Characterization of chondroitin sulfate lyase ABC from Bacteroides thetaiotaomicron WAL2926.
Chondroitin sulfate ABC lyase (ChonABC) is an enzyme with broad specificity that depolymerizes via beta-elimination chondroitin sulfate (CS) and dermatan sulfate (DS) glycosaminoglycans (GAGs). ChonABC eliminates the glycosidic bond of its GAG substrates on the nonreducing end of their uronic acid component. This lyase possesses the unusual ability to act on both epimers of uronic acid, either glucuronic acid present in CS or iduronic acid in DS. Recently, we cloned, purified, and determined the three-dimensional structure of a broad specificity chondroitin sulfate ABC lyase from Bacteroides thetaiotaomicron (BactnABC) and identified two sets of catalytic residues. Here, we report the detailed biochemical characterization of BactnABC together with extensive site-directed mutagenesis resulting in characterization of the previously identified active site residues. BactnABC's catalysis is stimulated by Ca(2+) and Mg(2+) cations, particularly against DS. It displays extremely low activity toward hyaluronic acid and no activity toward heparin/heparan sulfate. Degradation of CS and DS by BactnABC yields only disaccharide products, pointing to an exolytic mode of action. The kinetic evaluations of the active-site mutants indicate that CS and DS substrates bind in the same active site, which is accompanied by a conformational change bringing the two sets of active site residues together. Conservative replacements of key residues suggest that His345 plays the role of a general base, initiating the degradation by abstracting the C5 bound proton from DS substrates, whereas either Tyr461 or His454 perform the equivalent role for CS substrates. Tyr461 is proposed, as well, to serve as general acid, completing the degradation of both CS and DS by protonating the leaving group.
Department of Biochemistry, McGill University, Montreal, Quebec, Canada.
David
D
Shaya
Bum-Soo
BS
Hahn
Nam Young
NY
Park
Joon-Soo
JS
Sim
Yeong Shik
YS
Kim
Miroslaw
M
Cygler
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001439:Bacteroides / Q000201:enzymology*; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000737:chemistry / Q000494:pharmacology; D002240:Carbohydrate Sequence; D002384:Catalysis / Q000187:drug effects; D020134:Catalytic Domain / Q000235:genetics; D019765:Chondroitin ABC Lyase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002809:Chondroitin Sulfates / Q000737:chemistry / Q000378:metabolism; D006025:Glycosaminoglycans / Q000737:chemistry / Q000378:metabolism; D006639:Histidine / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry / Q000494:pharmacology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009994:Osmolar Concentration; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D013696:Temperature; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism
D001426:Bacterial Proteins; D006025:Glycosaminoglycans; D014443:Tyrosine; D006639:Histidine; D002809:Chondroitin Sulfates; D019765:Chondroitin ABC Lyase; D008274:Magnesium; D002118:Calcium