title: Structural and biochemical characterization of the broad substrate specificity of Bacteroides thetaiotaomicron commensal sialidase.
abstract: Sialidases release the terminal sialic acid residue from a wide range of sialic acid-containing polysaccharides. Bacteroides thetaiotaomicron, a symbiotic commensal microbe, resides in and dominates the human intestinal tract. We characterized the recombinant sialidase from B. thetaiotaomicron (BTSA) and demonstrated that it has broad substrate specificity with a relative activity of 97, 100 and 64 for 2,3-, 2,6- and 2,8-linked sialic substrates, respectively. The hydrolysis activity of BTSA was inhibited by a transition state analogue, 2-deoxy-2,3-dehydro-N-acetyl neuraminic acid, by competitive inhibition with a Ki value of 35μM. The structure of BSTA was determined at a resolution of 2.3Å. This structure exhibited a unique carbohydrate-binding domain (CBM) at its N-terminus (a.a. 23-190) that is adjacent to the catalytic domain (a.a. 191-535). The catalytic domain has a conserved arginine triad with a wide-open entrance for the substrate that exposes the catalytic residue to the surface. Unlike other pathogenic sialidases, the polysaccharide-binding site in the CBM is near the active site and possibly holds and positions the polysaccharide substrate directly at the active site. The structural feature of a wide substrate-binding groove and closer proximity of the polysaccharide-binding site to the active site could be a unique signature of the commensal sialidase BTSA and provide a molecular basis for its pharmaceutical application.
authors.affiliation: Korea Research Institute of Bioscience & Biotechnology, Daejeon, Republic of Korea.
authors.forename: Kwang-Hyun
authors.initials: KH
authors.lastname: Park
authors.forename: Min-Gyu
authors.initials: MG
authors.lastname: Kim
authors.forename: Hee-Jeong
authors.initials: HJ
authors.lastname: Ahn
authors.forename: Dae-Han
authors.initials: DH
authors.lastname: Lee
authors.forename: Jin-Hyo
authors.initials: JH
authors.lastname: Kim
authors.forename: Young-Wan
authors.initials: YW
authors.lastname: Kim
authors.forename: Eui-Jeon
authors.initials: EJ
authors.lastname: Woo
mesh_terms: D000595:Amino Acid Sequence; D001439:Bacteroides / Q000201:enzymology*; D001665:Binding Sites; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019158:N-Acetylneuraminic Acid / Q000031:analogs & derivatives* / Q000378:metabolism*; D009439:Neuraminidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013451:Sulfonic Acids / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D013451:Sulfonic Acids; C036137:2-deoxy-2,3-dehydro-N-acetylneuraminic acid; D009439:Neuraminidase; D019158:N-Acetylneuraminic Acid