Daimei Miura

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 Alcaligenes faecalis (AfBHBDh), a non-nicotinoprotein counterpart, were used as model enzymes. An insertion loop-deleted mutant, CADh Δ39–49, was constructed. An insertion loop from Mycobacterium paratuberculosis CADh (MpCADh) was introduced into AfBHBDh 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 AfBHBDh mutants showed no dye-mediated dehydrogenase activity. Moreover, the K_M and K_D 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 AfBHBDh. 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.

Although IgG-free immunosensors are in high demand owing to ethical concerns, the development of convenient immunosensors that alternatively integrate recombinantly produced antibody fragments, such as single-chain variable fragments (scFvs), remains challenging. The low affinity of antibody fragments, unlike IgG, caused by monovalent binding to targets often leads to decreased sensitivity. We improved the affinity owing to the bivalent effect by fabricating a bivalent antibody–enzyme complex (AEC) composed of two scFvs and a single glucose dehydrogenase, and developed a rapid and convenient scFv-employed electrochemical detection system for the C-reactive protein (CRP), which is a homopentameric protein biomarker of systemic inflammation. The development of a point-of-care testing (POCT) system is highly desirable; however, no scFv-based CRP-POCT immunosensors have been developed. As expected, the bivalent AEC showed higher affinity than the single scFv and contributed to the high sensitivity of CRP detection. The electrochemical CRP detection using scFv-immobilized magnetic beads and the bivalent AEC as capture and detection antibodies, respectively, was achieved in 20 min without washing steps in human serum and the linear range was 1–10 nM with the limit of detection of 2.9 nM, which has potential to meet the criteria required for POCT application in rapidity, convenience, and hand-held detection devices without employing IgGs.