Chapter 17 Type II NADH: quinone oxidoreductases of Plasmodium falciparum and Mycobacterium tuberculosis kinetic and high-throughput assays.

Type II NADH: quinone oxidoreductases (ndh) are flavoenzymes found in a broad range of organisms including plants, fungi, protozoa, and bacteria. The ndh enzymes catalyze the oxidation of NADH with concomitant reduction of quinone (Q). These membrane-bound respiratory enzymes differ from the canonical NADH: dehydrogenase (complex I), because they are not involved in the vectorial transfer of protons across membranes. In Plasmodium falciparum and Mycobacterium tuberculosis, causative agents of malaria and tuberculosis, respectively, ndhs have aroused interest because of the essential role played in maintaining a reduced Q-pool during infection. In this chapter, we present methods for the measurement of steady-state parameters for ndhs from both pathogens, highlighting best practices and caveats. In addition, owing to the interest in ndhs as potential chemotherapeutic targets, we describe a miniaturized endpoint assay that is validated for high-throughput screening (HTS) of chemical libraries.