RESUMEN
The addition or removal of hydroxy groups modulates the activity of many pharmacologically active biomolecules. It can be integral to the basic biosynthetic factory or result from associated tailoring steps. For the anti-MRSA antibiotic mupirocin, removal of a C8-hydroxy group late in the biosynthetic pathway gives the active pseudomonic acidâ A. An extra hydroxylation, at C4, occurs in the related but more potent antibiotic thiomarinolâ A. We report here in vivo and in vitro studies that show that the putative non-haem-iron(II)/α-ketoglutaratedependent dioxygenase TmuB, from the thiomarinol cluster, 4-hydroxylates various pseudomonic acids whereas C8-OH, and other substituents around the tetrahydropyran ring, block enzyme action but not substrate binding. Molecular modelling suggested a basis for selectivity, but mutation studies had a limited ability to rationally modify TmuB substrate specificity. 4-Hydroxylation had opposite effects on the potency of mupirocin and thiomarinol. Thus, TmuB can be added to the toolbox of polyketide tailoring technologies for the in vivo generation of new antibiotics in the future.
Asunto(s)
Antibacterianos/farmacología , Oxigenasas de Función Mixta/antagonistas & inhibidores , Sintasas Poliquetidas/efectos de los fármacos , Antibacterianos/química , Hidroxilación , Sintasas Poliquetidas/metabolismo , Especificidad por SustratoRESUMEN
The spread of antibiotic resistance is a major challenge for the treatment of Mycobacterium tuberculosis infections. In addition, the efficacy of drugs is often limited by the restricted permeability of the mycomembrane. Frontline antibiotics inhibit mycomembrane biosynthesis, leading to rapid cell death. Inspired by this mechanism, we exploited ß-lactones as putative mycolic acid mimics to block serine hydrolases involved in their biosynthesis. Among a collection of ß-lactones, we found one hit with potent anti-mycobacterial and bactericidal activity. Chemical proteomics using an alkynylated probe identified Pks13 and Ag85 serine hydrolases as major targets. Validation through enzyme assays and customized 13 C metabolite profiling showed that both targets are functionally impaired by the ß-lactone. Co-administration with front-line antibiotics enhanced the potency against M. tuberculosis by more than 100-fold, thus demonstrating the therapeutic potential of targeting mycomembrane biosynthesis serine hydrolases.