Uncovering the Key Determinants of NAD(H)/NADP(H) Cofactor Preference across Oxidoreductase Families

Nicotinamide adenine dinucleotide (NAD(H)) and nicotinamide adenine dinucleotide phosphate (NADP(H)) are essential cofactors in oxidoreductase-catalyzed reactions, mediating electron transfer in catabolism and anabolism, respectively. Understanding what determines enzyme preference for each cofactor has direct implications for cofactor engineering and metabolic engineering. In this study, we retrieved crystal structures of oxidoreductases co-crystallized with NAD(H) or NADP(H) from the RCSB PDB database and applied a series of curation steps to obtain a dataset of 917 entries, covering NAD(H)-preferring (429), NADP(H)-preferring (455), and dual-specificity (33) enzymes. We then examined the structural and chemical features of the binding pocket that govern this selectivity. Our results show that sequence similarity and overall structural similarity are poor predictors of cofactor preference. What matters instead is the chemical environment of the binding pocket. NAD(H)-preferring enzymes combine hydrophobic contacts with electrostatic repulsion driven by acidic residues (Asp/Glu), effectively excluding the 2'-phosphate group. NADP(H)-preferring enzymes rely on conserved Arg residues that recognize the 2'-phosphate group through electrostatic attraction and hydrogen bonding. Dual-specificity enzymes accommodate both cofactors by maintaining a mixture of positively and negatively charged residues in the pocket. Case studies of the SDR and ALDH families further show that, while the specific residues involved differ between families, the pocket chemistry determining cofactor preference follows a consistent cross-family logic. These findings provide a theoretical basis for predicting and engineering cofactor selectivity in oxidoreductases.