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.

Recombinant Snail Sialic Acid Aldolase is Promiscuous towards Aliphatic Aldehydes

“…To corroborate the observation that 1 a is not a substrate for sNPL, we compared the activity of sNPL and chNPL using a series of other NPL activity testing methods. The first method used was a coupled enzymatic assay which allows the oxidation of 2 a by a ManNAc‐dehydrogenase (NAMDH), [23] and the reduction of the co‐factor NAD + to NADH was detected in real‐time photometrically at 340 nm (Figure S3). NAMDH can be easily expressed in recombinant form in E. coli and proved to be very useful for selectively quantifying 2 a in the presence of N‐acetylglucosamine (GlcNAc), which are hard to distinguish by other colorimetric and chromatographic methods [24] .…”

Crystallographically correct but confusing presentation of structural models deposited in the Protein Data Bank

“…2(b) and 2(d), respectively. Sola-Carvajal et al (2014) clearly state that this enzyme always forms tetrameric oligomers, but the presentation in the PDB may suggest that the formation of complexes changes the oligomeric state of this enzyme. This is just one example illustrating why the optimal presentation of the structures in the PDB matters.…”