BPS2026 – Uncovering regulation of bifunctional enzymes with opposing activities

Cells maintain distinct reducing equivalent pools, with NADH primarily linked to energy-harvesting reactions and NADPH to energy-utilizing processes. Rebalancing these pools is essential for cellular adaptation and is of particular interest in metabolic engineering. Here, we characterize bifunctional NAD kinase/NADP phosphatase enzymes from extremophilic archaea that uniquely modulate both NAD(H) and NADP(H) levels. The enzymes were heterologously expressed in Escherichia coli, purified from inclusion bodies, and shown to possess NADP + phosphatase and NAD(H) kinase activities. Size-exclusion chromatography indicated an octameric quaternary structure. Ongoing studies include steady-state kinetic analysis and cryoEM structural determination to elucidate the molecular basis of bifunctionality and adaptation. This work represents the first structural and functional characterization of this enzyme class and establishes their potential as tools for reprogramming cellular redox balance in bioengineering applications.