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ANR funded project

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


NADBIO


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

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

Understand and inhibit synthase quinolinate
The nicotinamide adenosine dinucleotide (NAD) is synthesized in all living organisms from quinolinic acid (QA). However, the biosynthesis of QA differs between organisms. In eukaryotes it is produced via the degradation of tryptophan whereas in prokaryotes it is synthesized via two enzymes: L-aspartate oxidase (NadB) and quinolinate synthase (NadA),
a 4Fe-4S enzyme. In addition to the de novo pathway, most organisms possess a salvage pathway for synthesizing NAD from metabolites. In the pathogenic Mycobacterium leprae and Helicobacter pylori, this escape route is not present, making NadA an essential enzyme in these
bacteria and therefore a good target for the development of new antibacterial agents. There is no inhibitor (s) of NadA available and the molecular mechanism of QA formation is not known. The objectives of the NADBIO project were (1) to understand at a molecular level the
enzymatic mechanism NadA (including the intermediate species and the role of the iron-sulfur center in catalysis); (2) to find molecules to study the molecular mechanism and also to inhibit the enzyme in order to develop antibacterial against H. pylori and M. leprae and (3) to obtain the 3-dimensional structure of NadA with its Fe-S cluster.

A multidisciplinary approach
Substrates and intermediate analogues were used as probes to dissect the reaction mechanism. The role of the central Fe-S in catalysis as the Lewis acid was evaluated by combining spectroscopy Mössbauer and EPR using natural substrates of NadA and their analogues. The structure of NadA with its Fe-S, which provided information about the active site structure, and, consequently, shed light on the mechanism and design of inhibitors was obtained by Xray crystallography. The technique of high throughput screening on an E. coli strain was used to find molecules with an anti-NadA in vivo activity. In parallel, we also used a rational approach using substrates and intermediates analogues. Utilization of these various
approaches (chemistry, biochemistry, crystallography, spectroscopy and biology) allowed us to better understand the NAD biosynthetic pathway in prokaryotes and to design new anti-NadA antibacterial agents.

Results

1- Identification of the first inhibitor of quinolinate synthase: 4,5- dithiohydroxy phthalic acid (DTHPA). DTHPA is a good inhibitor of the quinolinate synthase (NadA) both in vitro and in vivo on bacteria (Publication 1). Identification also of DTHPA derivatives as good inhibitors
of NadA.
2- First NadA X-ray crystal structure in its active form (with its Fe-S center) at 1.6 Å resolution (Publication 2).
3- Mechanistic studies of QA formation: Elimination of one of the proposed mechanisms and demonstration that QA is formed by direct condensation of DHAP with IA (Publication 3). Crystal structure of a complex between a NadA active site variant and an intermediate resulting from the condensation of DHAP with IA (Publication 4).

Outlook

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Scientific outputs and patents

1- Studies of inhibitor binding to the [4Fe-4S] cluster of quinolinate synthase.
Chan A, Clémancey M, Mouesca JM, Amara P, Hamelin O, Latour JM, Ollagnier de Choudens
S. Angew Chem Int Ed Engl. 2012; 51:7711-4.
2- The crystal structure of Fe4S4 quinolinate synthase unravels an enzymatic dehydration mechanism that uses tyrosine and a hydrolase-type triad. Cherrier MV, Chan A, Darnault C, Reichmann D, Amara P, Ollagnier de Choudens S, Fontecilla-Camps JC. J Am Chem Soc. 2014 Apr 9; 136(14):5253-6.
3- Dual Activity of Quinolinate Synthase: Triose Phosphate Isomerase and Dehydration Activities Play Together To Form Quinolinate.Reichmann D, Couté Y, Ollagnier de Choudens S. Biochemistry. 2015 Oct 27; 54(42):6443-6.
4- Crystal Structures of Quinolinate Synthase in Complex with a Substrate analog, its First Condensation Intermediate and Substrate-derived Product.
Volbeda A, Darnault C, Renoux O, Reichmann D, Amara P, Ollagnier de Choudens S, C. Fontecilla-Camps J. J Am Chem Soc. 2016 (under revision).
Book: Fe4S4 Quinolinate Synthase (NadA).Cherrier MV, Ollagnier de Choudens S, Fontecilla-Camps JC Encyclopedia of Inorganic and Bioinorganic Chemistry 2016 (in press).

Partners

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

IBS/METALLO Institut de Biologie Structurale/ Groupe Métalloprotéines

INAC/SCIB Institut Nanosciences et Cryogénie

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

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

Submission abstract

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.

 

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.