title: Crystal structures and functional studies clarify substrate selectivity and catalytic residues for the unique orphan enzyme N-acetyl-D-mannosamine dehydrogenase.
abstract: NAMDH (N-acetyl-D-mannosamine dehydrogenase), from the soil bacteroidete Flavobacterium sp. 141-8, catalyses a rare NAD+-dependent oxidation of ManNAc (N-acetyl-D-mannosamine) into N-acetylmannosamino-lactone, which spontaneously hydrolyses into N-acetylmannosaminic acid. NAMDH belongs to the SDR (short-chain dehydrogenase/reductase) superfamily and is the only NAMDH characterized to date. Thorough functional, stability, site-directed mutagenesis and crystallographic studies have been carried out to understand better the structural and biochemical aspects of this unique enzyme. NAMDH exhibited a remarkable alkaline pH optimum (pH 9.4) with a high thermal stability in glycine buffer (Tm=64°C) and a strict selectivity towards ManNAc and NAD+. Crystal structures of ligand-free and ManNAc- and NAD+-bound enzyme forms revealed a compact homotetramer having point 222 symmetry, formed by subunits presenting the characteristic SDR α3β7α3 sandwich fold. A highly developed C-terminal tail used as a latch connecting nearby subunits stabilizes the tetramer. A dense network of polar interactions with the substrate including the encasement of its acetamido group in a specific binding pocket and the hydrogen binding of the sugar 4OH atom ensure specificity for ManNAc. The NAMDH-substrate complexes and site-directed mutagenesis studies identify the catalytic tetrad and provide useful traits for identifying new NAMDH sequences.
authors: *Department of Biochemistry and Molecular Biology-A, Faculty of Biology, Regional Campus of International Excellence "Campus Mare Nostrum", University of Murcia, Campus Espinardo, E-30100 Murcia, Spain.
authors: Agustín
authors: A
authors: Sola-Carvajal
authors: Fernando
authors: F
authors: Gil-Ortiz
authors: *Department of Biochemistry and Molecular Biology-A, Faculty of Biology, Regional Campus of International Excellence "Campus Mare Nostrum", University of Murcia, Campus Espinardo, E-30100 Murcia, Spain.
authors: Francisco
authors: F
authors: García-Carmona
authors: †Instituto de Biomedicina de Valencia-Consejo Superior de Investigaciones Científicas (IBV-CSIC) and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER-ISCIII), Valencia, Spain.
authors: Vicente
authors: V
authors: Rubio
authors: Álvaro
authors: Á
authors: Sánchez-Ferrer
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002237:Carbohydrate Dehydrogenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D003460:Crystallization; D018360:Crystallography, X-Ray; D005417:Flavobacterium / Q000201:enzymology / Q000235:genetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: D009243:NAD; D002237:Carbohydrate Dehydrogenases