title: Analysis of keystone enzyme in Agar hydrolysis provides insight into the degradation (of a polysaccharide from) red seaweeds.
abstract: Agars are abundant polysaccharides from marine red algae, and their chemical structure consists of alternating D-galactose and 3,6-anhydro-L-galactose residues, the latter of which are presumed to make the polymer recalcitrant to degradation by most terrestrial bacteria. Here we study a family 117 glycoside hydrolase (BpGH117) encoded within a recently discovered locus from the human gut bacterium Bacteroides plebeius. Consistent with this locus being involved in agarocolloid degradation, we show that BpGH117 is an exo-acting 3,6-anhydro-α-(1,3)-L-galactosidase that removes the 3,6-anhydrogalactose from the non-reducing end of neoagaro-oligosaccharides. A Michaelis complex of BpGH117 with neoagarobiose reveals the distortion of the constrained 3,6-anhydro-L-galactose into a conformation that favors catalysis. Furthermore, this complex, supported by analysis of site-directed mutants, provides evidence for an organization of the active site and positioning of the catalytic residues that are consistent with an inverting mechanism of catalysis and suggests that a histidine residue acts as the general acid. This latter feature differs from the vast majority of glycoside hydrolases, which use a carboxylic acid, highlighting the alternative strategies that enzymes may utilize in catalyzing the cleavage of glycosidic bonds.
authors.affiliation: Department of Biochemistry and Microbiology, University of Victoria, PO Box 3055 STN CSC, Victoria, British Columbia V8W 3P6, Canada.
authors.forename: Jan-Hendrik
authors.initials: JH
authors.lastname: Hehemann
authors.forename: Leo
authors.initials: L
authors.lastname: Smyth
authors.forename: Anuj
authors.initials: A
authors.lastname: Yadav
authors.forename: David J
authors.initials: DJ
authors.lastname: Vocadlo
authors.forename: Alisdair B
authors.initials: AB
authors.lastname: Boraston
mesh_terms: D000362:Agar / Q000737:chemistry*; D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain; D002855:Chromatography, Thin Layer / Q000379:methods; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray / Q000379:methods; D006026:Glycoside Hydrolases / Q000737:chemistry*; D006868:Hydrolysis; D008958:Models, Molecular; D009841:Oligonucleotides / Q000737:chemistry; D011134:Polysaccharides / Q000737:chemistry; D011487:Protein Conformation; D017510:Protein Folding; D012624:Seaweed / Q000737:chemistry*; D013379:Substrate Specificity
chemical_list: D009841:Oligonucleotides; D011134:Polysaccharides; D000362:Agar; D006026:Glycoside Hydrolases