Characterisation of methionine adenosyltransferase from Mycobacterium smegmatis and M. tuberculosis

The gene encoding methionine adenosyltransferase has been cloned from Mycobacterium tuberculosis and the model organism M. smegmatis. Both enzymes retained all amino acids known to be involved in catalysing the reaction. While the M. smegmatis enzyme could be functionally expressed, the M. tuberculosis homologue was insoluble and inactive under a large variety of expression conditions. For the M. smegmatis enzyme, the Vmax for S-adenosylmethionine formation was 1.30 μmol/min/mg protein and the Km for methionine and ATP was 288 μM and 76 μM respectively. In addition, the enzyme was competitively inhibited by 8-azaguanine and azathioprine with a Ki of 4.7 mM and 3.7 mM respectively. Azathioprine inhibited the in vitro growth of M. smegmatis with a minimal inhibitory concentration (MIC) of 500 μM, while the MIC for 8-azaguanine was >1.0 mM.The methionine adenosyltransferase from both organisms had a primary structure very similar those previously characterised in other prokaryotic and eukaryotic organisms. The kinetic properties of the M. smegmatis enzyme were also similar to known prokaryotic methionine adenosyltransferases. Inhibition of the enzyme by 8-azaguanine and azathioprine provides a starting point for the synthesis of higher affinity purine-based inhibitors.Tuberculosis represents one of the world's greatest sources of mortality and morbidity, with approximately 8 million new infections and 2 million deaths per year [1]. The situation regarding the control of tuberculosis has significantly worsened over the last decade, with the spread of strains resistant to multiple antimycobacterial agents. There is a profound need for the identification and development of novel chemotherapeutic compounds against tuberculosis. The characterisation of mycobacterial biochemical pathways aids this process through the identification of enzymes amenable to therapeutic inhibition.Mycobacterium tuberculosis is difficult to kill for a number of reasons. The organism is surrounded