The goal of this research is to develop novel, more effective therapies for the treatment of tuberculosis, including multidrug-resistant tuberculosis and related atypical mycobacterial infections. The complex cell wall of these gram-positive actinomycetes is their most characteristic feature and its biosynthesis is the target of some of the most effective antimycobacterial agents. A better understanding of the biochemical transformations used in cell wall elaboration will allow us to design novel interventions through the use of the tools of rational drug design and combinatorial chemistry. This project has focused on understanding the post-fatty acid synthetic modifications which occur to lipids comprising the outermost layer of the mycobacterial cell wall, the mycolic acids. These lipids are alpha-branched, beta-hydroxy fatty acids that are unique to mycobacteria which are heavily modified by a variety of functional groups. Modification reactions of mycolic acids are essential to proper functioning of the cell wall as a permeability barrier and as a mediator of mycobacteria-host cell interactions. A certain subset of these modifications are unique to the pathogenic mycobacteria and we have shown that these are associated with various pathogenic properties of these bacteria (for example, the ability to withstand an oxidative burst). We have identified a family of six highly-related enzymes which are responsible for introducing these modifications and characterized their function. Surprisingly all of these enzymes appear to utilize a common chemical mechanism involving initial cation formation following methyl group addition to an olefinic mycolate precursor. This common intermediate offers a uniquely vulnerable target for intervention since inhibiting it would affect six enzymes simultaneously, making the emergence of drug resistance very unlikely. We have cloned, overexpressed, and purified the enzymes which catalyze these reactions, characterized their activity in vitro and begun characterizing the substrate, product, and acyl carrier involved in these reactions. These studies have led to a detailed picture of how mycolic acids are biosynthesized and the point at which the modification reactions take place, both previously unknown. Our future studies will use these enzymes, the corresponding in vitro assays, and the precise structures of the chemical products and substrates to guide the design of novel chemotherapeutic agents.
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