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Crystal structures and functional studies clarify substrate selectivity and catalytic residues for the unique orphan enzyme N-acetyl-D-mannosamine dehydrogenase

DOI: 10.1042/BJ20140266 DOI Help
PMID: 24969681 PMID Help

Authors: Agustan Sola-Carvajal (University of Murcia) , Fernando Gilâ-Ortiz (Instituto de Biomedicina de Valencia-Consejo Superior de Investigaciones Científicas (IBV-CSIC) and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER-ISCIII)) , Francisco Garcã­-Carmona (University of Murcia) , Vicente Rubio (Instituto de Biomedicina de Valencia-Consejo Superior de Investigaciones Científicas (IBV-CSIC) and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER-ISCIII)) , Alvaro Sanchez-Ferrer (University of Murcia)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Biochemical Journal , VOL 462 , PAGES 499 - 511

State: Published (Approved)
Published: September 2014
Diamond Proposal Number(s): 8035

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.

Journal Keywords: Binding; Carbohydrate; Catalysis; Crystallization; Crystallography; X-Ray; Flavobacterium; Models; Molecular; Mutagenesis; Site-Directed; NAD; Sequence; Substrate Specificity

Diamond Keywords: Enzymes; Bacteria

Subject Areas: Biology and Bio-materials


Instruments: I24-Microfocus Macromolecular Crystallography

Other Facilities: ID23-1, ID23-2 at ESRF

Added On: 25/09/2014 08:49

Discipline Tags:

Structural biology Life Sciences & Biotech

Technical Tags:

Diffraction Macromolecular Crystallography (MX)