Abstract
Pyruvyl modification of oligosaccharides is widely seen in both prokaryotes and eukaryotes. Although the biosynthetic mechanisms of pyruvylation have been investigated, enzymes that metabolize and degrade pyruvylated oligosaccharides are not well known. Here, we searched for a pyruvylated galactose (PvGal)-releasing enzyme by screening soil samples. We identified a Bacillus strain, as confirmed by the 16S ribosomal RNA gene analysis, that exhibited PvGal-ase activity toward p-nitrophenyl-β-D-pyruvylated galactopyranose (pNP-β-D-PvGal). Draft genome sequencing of this strain, named HMA207, identified three candidate genes encoding potential PvGal-ases, among which only the recombinant protein encoded by ORF1119 exhibited PvGal-ase activity. Although ORF1119 protein displayed broad substrate specificity for pNP sugars, pNP-β-D-PvGal was the most favorable substrate. The optimum pH for the ORF1119 PvGal-ase was determined as 7.5. A BLAST search suggested that ORF1119 homologs exist widely in bacteria. Among two homologs tested, BglC from Clostridium but not BglH from Bacillus showed PvGal-ase activity. Crystal structural analysis together with point mutation analysis revealed crucial amino acids for PvGal-ase activity. Moreover, ORF1119 protein catalyzed the hydrolysis of PvGal from galactomannan of Schizosaccharomyces pombe, suggesting that natural polysaccharides might be substrates of the PvGal-ase. This novel PvGal-catalyzing enzyme might be useful for glycoengineering projects to produce new oligosaccharide structures.
Highlights
Pyruvyl modification of oligosaccharides is widely seen in both prokaryotes and eukaryotes
0.5 mg of recombinant fission yeast pyruvyltransferase Pvg1p was incubated with 40 mM PEP monopotassium salt and 1 mg of pNP-β-Gal in 150 μl of 0.2 M MOPS-NaOH buffer at 37 °C overnight. pNP-β-D-pyruvylated galactose (PvGal) was collected by HPLC using a GL-7400 system equipped with a UV spectrophotometer (GL sciences) and a Cosmosil 5C18 reverse-phase column (Nacalai tesque, 4.6 × 150 nm) set at 30 °C
To search for a PvGal-ase, we isolated more than 200 bacterial strains from soil samples
Summary
Pyruvyl modification of oligosaccharides is widely seen in both prokaryotes and eukaryotes. ORF1119 protein catalyzed the hydrolysis of PvGal from galactomannan of Schizosaccharomyces pombe, suggesting that natural polysaccharides might be substrates of the PvGal-ase. This novel PvGal-catalyzing enzyme might be useful for glycoengineering projects to produce new oligosaccharide structures. Pyruvylation of Gal (PvGal) leads to a negative charges on the cell surface, which is important for intercellular interactions in the marine sponge Microciona prolifera[13] and in S. pombe[14,15]. There are several reports about the existence of PvGal in bacteria; here, we screened soil samples for microorganisms that exhibit PvGal-ase activity to release PvGal. As a result, we identified a novel gene that encodes a PvGal-ase and characterized its enzymatic activity
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