a β‐1,4 galactosyltransferase and generation of mutants with altered lipopolysaccharide , 2000, Molecular microbiology.

[20]  Chi-Huey Wong,et al.  Regeneration of sugar nucleotide for enzymic oligosaccharide synthesis: use of Gal-1-phosphate uridyltransferase in the regeneration of UDP-galactose, UDP-2-deoxygalactose, and UDP-galactosamine , 1992 .

[21]  Shin-Ichiro Nishimura,et al.  Construction and structural characterization of versatile lactosaminoglycan-related compound library for the synthesis of complex glycopeptides and glycosphingolipids. , 2006, The Journal of organic chemistry.

[22]  S. Tsuji,et al.  Biologically Active Glycosides from Asteroidea, XXXI. Glycosphingolipids from the Starfish Asterias amurensis versicolor sladen, 2. Structure and Biological Activity of Ganglioside Molecular Species , 1993 .

[23]  Huilei Yu,et al.  Chemoenzymatic synthesis of CMP-sialic acid derivatives by a one-pot two-enzyme system: comparison of substrate flexibility of three microbial CMP-sialic acid synthetases. , 2004, Bioorganic & medicinal chemistry.

[24]  Yanhong Li,et al.  One-pot three-enzyme synthesis of UDP-GlcNAc derivatives. , 2011, Chemical communications.

[25]  T. Saito,et al.  Chemical structure of three neutral trisaccharides isolated in free form from bovine colostrum. , 1987, Carbohydrate research.

[26]  K. Moremen,et al.  Enzymatic synthesis of natural and 13C enriched linear poly-N-acetyllactosamines as ligands for galectin-1. , 1999, Glycobiology.

[27]  Li Ding,et al.  Efficient one-pot multienzyme synthesis of UDP-sugars using a promiscuous UDP-sugar pyrophosphorylase from Bifidobacterium longum (BLUSP). , 2012, Chemical communications.

[28]  Chun‐Cheng Lin,et al.  Sequential one-pot enzymatic synthesis of oligo-N-acetyllactosamine and its multi-sialylated extensions. , 2014, Chemical communications.

[29]  P. Wang,et al.  Helicobacter hepaticus Hh0072 gene encodes a novel alpha1-3-fucosyltransferase belonging to CAZy GT11 family. , 2010, Glycobiology.

[30]  S. Withers,et al.  A β‐1,4‐Galactosyltransferase from Helicobacter pylori is an Efficient and Versatile Biocatalyst Displaying a Novel Activity for Thioglycoside Synthesis , 2008, Chembiochem : a European journal of chemical biology.

[31]  Kam Y. Lau,et al.  Highly efficient chemoenzymatic synthesis of beta1-4-linked galactosides with promiscuous bacterial beta1-4-galactosyltransferases. , 2010, Chemical communications.

[32]  Yuyang Wu,et al.  Novel biosynthetic functions of lipopolysaccharide rfaJ homologs from Helicobacter pylori. , 2005, Glycobiology.

[33]  K. Fukuda,et al.  Chemical characterization of milk oligosaccharides of the eastern quoll (Dasyurus viverrinus) , 2015, Glycoconjugate Journal.

[34]  T. Yamakawa,et al.  Characterization of glycolipids from rat granuloma and macrophage. , 1978, Journal of biochemistry.

[35]  Ola Blixt,et al.  Chemoenzymatic synthesis of glycan libraries. , 2006, Methods in enzymology.

[36]  Corwin M. Nycholat,et al.  Helicobacter pylori β1,3-N-acetylglucosaminyltransferase for versatile synthesis of type 1 and type 2 poly-LacNAcs on N-linked, O-linked and I-antigen glycans. , 2012, Glycobiology.

[37]  P. Wang,et al.  Wide sugar substrate specificity of galactokinase from Streptococcus pneumoniae TIGR4. , 2011, Carbohydrate research.

[38]  A. Kuno,et al.  Polylactosamine on glycoproteins influences basal levels of lymphocyte and macrophage activation , 2007, Proceedings of the National Academy of Sciences.

[39]  P. V. Balaji,et al.  Expression of deletion constructs of bovine beta-1,4-galactosyltransferase in Escherichia coli: importance of Cys134 for its activity. , 1993, Protein engineering.

[40]  Ola Blixt,et al.  Large-scale chemoenzymatic synthesis of blood group and tumor-associated poly-N-acetyllactosamine antigens. , 2006, Carbohydrate research.

[41]  E. Samain,et al.  Synthesis of α-galactosyl epitopes by metabolically engineered Escherichia coli. , 2012, Carbohydrate research.

[42]  W. Wakarchuk,et al.  Role of paired basic residues in the expression of active recombinant galactosyltransferases from the bacterial pathogen Neisseria meningitidis. , 1998, Protein engineering.

[43]  O. Renkonen.  Enzymatic in vitro synthesis of I-branches of mammalian polylactosamines: generation of scaffolds for multiple selectin-binding saccharide determinants , 2000, Cellular and Molecular Life Sciences CMLS.

[44]  S. Koizumi,et al.  Cloning and expression of beta1,4-galactosyltransferase gene from Helicobacter pylori. , 2000, Glycobiology.

[45]  Lothar Elling,et al.  Chemo-enzymatic synthesis of poly-N-acetyllactosamine (poly-LacNAc) structures and their characterization for CGL2-galectin-mediated binding of ECM glycoproteins to biomaterial surfaces , 2009, Glycoconjugate Journal.

[46]  T. Saito,et al.  New galactosyllactose containing alpha-glycosidic linkage isolated from ovine (Booroola dorset) colostrum. , 1989, Biochimica et biophysica acta.

[47]  M. Palcic,et al.  Transfer of D-galactosyl groups to 6-O-substituted 2-acetamido-2-deoxy-D-glucose residues by use of bovine D-galactosyltransferase. , 1987, Carbohydrate research.

[48]  W. Wakarchuk,et al.  Efficient preparation of natural and synthetic galactosides with a recombinant beta-1,4-galactosyltransferase-/UDP-4'-gal epimerase fusion protein. , 2001, The Journal of organic chemistry.

[49]  Corwin M. Nycholat,et al.  Synthesis of biologically active N- and O-linked glycans with multisialylated poly-N-acetyllactosamine extensions using P. damsela α2-6 sialyltransferase. , 2013, Journal of the American Chemical Society.

[50]  I. Brockhausen,et al.  UDP-Gal: GlcNAc-R β1,4-galactosyltransferase—a target enzyme for drug design. Acceptor specificity and inhibition of the enzyme , 2006, Glycoconjugate Journal.

[51]  T. Saito,et al.  Chemical characterisation of six oligosaccharides in a sample of colostrum of the brown capuchin, Cebus apella (Cebidae: primates). , 1999, Comparative biochemistry and physiology. Part C, Pharmacology, toxicology & endocrinology.

[52]  B. Ramakrishnan,et al.  alpha-Lactalbumin (LA) stimulates milk beta-1,4-galactosyltransferase I (beta 4Gal-T1) to transfer glucose from UDP-glucose to N-acetylglucosamine. Crystal structure of beta 4Gal-T1 x LA complex with UDP-Glc. , 2001, The Journal of biological chemistry.

[53]  J. Collins,et al.  Structure of a marsupial-milk trisaccharide. , 1980 .

[54]  Karl F Johnson,et al.  Synthesis of oligosaccharides by bacterial enzymes , 1999, Glycoconjugate Journal.

[55]  W. Wakarchuk,et al.  Functional Relationships of the Genetic Locus Encoding the Glycosyltransferase Enzymes Involved in Expression of the Lacto-N-neotetraose Terminal Lipopolysaccharide Structure in Neisseria meningitidis* , 1996, The Journal of Biological Chemistry.