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.
- Flavobacterium sp. 141-8
- N-acetyl-D-mannosamine dehydrogenase
- short-chain dehydrogenase/reductase
- sialic acid
- substrate selectivity
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
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