Unusual conformation of nicotinamide adenine dinucleotide (NAD) bound to diphtheria toxin: A comparison with NAD bound to the oxidoreductase enzymes

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Abstract
The conformation of NAD bound to diphtheria toxin (DT), an ADP‐ribosylating enzyme, has been compared to the conformations of NAD(P) bound to 23 distinct NAD(P)‐binding oxidoreductase enzymes, whose structures are available in the Brookhaven Protein Data Bank. For the oxidoreductase enzymes, NAD(P) functions as a cofactor in electron transfer, whereas for DT, NAD is a labile substrate in which the N‐glycosidic bond between the nicotinamide ring and the N‐ribose is cleaved. All NAD(P) conformations were compared by (1) visual inspection of superimposed molecules, (2) RMSD of atomic positions, (3) principal component analysis, and (4) analysis of torsion angles and other conformational parameters. Whereas the majority of oxidoreductase‐bound NAD(P) conformations are found to be similar, the conformation of NAD bound to DT is found to be unusual. Distinctive features of the conformation of NAD bound to DT that may be relevant to DT's function as an ADP‐ribosylating enzyme include (1) an unusually short distance between the PN and N1N atoms, reflecting a highly folded conformation for the nicotinamide mononucleotide (NMN) portion of NAD, and (2) a torsion angle XN ∼ 0° about the scissile N‐glycosidic bond, placing the nicotinamide ring outside of the preferred anti and syn orientations. In NAD bound to DT, the highly folded NMN conformation and torsion angle XN ∼ 0° could contribute to catalysis, possibly by orienting the C1′N atom of NAD for nucleophilic attack, or by placing strain on the N‐glycosidic bond, which is cleaved by DT. The unusual overall conformation of NAD bound to DT is likely to reflect the structure of DT, which is unusual among NAD(P)‐binding enzymes. In DT, the NAD binding site is formed at the junction of two antiparallel β‐sheets. In contrast, although the 24 oxidoreductase enzymes belong to at least six different structural classes, almost all of them bind NAD(P) at the C‐terminal end of a parallel β‐sheet. The structural alignments and principal component analysis show that enzymes of the same structural class bind to particularly similar conformations of NAD(P), with few exceptions. The conformation of NAD bound to DT superimposes closely with that of an NAD analogue bound to Pseudomonas exotoxin A, an ADP‐ribosylating toxin that is structurally homologous to DT. This suggests that all of the ADP‐ribosylating enzymes that are structurally homologous to DT and ETA will bind a highly similar conformation of NAD.