Biochemical characterisation of pivotal enzymes involved in mycobacterial mycolic acid biosynthesis

Tuberculosis (TB) is a life?threatening condition that can last for several years during which patients are debilitated and may disseminate the bacterium that causes the disease, Mycobacterium tuberculosis (Mtb). At least 30 million individuals worldwide will have died from TB in the last decade of the 20th century. In the UK the steady decline in TB cases over the whole of the last century halted in the mid 1980s and there has been alarming signs of increased numbers of cases in certain communities. The situation is compounded by the AIDS epidemic and by the emergence of Mtb strains that are resistant to virtually all the drugs that would normally be used to treat TB. It can be argued that, globally, Mtb is the single most important infectious agent affecting mankind. All bacteria have cells that, like plants, are enclosed in a cell wall. This protects the organism from its immediate environment and, fortuitously, presents an important target for drugs, like penicillin, that can be used to treat bacterial infections. However, Mtb has a distinctive cell wall that differs in composition from that of other bacteria; in particular it contains an exceptional amount of unique lipids (fats) and sugars. Although there are drugs that affect the unique Mtb cell wall, the current treatment for tuberculosis lasts 6 months and is potentially toxic to patients who often cease treatment early. Moreover, the efficacy of treatment is threatened by the emergence of drug-resistant strains of Mtb. There is a great need for new and better drugs to treat TB. The work proposed here therefore fulfils the clear need to extend our understanding of the bacterial physiology of the tubercle bacillus in the hope of priming novel therapeutic approaches to this ancient human adversary.

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Mycolic acids are essential components of the cell envelope of Mycobacterium tuberculosis and access to comprehensive knowledge of the underlying genetic and biochemical profiles of key enzymes in mycolate synthesis is urgently needed. The following pivotal enzymes, identified in our previous studies will be investigated: (a) the search for an ?alternative? Mt-FabH-like enzyme involved in mycolate biosynthesis and the biochemical/structural characterisation of inhibition of beta-ketoacyl synthase (KAS) enzymes by platensimycin and platensin; (b) determination of the Mt-Pks13 crystal structure, in silico screening for KAS inhibitors and the role of Rv3802c as the mycolyltransferase-II involved in mycolic acid biosynthesis; (c) the role of Mt-MycR in regulation of mycolate biosynthesis; (d) identification and characterisation of other target(s) of the anti-mycobacterial drug isoxyl; and (e) the role of FadB homologues in fatty acid/mycolic acid degradation in M. tuberculosis. Full characterisation of the properties and the role of the above enzymes will be a major advance in mycobacterial cell wall assembly.