Mechanistically Distinct Nonribosomal Peptide Synthetases Assemble the Structurally Related Antibiotics Viomycin and Capreomycin

Tuberculosis (TB) is a worldwide burden for human health. More than 1.8 million people succumbed to TB infection in 2008 alone, and as many as two billion people may be passively infected by Mycobacterium tuberculosis, the causative agent of TB.[1] One of the challenges facing the successful treatment of M. tuberculosis infections is the development of strains that are resistant to many of the antibiotics used in the clinic. Cases of multidrug-resistant TB (MDR-TB), defined as TB caused by a M. tuberculosis strain that is resistant to both rifampin and isoniazid, have been identified in nearly every country surveyed.[1, 2] Extensively drug-resistant TB (XDR-TB), defined as MDR-TB that is also resistant to a flouroquinolone and at least one of three injectables (capreomycin, streptomycin, or amikacin), has spread to more than 45 countries, including the United States.[1, 2] The development and spread of drug-resistant strains of M. tuberculosis has put a high priority on the development of new antituberculosis drugs and the generation of derivatives of known drugs that regain their antibiotic activity against resistant strains. The tuberactinomycin family of antituberculosis drugs are important components of our drug arsenal against drug-resistant M. tuberculosis. The most prominent member of this family is capreomycin (CMN), which is a key drug in the treatment of MDR-TB based on its inclusion on the World Health Organization’s “Model List of Essential Medicines.”[3] Furthermore, if CMN is the injectable that XDR-TB infection is resistant to, it almost guarantees treatment failure.[4] Based on its level of importance in treating drug-resistant forms of TB, it is important that new CMN derivatives be developed that regain activity against resistant M. tuberculosis strains. Synthetic procedures to synthesize derivatives have been difficult.[5, 6] More success has been accomplished by semisynthetic approaches,[7–9] but the derivatives are limited by the functional groups present on the cyclic pentapeptide core. Complementing these approaches with metabolic engineering of the enzymology that produces the cyclic pentapeptide core has the potential of enabling further structural diversification. Our focus is on the metabolic engineering aspect of drug development. Harnessing the full potential of metabolic engineering to generate new drug derivatives requires a complete understanding of how the targeted drug is biosynthesized by the producing organism. To this end, we have focused on understanding tuberactinomycin biosynthesis at the molecular and biochemical level by using CMN and the structural analog viomycin (VIO) as model systems. Our focus on both CMN and VIO was based on our hypothesis that by studying the biosynthesis of two structurally related molecules (Scheme 1), we would gain considerable insights into how the tuberactinomycins are biosynthesized and structurally modified by the natural enzymology. To date, we have sequenced and annotated the CMN and VIO biosynthesis gene clusters,[10, 11] reconstituted CMN and VIO production in the heterologous host Streptomyces lividans,[12] biochemically characterized L-capreomycidine formation,[13] and identified the amino acids activated by each of the adenylation (A) domains of the CMN nonribosomal peptide synthetases (NRPS) and some of the A domains of the VIO NRPS (Scheme 2).[14] Zabriskie and colleagues have also contributed important genetic and biochemical information that enable more refined models of tuberactinomycin biosynthesis to be developed.[15–18] Open in a separate window Scheme 1 Chemical structures of viomycin (VIO), tuberactinamine A (TMN A; des-β-lysine VIO) and the four derivatives that make up capreomycin (CMN). The numbering within the cyclic pentapeptide cores of the antibiotics is used to identify positions noted in the text.