NAD-binding proteins bind the dinucleotide NAD (nicotinamide adenine dinucleotide) and generally are enzymes that catalyze a redox reaction in which a proton is transferred from the carbon of a substrate alcohol group to NAD, thereby oxidizing the substrate and reducing NAD to NADH (Fig. 1). Examples of NAD-binding proteins include lactate dehydrogenase, alcohol dehydrogenase, and malate dehydrogenase. These different proteins catalyze the same basic reaction, but on different substrates, simply as a result of having differing substrate specificities.
Figure 1. The reaction catalyzed by the NAD-binding proteins. Rx and R2, vary depending on the substrate specificity of the enzyme. For malate dehydrogenase, the substrate malate has Rx = COOH and R2 = CH2-COOH. For lactate 
dehydrogenase, the substrate lactate has Rx = COOH and R2 = CH3. For alcohol dehydrogenase, the substrate ethanol 
has R1 = CH3 and R2 = H. 
substrate, and the other binds NAD. The substrate-binding domains of the different proteins are unrelated, but their NAD-binding domains have a common structural fold. The NAD-binding domain is a symmetrical 6-stranded b-sheet with a-helices on both sides, formed from two b-a-b-a-b protein motifs, called mononucleotide-binding motifs or Rossmann folds. Each of the two mononucleotide-binding motifs binds one-half of the dinucleotide; the adenine moiety of NAD binds to the first of these structural motifs, and nicotinamide binds to the second. The first three elements of secondary structure in the common adenine-binding motif, b1-aA-b2, have a similar sequence amongst the NAD-binding proteins (1). This fingerprint sequence has small hydrophobic residues conserved in b1, followed by a glycine-rich region (-Gly-X-Gly-X-X-Gly, where X is any residue), additional small hydrophobic residues in the aA-helix, and an Asp/Glu at the end of helix aA. The glycine residues adopt conformations forbidden to other residues and allow close packing of the strands and helix, plus a close approach between the adenine pyrophosphate and the N-terminal end of the aA-helix. The Asp/Glu residue interacts with the ribose hydroxyl. This signature sequence of ~30 residues was derived from the tertiary structures of known NAD-binding proteins and can be used to predict NAD-binding in proteins from simply their primary structure.
