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Biochemical characterization of quinolinic acid phosphoribosyltransferase from Mycobacterium tuberculosis H37Rv and inhibition of its activity by pyrazinamide.

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.
AuthorsHyun Kim, Keigo Shibayama, Emiko Rimbara, Shigetarou Mori
JournalPloS one (PLoS One) Vol. 9 Issue 6 Pg. e100062 ( 2014) ISSN: 1932-6203 [Electronic] United States
PMID24949952 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
Chemical References
  • Enzyme Inhibitors
  • Pyrazinamide
  • pyrazinoic acid
  • Pentosyltransferases
  • nicotinate-nucleotide diphosphorylase (carboxylating)
  • Quinolinic Acid
Topics
  • Enzyme Inhibitors (pharmacology)
  • Kinetics
  • Molecular Docking Simulation
  • Mutagenesis, Site-Directed
  • Mutation
  • Mycobacterium tuberculosis (enzymology)
  • Pentosyltransferases (antagonists & inhibitors, chemistry, genetics, metabolism)
  • Protein Conformation
  • Pyrazinamide (analogs & derivatives, pharmacology)
  • Quinolinic Acid (metabolism)

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