Structure and mechanism of a key enzyme in M. tuberculosis cell envelope biogenesis

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Mycobacterium tuberculosis (Mtb) is a pathogenic bacterium that causes tuberculosis (TB) and which is currently a major cause of concern for the World Health Organization, due to the huge numbers of humans infected worldwide (about one third of the world's population) and to the proliferation of types (strains) of the bacterium that are widely resistant to existing antibiotics. This is an often inevitable consequence of the overuse of antibiotics, and means that a continual new pipeline of therapeutic drugs must be produced. In the case of Mtb, targeting its complicated fatty acid metabolism pathways and cell wall structure have traditionally been very effective ways of disabling and killing the bacterium. Mtb has a dense outer layer (termed the envelope or wall) which contains a complex cross-linked mixture of carbohydrates and lipids that provide a formidable barrier around the bacterium and that help to protect it from the immune system, and to survive in the human body in the infective state. Understanding how the complicated envelope is constructed could hold the key to combating Mtb, as well as providing interesting new data on the novel biochemistry involved. In this respect, it was shown recently that two different types of drugs (benzothiazinones and dinitrobenzamides) both act on Mtb by inhibiting an enzyme system that is responsible for providing some of the key 'building blocks' for the cell envelope. This system comprises two proteins named DprE1 and DprE2, whose role is to convert a sugar-linked lipid from one conformation to another, to enable it to be used as an 'anchor' by which peculiar long chain fatty acids unique to the mycobacteria (mycolipids) can be attached to the core of the envelope. This becomes a major structural feature of the envelope that is critical for the bacteria to remain viable and to sustain an infective state. However, until recently the DprE1 and DprE2 proteins had proven almost impossible to produce in a soluble form that would be appropriate for studying their 3-dimensional structure and for interrogating their reaction mechanism. In advance of this application, we have overcome these problems by producing soluble DprE1/E2 enzymes cloned from a related bacterium (Mycobacterium smegmatis) and also by producing more stable, soluble forms of Mtb DprE1/E2 by fusing these proteins together at the genetic level. We are now able to make large amounts of the relevant proteins, and in this proposed programme of research we will exploit these breakthroughs to solve the structures of the DprE1/DprE2 proteins (using the technique of X-ray crystallography, where X-ray irradiation of crystals of the target protein produces a specific diffraction pattern that can pinpoint the locations of the component atoms and allow the structure to be built) and will also perform a careful characterization of their mechanisms and their individual roles in the generation of the key 'building blocks' for cell envelope assembly. In this way, we will provide important new information on an enzyme system crucial for the viability of the TB-causing bacterium, enabling further strategies to target this DprE1/E2 system with novel antibiotics.