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.

Pyrazinamide Is Active against Mycobacterium tuberculosis Cultures at Neutral pH and Low Temperature

“…Later, Zhang and Mitchison (4) described a model for the antimycobacterial effect of PZA, in which extracellular protonation of POA facilitates its reentry into the mycobacteria, causing membrane damage and acidification of the cytoplasm. This model is not regarded as complete, and alternative mechanisms and targets, e.g., ATP depletion (19), inhibition of ribosomal protein S1 (20), aspartate decarboxylase (21), and quinolinic acid phosphoribosyltransferase (22) have been proposed, but the requirement for low pH has hardly ever been questioned. The data presented here, together with the historical data (18) and those of the recent study of Peterson et al (17), where antimicrobial activity of PZA at neutral pH was detected in starved cultures in vitro, clearly demonstrate that an extracellular acidic pH is not a prerequisite.…”

Update of Antitubercular Prodrugs from a Molecular Perspective: Mechanisms of Action, Bioactivation Pathways, and Associated Resistance

“…Outside the cell, in an acidic environment as in macrophages, as mallp ortion of the POA anion is protonated into POA, whichi sr eabsorbed by passive diffusion, bringing protons into the cell and causing cytoplasm acidification. [133] ChemMedChem 2017, 12,1657 -1676 www.chemmedchem.org 2017 Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim As ap rodrug, the active metabolite POA could also disrupt many functions inside the bacterium (Scheme1 1). It is also responsible for the decrease of the membrane potentiala nd collapse of the proton motivef orce that could also affect the membrane transport function.…”

“…Reactioncatalyzed by QAPRTase in the de novo pathway of NAD biosynthesis, adapted from reference [133] (PPi:i norganic pyrophosphate). Reactioncatalyzed by QAPRTase in the de novo pathway of NAD biosynthesis, adapted from reference [133] (PPi:i norganic pyrophosphate).…”

“…[123,[130][131][132] In 2014, Kim et al proposed that PZA or POA disrupts the de novo NAD biosynthesis pathway by targeting the quinolinic acid phosphoribosyl transferase (QAPR-Ta se, encoded by nadC)( Scheme 12). [133] As ap rodrug, the active metabolite POA could also disrupt many functions inside the bacterium (Scheme1 1). Ther ibosomal protein S1 (RpsA), which is involved in the process of trans-translation, was recently identified as an ew target of POA.…”

Pyrazinoic Acid Inhibits Mycobacterial Coenzyme A Biosynthesis by Binding to Aspartate Decarboxylase PanD

“…Largely on the basis of biochemical evidence, two pathways have been proposed to be targeted by POA: fatty acid synthesis via targeting fatty acid synthetase FAS I 12 and NAD + biosynthesis via targeting quinolinic acid phosphoribosyltransferase QAPRTase. 13 Furthermore, the ribosomal protein S1 14 and Rv2783, a protein involved in RNA and DNA metabolism, 15 were proposed as targets. Recent evidence demonstrates that the antimycobacterial activity of PZA/POA is independent of trans -translation and RpsA.…”