Investigation of ( S)-(-)-Acidomycin: A Selective Antimycobacterial Natural Product That Inhibits Biotin Synthase.

The synthesis, absolute stereochemical configuration, complete biological characterization, mechanism of action and resistance, and pharmacokinetic properties of ( S)-(-)-acidomycin are described. Acidomycin possesses promising antitubercular activity against a series of contemporary drug susceptible and drug-resistant M. tuberculosis strains (minimum inhibitory concentrations (MICs) = 0.096-6.2 µM) but is inactive against nontuberculosis mycobacteria and Gram-positive and Gram-negative pathogens (MICs > 1000 µM). Complementation studies with biotin biosynthetic pathway intermediates and subsequent biochemical studies confirmed acidomycin inhibits biotin synthesis with a Ki of approximately 1 µM through the competitive inhibition of biotin synthase (BioB) and also stimulates unproductive cleavage of S-adenosyl-l-methionine (SAM) to generate the toxic metabolite 5'-deoxyadenosine. Cell studies demonstrate acidomycin selectively accumulates in M. tuberculosis providing a mechanistic basis for the observed antibacterial activity. The development of spontaneous resistance by M. tuberculosis to acidomycin was difficult, and only low-level resistance to acidomycin was observed by overexpression of BioB. Collectively, the results provide a foundation to advance acidomycin and highlight BioB as a promising target.

Purification, Characterization, and Biochemical Assays of Biotin Synthase From Escherichia coli.

Biotin synthase (BioB) catalyzes the oxidative insertion of a sulfur atom between the C6 methylene and the C9 methyl positions in dethiobiotin. The enzyme couples oxidation of each carbon position to reduction of the S-adenosyl-l-methionine (SAM) sulfonium center, generating 5'-deoxyadenosine and l-methionine, products that are characteristic of enzymes from the radical SAM superfamily. In bacteria, biotin biosynthesis is tightly regulated by the dual-function BirA repressor/holocarboxylase synthetase, resulting in very low levels of all biotin biosynthetic enzymes such that activity-based purification of BioB from the native organism is virtually impossible. However, overexpression and purification of recombinant BioB from E. coli are straight forward and, in contrast with many radical SAM enzymes, can be carried out under aerobic conditions. The active enzyme contains two iron-sulfur clusters, and the characterization and manipulation of these clusters are essential for a thorough understanding of enzyme catalysis and stability. An optimized in vitro assay for BioB is described herein that requires use of an auxiliary protein reducing system and must be carried out under anaerobic conditions to prevent oxidative damage to the reduced iron-sulfur clusters. Three methods for detection of biotin are described, with discussion of the advantages and limitations of each method. Challenges that may be encountered in adapting these assays to other organisms are also discussed.