Crystal structures and functional studies clarify substrate selectivity and catalytic residues for the unique orphan enzyme N-acetyl-D -mannosamine dehydrogenase
| Autor: | Agustín Sola-Carvajal, Fernando Gil-Ortiz, Álvaro Sánchez-Ferrer, Vicente Rubio, Francisco García-Carmona |
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| Rok vydání: | 2014 |
| Předmět: |
Models
Molecular Stereochemistry Dehydrogenase Biology Reductase Crystallography X-Ray Flavobacterium Biochemistry Catalysis Substrate Specificity Tetramer Oxidoreductase Amino Acid Sequence Binding site Molecular Biology chemistry.chemical_classification Binding Sites Cell Biology NAD Enzyme chemistry Mutagenesis Site-Directed Carbohydrate Dehydrogenases NAD+ kinase Crystallization Sequence Alignment Homotetramer |
| Zdroj: | Biochemical Journal. 462:499-511 |
| ISSN: | 1470-8728 0264-6021 |
| DOI: | 10.1042/bj20140266 |
| Popis: | 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 ( T m=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. Abbreviations: AldT, aldohexose dehydrogenase; CCD, charge-coupled device; C-tail, C-terminal tail; FspNAMDH, NAMDH from Flavobacterium sp. 141-8; 3β/17β-HSD, 3β/17β-hydroxysteroid dehydrogenase; ManNAc, N-acetyl-D-mannosamine; NAMDH, N-acetyl-D-mannosamine dehydrogenase; Neu5Ac, N-acetylneuraminic acid; RsGDH, Rhodobacter sphaeroides galactitol dehydrogenase; SDR, short-chain dehydrogenase/reductase; TLS, translation–libration–screw–rotation |
| Databáze: | OpenAIRE |
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