ubiquinone and Tuberculosis

MenJ, annotated as an oxidoreductase, was recently demonstrated to catalyze the reduction (saturation) of a single double bond in the isoprenyl side-chain of mycobacterial menaquinone. This modification was shown to be essential for bacterial survival in J774A.1 macrophage-like cells, suggesting that MenJ may be a conditional drug target in Mycobacterium tuberculosis and other pathogenic mycobacteria. Recombinant protein was expressed in a heterologous host, and the activity was characterized. Although highly regiospecific in vivo, the activity is not absolutely regiospecific in vitro; in addition, the enzyme is not specific for naphthoquinones vs benzoquinones. Coenzyme Q-1 (a benzoquinone, UQ-1) was used as the lipoquinone substrate, and NADH oxidation was followed spectrophotometrically as the activity readout. NADPH could not be substituted for NADH in the reaction mixture. The enzyme contains a FAD binding site that was 72% occupied in the purified recombinant protein. Enzyme activity was maximal at 37 °C and pH 7.0; addition of divalent cations, EDTA, and reducing agents such as dithiothreitol to the reaction mixture had no effect on activity. The addition of detergents did not stimulate activity, and addition of saturating levels of FAD had relatively little effect on the observed kinetic parameters. These properties allowed the development of a facile assay needed to study this potential drug target, which is also amenable to high throughput screening. The K

Topics: Bacterial Proteins; Biosynthetic Pathways; Flavin-Adenine Dinucleotide; Humans; Mycobacterium Infections, Nontuberculous; Mycobacterium smegmatis; Mycobacterium tuberculosis; NAD; Oxidation-Reduction; Oxidoreductases; Recombinant Proteins; Tuberculosis; Ubiquinone; Vitamin K 2

Acetohydroxyacid synthase (AHAS) catalyzes the first step of branched-chain amino acid (BCAA) biosynthesis, a pathway essential to the lifecycle of plants and microorganisms. This enzyme is of high interest because its inhibition is at the base of the exceptional potency of herbicides and potentially a target for the discovery of new antimicrobial drugs. The enzyme has conserved attributes from its predicted ancestor, pyruvate oxidase, such as a ubiquinone-binding site and the requirement for FAD as cofactor. Here, we show that these requirements are linked to the regulation of AHAS, in relationship to its anabolic function. Using various soluble quinone derivatives (

Topics: Acetolactate Synthase; Benzoquinones; Flavin-Adenine Dinucleotide; Humans; Models, Molecular; Mycobacterium tuberculosis; Oxidation-Reduction; Saccharomyces cerevisiae; Tuberculosis; Ubiquinone

Topics: Animals; Cytochromes; Electron Transport; Glycolipids; Liver; Mice; Mycobacterium tuberculosis; Oxidoreductases; Succinate Dehydrogenase; Toxins, Biological; Tuberculosis; Ubiquinone