Abstract
Tuberculosis (TB), which is caused by the pathogen Mycobacterium tuberculosis (Mtb) has rapidly acquired drug resistance to many front-line antibiotics. This has led to the World Health Organisation declaring TB a public health crisis. The proline metabolism pathway has recently come into light as a putative drug target against Mtb. Four out of the five proline biosynthetic enzymes found in Mtb are entirely essential for the survival of the pathogen. Proline dehydrogenase (ProDH) and pyrroline-5- carboxylate reductase (P5CR) are two enzymes that are not well characterised in Mtb and are involved with the formation and utilisation of pryrroline-5-carboxylate (P5C) respectively. It has been suggested that Mtb requires this intermediate of proline metabolism, P5C to neutralise methylglyoxal, an intracellular cytotoxin. By dissecting the biophysical properties of these two enzymes, there is potential for an insight into the enzymes’ role in proline and methylglyoxal biochemistry, potentially giving rise to new methods to treat latent TB. Transforming the overexpression cell line (Mycobacterium smegmatis mc2 4517) with a plasmid bearing either P5CR or ProDH led to the production of soluble ProDH at 28 ⁰C and P5CR at 37 ⁰C in as little as six hours. To date, we have achieved partially purified preparations of ProDH and preparations of P5CR at more than 90 % purity. Following expression, P5CR was successfully crystallised, and preliminary diffraction studies revealed some diffraction patterns, extending to 6.33 Å. Kinetic studies of P5CR show that its Km for P5C is 55.26 (± 21.5) μM and its kcat for P5C is 0.133 s-1.