Even in the absence of a template, glycosyltransferases can catalyze the synthesis of carbohydrate polymers of specific sequence. The paradigm has been that one enzyme catalyzes the formation of one type of glycosidic linkage, yet certain glycosyltransferases generate polysaccharide sequences composed of two distinct linkage types. In principle, bifunctional glycosyltransferases can possess separate active sites for each catalytic activity or one active site with dual activities. We encountered the fundamental question of one or two distinct active sites in our investigation of the galactosyltransferase GlfT2. GlfT2 catalyzes the formation of mycobacterial galactan, a critical cell-wall polymer composed of galactofuranose residues connected with alternating, regioisomeric linkages. We found that GlfT2 mediates galactan polymerization using only one active site that manifests dual regioselectivity. Structural modeling of the bifunctional glycosyltransferases hyaluronan synthase and cellulose synthase suggests that these enzymes also generate multiple glycosidic linkages using a single active site. These results highlight the versatility of glycosyltransferases for generating polysaccharides of specific sequence. We postulate that a hallmark of processive elongation of a carbohydrate polymer by a bifunctional enzyme is that one active site can give rise to two separate types of glycosidic bonds.
glycosyltransferases, Substrate Specificity, galactosyltransferase, Galactan, modeling, hyaluronan, polymerization
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The molecular structure of the wild strain of the lipopolysaccharide core of Aeromonas salmonicida, ssp salmonicida has been sequenced using tandem mass spectrometry. The core oligosaccharide was determined to contain an O-4 phosphorylated and O-5 substituted Kdo reducing group, and its structure is proposed as the follows: R-L-α-D-Hepp-(1→2)-L-α-D-Hepp-(1→3)-L-α-D-Hepp-(1→5)-α-Kdop-(2→lipid A) α-D-GlcpN-(1→7)-L-β--D-Hepp R= α-D-Galp-(1→4)-α-D-GalpNAc-(1→6)-α-D-Glcp-(1→4) After the core oligosaccharide of LPS was released from the lipid A portion by conventional treatment with 1% acetic acid, we demonstrated the existence of a homogeneous mixture composed mainly of the native core oligosaccharide containing the Kdo with its O-4 phosphate group intact, and a degraded core oligosaccharide mixture, which eliminated the O-4 phosphate group with extreme facility. The precise molecular structure and glycone sequence of the homogeneous mixture of phosphorylated and dephosphorylated core oligosaccharides was determined by electrospray ionization (ESI) mass spectrometry and tandem mass spectrometric analysis. CID-MS/MS of the homogeneous mixture of permethylated core oligosaccharides afforded a series of diagnostic product ions which confirmed the established sequence of the glycones to be determined. Matrix-assisted laser desorption/ionization (MALDI) tandem mass spectrometry reconfirmed the molecular structure of the dephosphorylated homogeneous permethylated mixture of the core oligosaccharides containing the diastereomeric 4,8- and 4,7-anhydro-α-keto acids.
Aeromonas salmonicida ssp salmonicida, native phosphorylated core oligosaccharide, degraded dephosphorylated core oligosaccharide, electrospray and matrix-assisted laser desorption/ionization, mass spectrometry and tandem mass spectrometry, structural investigation
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The molecular structure of the mutant form of the lipopolysaccharide of Aeromonas salmonicida was determined to contain an O-4 phosphorylated and O-5 substituted Kdo reducing group, and is proposed as the following: L-α-D-Hepp-(1→2)-L-α-D-Hepp-(1→3)-L-α-D-Hepp-(1→5)-α-Kdop-(2→Lipid A)It was established that during the cleavage of this LPS with 1% acetic acid, to release the core oligosaccharide from the Lipid A portion, we obtained a degraded core oligosaccharide which eliminated its phosphate group with extreme facility. The precise molecular structure of this dephosphorylated core was deduced by electrospray mass spectrometry and is proposed as the following: L-α-D-Hepp-(1→2)-L-α-D-Hepp-(1→3)-L-α-D-Hepp-(1→5)-α-D-Kdop-(4,7-anhydro)-α-keto acid Low energy collision ESI-QqTOF-MS/MS analysis of the dephosphorylated core oligosaccharide confirmed the presence of the O-5 glycosylated 4,8- and 4,7-anhydro derivatives of the enolizable α-keto-acids. The CID tandem mass spectrometric analysis of the heterogeneous mixture of the permethylated core oligosaccharide established the unreported methylation reaction on the diastereomeric 4,8- and 4,7-anhydro α-keto-acids and the complete permethylation and addition reaction of the O-5 glycosylated open chain reducing end terminal D-arabino-3-en-2-ulonic acid. The stereo-specific fragmentation routes obtained during the tandem mass spectrometric analysis permitted the precise sequencing of this dephosphorylated rough core oligosaccharide of the mutant LPS of A. salmonicida.
Lipopolysaccharide, Aeromonas salmonicida, structural investigation, dephosphorylated core oligosaccharide, electrospray tandem mass spectrometry
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