Combining carbochips and mass spectrometry to study the donor specificity for the Neisseria meningitidis β1,3-N-acetylglucosaminyltransferase LgtA

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

“…UDP-GlcNTFA ( 2 ) containing an N -trifluoroacetyl group was an excellent donor substrate for both β3GlcNAcTs. In contrary to a previous report indicating UDP-GlcNAcN 3 ( 3 ) containing an N -azidoacetyl group as an intolerable donor for NmLgtA, 19 the compound was confirmed by both HRMS and CE assays to be an acceptable donor substrate for both β3GlcNAcTs although the yields were not high.…”

“…It was quite exciting to notice the well tolerance of UDP-GlcNAc6S ( 16 ) which contains a relatively large and negatively charged O -sulfate group at C-6 of GlcNAc as the donor substrate by Hpβ3GlcNAcT. In consistent with previous reports, 15, 19 UDP-GalNAc ( 23 ) was an excellent donor substrate for NmLgtA. The compound, however, was not a donor for Hpβ3GlcNAcT.…”

“…1 Regarding donor substrate specificity, only a limited number of UDP-sugars have been tested for NmLgtA which was shown to tolerate uridine 5'-diphosphate- N -acetylglucosamine (UDP-GlcNAc), uridine 5'-diphosphate- N -acetylgalactosamine (UDP-GalNAc), UDP-4-deoxy-GalNAc, UDP-6-deoxy-GalNAc/GlcNAc, and UDP- N -propanoylglucosamine. 15, 19 The donor substrate specificity of Hpβ3GlcNAcT has not been explored in detail.…”

Synthetic Disialyl Hexasaccharides Protect Neonatal Rats from Necrotizing Enterocolitis

“…To obtain lacto-N-neotetraose (LNnT, 3), a common human milk tetrasaccharide, the Lc 3 trisaccharide (GlcNAcb1-3Galb1-4Glc, 2; Scheme 1) was synthesized from the inexpensive disaccharide lactose (1) and the monosaccharide GlcNAc by using a one-pot four-enzyme GlcNAc activation and transfer system containing Bifidobacterium longum (strain ATCC 55813) N-acetylhexosamine-1-kinase (NahK), [13] Pasteurella multocida N-acetylglucosamine uridyltransferase (PmGlmU), [14] Pasteurella multocida inorganic pyrophosphatase (PmPpA), [12] and Neisseria meningitidis b1-3-N-acetylglucosaminyltransferase (NmLgtA). [15] In this system, adenosine 5'-triphosphate (ATP) and GlcNAc were used in an NahK-catalyzed reaction to form N-acetylglucosamine-1-phosphate (GlcNAc-1-P), which was used with uridine 5'-triphosphate (UTP) by PmGlmU to form uridine 5'-diphosphate (UDP)-GlcNAc, the sugar nucleotide donor for NmLgtA for the production of Lc 3 from lactose. All four enzymes were quite active in tris(hydroxymethyl)aminomethane (Tris)-HCl buffer at pH 8.0, and Lc 3 trisaccharide (1.36 g) was obtained in an excellent yield (95 %) by incubation at 37 8C for 2 days.…”

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2011–2012

“…To study the donor specificity for the Neisseria meningitidis 1,3-N-acetylglucosaminyl-transferase LgtA (Guan et al, 2011) Neu5Ac-GalGlcNAc TOF Comparison of MALDI-TOF and DDI array method for influenza neuraminidase activity (see text) (Pourceau, et al, 2011) Phosphorylated high-mannose glycans TOF/TOF (DHB/TFA) For analysis of P-type lectins Plant cell wall polysaccharides TOF Glycans immobilized via hydrophobic interactions on an alkylthiol functionalised gold sample plate. Used to screen for glycosidase specificity in environmental samples (Beloqui et al, 2012) Various Monolayer on gold, TOF For study of glycosyltransferase activity (Gao et al, 2012a) yields.…”