An engineered enzyme that targets circulating lactate to alleviate intracellular NADH:NAD+ imbalance

Abstract

An elevated intracellular NADH:NAD⁺ ratio, or ‘reductive stress’, has been associated with multiple diseases, including disorders of the mitochondrial electron transport chain. As the intracellular NADH:NAD⁺ ratio can be in near equilibrium with the circulating lactate:pyruvate ratio, we hypothesized that reductive stress could be alleviated by oxidizing extracellular lactate to pyruvate. We engineered LOXCAT, a fusion of bacterial lactate oxidase (LOX) and catalase (CAT), which irreversibly converts lactate and oxygen to pyruvate and water. Addition of purified LOXCAT to the medium of cultured human cells with a defective electron transport chain decreased the extracellular lactate:pyruvate ratio, normalized the intracellular NADH:NAD⁺ ratio, upregulated glycolytic ATP production and restored cellular proliferation. In mice, tail-vein-injected LOXCAT lowered the circulating lactate:pyruvate ratio, blunted a metformin-induced rise in blood lactate:pyruvate ratio and improved NADH:NAD⁺ balance in the heart and brain. Our study lays the groundwork for a class of injectable therapeutic enzymes that alleviates intracellular redox imbalances by directly targeting circulating redox-coupled metabolites.