Ricerc@Sapienza

Mycobacterium tuberculosis is the world's most deadly bacterial pathogen. In 2015, the World Health Organization (WHO) reported
that more than 9 million people were newly diagnosed with the disease, and that 1.6 million people died from Tuberculosis (TB). The
recent emergence of multi-drug resistant (MDR) strains of M. tuberculosis requires patients diagnosed with MDR-TB to be treated with second-line, toxic drugs for 20 months. Even so, almost half of MDR-TB patients died last year (190,000 of 480,000 patients). The need for new drugs to treat TB is a clear global priority.
Recently, it was shown that M. tuberculosis lacks a complete TCA cycle; specifically that the gene annotated as alpha-ketoglutarate
decarboxylase does not possess that activity. It was suggested instead that alpha-ketoglutarate was converted to L-glutamate and then
to gamma-aminobutyric acid (GABA), and finally to succinate semialdehyde by glutamate decarboxylase (Gad) and GABA
transaminase, respectively. Orthologues of each of these enzymes have been identified in the genome of M. tuberculosis, but none
have been expressed or functionally characterized. The only of the "GABA shunt" enzymes to have been enzymatically characterized
is succinate semialdehyde dehydrogenase, which generates succinate, the TCA cycle intermediate, and NADH.
All L-glutamate decarboxylases characterized so far contain the organic cofactor pyridoxal phosphate (PLP) to which the substrate
covalently binds and is subsequently chemically converted to product. We propose to heterologously express M. tuberculosis Gad,
purify the protein to homogeneity, determine the chemical state of bound PLP spectroscopically and spectrofluorimetrically, assess the
pH-dependent enzymatic activity and its oligomeric state.
This knowledge will provide a platform for the later examination of potential inhibitors of the enzyme as a "druggable target" in M.
tuberculosis.