Abstract
Quinolinic acid phosphoribosyltransferase (QAPRTase, EC 2.4.2.19) is a key enzyme in the de novo pathway of nicotinamide adenine dinucleotide (NAD) biosynthesis and a target for the development of new anti-tuberculosis drugs. QAPRTase catalyzes the synthesis of nicotinic acid mononucleotide from quinolinic acid (QA) and 5-phosphoribosyl-1-pyrophosphate (PRPP) through a phosphoribosyl transfer reaction followed by decarboxylation. The crystal structure of QAPRTase from Mycobacterium tuberculosis H37Rv (MtQAPRTase) has been determined; however, a detailed functional analysis of MtQAPRTase has not been published. Here, we analyzed the enzymatic activities of MtQAPRTase and determined the effect on catalysis of the anti-tuberculosis drug pyrazinamide (PZA). The optimum temperature and pH for MtQAPRTase activity were 60°C and pH 9.2. MtQAPRTase required bivalent metal ions and its activity was highest in the presence of Mg2+. Kinetic analyses revealed that the Km values for QA and PRPP were 0.08 and 0.39 mM, respectively, and the kcat values for QA and PRPP were 0.12 and 0.14 [s-1], respectively. When the amino acid residues of MtQAPRTase, which may interact with QA, were substituted with alanine residues, catalytic activity was undetectable. Further, PZA, which is an anti-tuberculosis drug and a structural analog of QA, markedly inhibited the catalytic activity of MtQAPRTase. The structure of PZA may provide the basis for the design of new inhibitors of MtQAPRTase. These findings provide new insights into the catalytic properties of MtQAPRTase.
Highlights
Tuberculosis (TB) is a chronic infectious disease, caused by the intracellular pathogen Mycobacterium tuberculosis, with an estimated 8.7 million cases and 1.4 million deaths each year according to a 2012 World Health Organization (WHO) Report [1]
Expression and purification of MtQAPRTase Full-length nadC from M. tuberculosis H37Rv was inserted into the expression vector pET-30a downstream of the T7 promoter to express a His-tagged recombinant MtQAPRTase
This analysis shows that the Histag could be located outside the active site of MtQAPRTase (Fig. S1), suggesting that the His-tag does not interfere with the activity of MtQAPRTase
Summary
Tuberculosis (TB) is a chronic infectious disease, caused by the intracellular pathogen Mycobacterium tuberculosis, with an estimated 8.7 million cases and 1.4 million deaths each year according to a 2012 World Health Organization (WHO) Report [1]. There is an urgent need for new countermeasures against TB. To address this issue, the aim of the present study was to define the functions of poorly characterized enzymes that may provide targets for designing new drugs to eradicate M. tuberculosis infections. Quinolinic acid phosphoribosyltransferase (QAPRTase; EC 2.4.2.19) is encoded by nadC and is a key enzyme in the de novo pathway of nicotinamide adenine dinucleotide (NAD) biosynthesis [5,6,7]. NAD biosynthesis is essential for cell survival and viability [9], which makes it an attractive target for the development of new antibacterial drugs, with steps shared by de novo and recycling pathways as a source of candidate enzymes for therapeutic intervention [5,10,11,12]
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