Location and conformation of pantothenate and its derivatives in Mycobacterium tuberculosis pantothenate kinase: insights into enzyme action

Previous studies of complexes of Mycobacterium tuberculosis PanK (MtPanK) with nucleotide diphosphates and non-hydrolysable analogues of nucleoside triphosphates in the presence or the absence of pantothenate established that the enzyme has dual specificity for ATP and GTP, revealed the unusual movement of ligands during enzyme action and provided information on the effect of pantothenate on the location and conformation of the nucleotides at the beginning and the end of enzyme action. The X-ray analyses of the binary complexes of MtPanK with pantothenate, pantothenol and N-nonylpantothenamide reported here demonstrate that in the absence of nucleotide these ligands occupy, with a somewhat open conformation, a location similar to that occupied by phosphopantothenate in the `end' complexes, which differs distinctly from the location of pantothenate in the closed conformation in the ternary `initiation' complexes. The conformation and the location of the nucleotide were also different in the initiation and end complexes. An invariant arginine appears to play a critical role in the movement of ligands that takes place during enzyme action. The work presented here completes the description of the locations and conformations of nucleoside diphosphates and triphosphates and pantothenate in different binary and ternary complexes, and suggests a structural rationale for the movement of ligands during enzyme action. The present investigation also suggests that N-alkylpantothenamides could be phosphorylated by the enzyme in the same manner as pantothenate.

Mycobacterium tuberculosis mutT1 (Rv2985) and ADPRase (Rv1700) proteins constitute a two-stage mechanism of 8-Oxo-dGTP and 8-Oxo-GTP detoxification and adenosine to cytidine mutation avoidance

Approximately one third of the world population is infected with Mycobacterium tuberculosis, the causative agent of tuberculosis. A better understanding of the pathogen biology is crucial to develop new tools/strategies to tackle its spread and treatment. In the host macrophages, the pathogen is exposed to reactive oxygen species, known to damage dGTP and GTP to 8-oxo-dGTP and 8-oxo-GTP, respectively. Incorporation of the damaged nucleotides in nucleic acids is detrimental to organisms. MutT proteins, belonging to a class of Nudix hydrolases, hydrolyze 8-oxo-G nucleoside triphosphates/diphosphates to the corresponding nucleoside monophosphates and sanitize the nucleotide pool. Mycobacteria possess several MutT proteins. However, a functional homolog of Escherichia coli MutT has not been identified. Here, we characterized MtuMutT1 and Rv1700 proteins of M. tuberculosis. Unlike other MutT proteins, MtuMutT1 converts 8-oxo-dGTP to 8-oxo-dGDP, and 8-oxo-GTP to 8-oxo-GDP. Rv1700 then converts them to the corresponding nucleoside monophosphates. This observation suggests the presence of a two-stage mechanism of 8-oxo-dGTP/8-oxo-GTP detoxification in mycobacteria. MtuMutT1 converts 8-oxo-dGTP to 8-oxo-dGDP with a K-m of similar to 50 mu M and V-max of similar to 0.9 pmol/min per ng of protein, and Rv1700 converts 8-oxo-dGDP to 8-oxo-dGMP with a K-m of similar to 9.5 mu M and V-max of similar to 0.04 pmol/min per ng of protein. Together, MtuMutT1 and Rv1700 offer maximal rescue to E. coli for its MutT deficiency by decreasing A to C mutations (a hallmark of MutT deficiency). We suggest that the concerted action of MtuMutT1 and Rv1700 plays a crucial role in survival of bacteria against oxidative stress.