Temperature stability of proteins essential for the intracellular survival of Mycobacterium tuberculosis
Résumé
In Mycobacterium tuberculosis the genes hsaD (2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid hydrolase) and nat (arylamine N-acetyltransferase) are essential for survival inside of host macrophages. These genes act as an operon and have been suggested to be involved in cholesterol metabolism. However, the role of NAT in this catabolic pathway has not been determined. In an effort to better understand the function of these proteins, we have expressed, purified and characterized TBNAT and HsaD from M. tuberculosis. Both proteins demonstrated remarkable heat stability with TBNAT and HsaD retaining >95% of their activity following incubation at 60ºC for 30 min. The first and second domain of TBNAT was demonstrated to be very important to the heat stability of the protein, as the transfer of these domains caused a dramatic reduction in the heat stability. The specific activity of TBNAT was tested against a broad range of acyl CoA cofactors using hydralazine as a substrate. TBNAT was found to be able to utilize not just acetyl CoA, but also n-propionyl CoA and acetoacetyl CoA, though at a slower rate. As propionyl CoA is a product of cholesterol catabolism, we propose NAT could have a role in the utilization of this important cofactor.
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