A Unique Insertion Loop Facilitates Tight NAD+ Binding in Nicotinoprotein: Insights from In Vitro Loop Engineering and In Silico Studies

Nicotinoproteins are a group of NAD⁺-dependent dehydrogenases that bind NAD⁺ tightly and catalyze reactions without using free NAD⁺. In this study, we investigated the role of the unique insertion loop in nicotinoproteins. Carveol dehydrogenase (CADh), a short-chain dehydrogenase/reductase (SDR) nicotinoprotein, and β-hydroxybutyrate dehydrogenase from <i>Alcaligenes faecalis</i> (<i>Af</i>BHBDh), a non-nicotinoprotein counterpart, were used as model enzymes. An insertion loop-deleted mutant, CADh Δ39-49, was constructed. An insertion loop from <i>Mycobacterium paratuberculosis</i> CADh (<i>Mp</i>CADh) was introduced into <i>Af</i>BHBDh to generate the two mutants. The results showed that CADh Δ39-49 lost NAD⁺ tight binding capacity and could not utilize free NAD⁺. In contrast, the <i>Af</i>BHBDh mutants showed no dye-mediated dehydrogenase activity. Moreover, the <i>K</i>_M and <i>K_D</i> values for NAD⁺ were higher than those of the wild-type enzyme. Docking simulations revealed a stronger binding affinity between NAD⁺ and the mutants than with the wild-type <i>Af</i>BHBDh. Taken together, these results suggest that the insertion loop interferes with NAD⁺ entry into the active site of the enzyme while creating a more energetically favorable binding environment. This loop is necessary but alone is insufficient to achieve NAD⁺ tight binding. This study deepens understanding of NAD⁺ binding in SDR nicotinoproteins and provides insights for SDR enzyme engineering.