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ANR funded project

Blanc - SIMI 7 - Chimie moléculaire, organique, de coordination, catalyse et chimie biologique (Blanc SIMI 7) 2012
Projet NADBIO

Quinolinate synthase, an iron-sulfur protein as a key target for the design of antibacterial agents

Nicotinamide adenine dinucleotide (NAD) plays a crucial role as a cofactor in numerous essential redox biological reactions. In fact, in all living organisms, NAD derives from quinolinic acid, the biosynthetic pathway of which differs among organisms. In most eukaryotes, quinolinic acid (QA) is produced via the degradation of tryptophan. Alternatively, in pathogenic bacteria such as Mycobacterium leprae and Helicobacter pylori or in the opportunistic bacteria Escherichia coli, quinolinic acid is synthesized via an unique condensation reaction between iminoaspartate and dihydroxyacetone phosphate as the result of the concerted action of two enzymes, L-aspartate oxidase encoded by the nadB gene, and quinolinate synthase, encoded by the nadA gene. Besides the de novo synthesis of NAD, a salvage pathway may exist that enables NAD to be recycled from diverse metabolites. M. leprae and H. pylori, pathogens causing leprosy and certain stomach cancers, respectively, do not have a salvage pathway and thus cannot recycle NAD. The presence of different pathways for the biosynthesis of quinolinic acid in most prokaryotes and eukaryotes, in addition to the absence of the salvage pathway in some microorganisms, make of NadA a novel target for the development of new specific antibacterial drugs. So far no quinolinate synthase inhibitors have been described. NadA is a universal metalloenzyme containing a 4Fe/4S cluster coordinated by three cysteine residues that is thought to play an essential role in catalysis. The formation of quinolinic acid, the precursor to the pyridine ring of NAD, is a long-standing unsolved problem in biosynthesis of cofactors like thiamin, molybdopterin, pyridoxal phosphate and vitamin B12. Indeed, the reaction catalyzed by NadA constitutes the only step whose mechanism is unknown in the NAD biosynthetic pathway. Two mechanisms that account for the synthesis of quinolinic acid have been advanced in the literature but neither one has been tested. In addition, the role of the 4Fe/4S in the catalysis has not been demonstrated. There are two possible explanations: the complexity of the reaction, which is bi-molecular, and the oxygen sensitivity of the 4Fe/4S cluster. Unravel the mechanism of the reaction catalyzed by NadA thus constitutes a real challenge in biological chemistry. The goal of this project is exactly to understand at a molecular level the enzymatic mechanism of NadA and to identify molecules that will allow its study and inhibition. This will be rendered possible by a permanent exchange of information between chemists, biochemists, biophysicists, microbiologists, crystallographers and technologues. More precisely, substrate and intermediate analogs will be used as molecular probes for the study of the mechanism. We will take advantage of the ability of these molecules to inhibit NadA in vitro to design antibacterial agents. The role of the Fe/S cluster as a Lewis acid in the catalysis will be investigated by classical and advanced EPR spectroscopy as well as Mössbauer spectroscopy using either natural substrates or analogs. We will attempt to solve the crystal structure of NadA containing its Fe/S center in order to identify the catalytic amino acids at the active site and postulate a reaction mechanism. This approach should also lead to the design of inhibitors. Still, a high-throughput screening method will be used as the main approach to identify molecules displaying anti-NadA activity. Inhibitory activity of interesting molecules will be confirmed in vitro on NadA from M. leprae and H. pylori and then assayed in vivo on these pathogens in collaboration with the Institut Pasteur in Paris and the Ecole Polytechnique Fédérale de Lausanne in Switzerland. We expect from this project to fully understand the NAD biosynthetic pathway in bacteria, through formation of quinolinic acid, and to develop new antibacterial agents against NadA.

Partners

IBS/METALLO Institut de Biologie Structurale/ Groupe Métalloprotéines

iRTSV/CBM/PMB Institut de Recherche en Technologies et Sciences pour le vivant/Laboratoire de Chimie et Biologie des Métaux (PMB)

INAC/SCIB Institut Nanosciences et Cryogénie

CEA/CNRS/UJF Laboratoire de Chimie et Biologie des Métaux (BioCat)

ANR grant: 550 000 euros
Beginning and duration: janvier 2013 - 36 mois

 

ANR Programme: Blanc - SIMI 7 - Chimie moléculaire, organique, de coordination, catalyse et chimie biologique (Blanc SIMI 7) 2012

Project ID: ANR-12-BS07-0018

Project coordinator:
Madame Sandrine Ollagnier-de Choudens (Laboratoire de Chimie et Biologie des Métaux (BioCat))
sollagnier@nullcea.fr

 

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The project coordinator is the author of this abstract and is therefore responsible for the content of the summary. The ANR disclaims all responsibility in connection with its content.