Arylamine N-acetyltransferase of Mycobacterium marinum

• Main Author: Fullam, Elizabeth
• Published: University of Oxford 2007
• Subjects: 615.1
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.491495

Arylamine N-acetyltransferases (NATs, E.C. 2.1.3.5) are enzymes which are found in both eukaryotic and prokaryotic species and are responsible for catalysing the transfer of an acetyl group from the cofactor acetyl CoA to the terminal nitrogen of arylamine, arylhydrazine and arylhydrazide molecules. NAT has recently been identified in Mycobacterium tuberculosis, the causative agent of TB. When the nat gene is deleted from Mycobacterium bovis BCG (~nat) the phenotypic changes associated with the organism included very low mycolic acid levels, an alteration in the cell wall architecture and the ~nat organism is killed more easily within macrophages. NAT has therefore been identified as a putative target for antitubercular therapy. Mycobacterium marinum is a close relative of M tuberculosis and M marinum infection of zebrafish is increasingly being used as an in vivo model of TB infection, The work in this thesis describes the cloning and expression of the nat gene from M marinum (mmnat), the production and purification of the NAT protein from M marinum (MMNAT) and the characterisation of the enzymic activity of MMNAT. Genetically engineered derivatives of mnmat were created in order to investigate the function of the third domain of the protein and the effect of single-site directed mutants ofMMNAT were also investigated. Structural studies of MMNAT were undertaken and the X-ray structure of the native enzyme was determined at a resolution of 1.9A. Additionally, the crystal structures were determined for MMNAT in complex with the substrate isoniazid at a resolution of 1.9A and MMNAT in complex with CoA at a resolution of 2.4A. The determination of binding site of CoA in MMNAT has shed the first light on cofactor recognition in prokaryotic NAT enzymes and allowed for a direct comparison of the mode of acetyl coenzyme A recognition in mammalian NATs, which differs considerably. Further to these studies, a synthetic chemistry program was undertaken to develop inhibitors of prokaryotic NATs, in particular MMNAT. A high-throughput screen has been carried out previously which identified six compounds that are specific inhibitors of prokaryotic NAT enzymes. Three of the compounds were selected for further investigation in this study. These hit compounds and analogues thereof have been resynthesised and tested for inhibitory activity against the MMNAT protein, which was not available at the time of the highthroughput screen. One compound in particular was found to be more potent against MMNAT (ICso 6J.lM), and also inhibit the growth of M marinum, M bovis BCG and M tuberculosis.