Taking control of cellular NAD ⁺ concentrations: Cellular concentrations of the nicotinamide adenine dinucleotide (NAD ⁺ ) are critical for proper metabolism and are often altered in aging and disease. To enable better understanding of these processes, Titov et al. altered the concentration of NAD ⁺ in particular cellular compartments. They did this through expression of a bacterial enzyme targeted to specific compartments of human cells in culture. Their experiments emphasize the important role of the electron transport chain in redox transfer of electrons to NADH, rather than proton pumping, in mitochondrial pathogenesis. Science , this issue p. 231

Structure-guided engineering of an NADH oxidase switches its cofactor preference, thus yielding an NADPH oxidase that can be used to tune the cellular NADP+/NADPH ratio and to examine the links between mitochondrial NADH and NADPH pools. The redox coenzymes NADH and NADPH are broadly required for energy metabolism, biosynthesis and detoxification. Despite detailed knowledge of specific enzymes and pathways that utilize these coenzymes, a holistic understanding of the regulation and compartmentalization of NADH- and NADPH-dependent pathways is lacking, partly because of a lack of tools with which to investigate these processes in living cells. We have previously reported the use of the naturally occurring Lactobacillus brevis H2O-forming NADH oxidase (LbNOX) as a genetic tool for manipulation of the NAD+/NADH ratio in human cells. Here, we present triphosphopyridine nucleotide oxidase (TPNOX), a rationally designed and engineered mutant of LbNOX that is strictly specific to NADPH. We characterized the effects of TPNOX expression on cellular metabolism and used it in combination with LbNOX to show how the redox states of mitochondrial NADPH and NADH pools are connected.

We recently identified meclizine, an over-the-counter drug, as an inhibitor of mitochondrial respiration. Curiously, meclizine blunted respiration in intact cells but not in isolated mitochondria, suggesting an unorthodox mechanism. Using a metabolic profiling approach, we now show that treatment with meclizine leads to a sharp elevation of cellular phosphoethanolamine, an intermediate in the ethanolamine branch of the Kennedy pathway of phosphatidylethanolamine biosynthesis. Metabolic labeling and in vitro enzyme assays confirmed direct inhibition of the cytosolic enzyme CTP:phosphoethanolamine cytidylyltransferase (PCYT2). Inhibition of PCYT2 by meclizine led to rapid accumulation of its substrate, phosphoethanolamine, which is itself an inhibitor of mitochondrial respiration. Our work identifies the first pharmacologic inhibitor of the Kennedy pathway, demonstrates that its biosynthetic intermediate is an endogenous inhibitor of respiration, and provides key mechanistic insights that may facilitate repurposing meclizine for disorders of energy metabolism.