Quinolinate synthase, an iron-sulfur enzyme in NAD biosynthesis

International audienceNicotinamide adenine dinucleotide (NAD) plays a crucial role as a cofactor in numerous essential redox biological reactions. NAD derives from quinolinic acid which is synthesized in E. coli from L-aspartate and dihydroxyacetone phosphate (DHAP) as the result of the concerted action of two enzymes, L-aspartate oxidase (NadB) and quinolinate synthetase (NadA). We report here the characterization of NadA protein from E. coli. When anaerobically purified, the isolated soluble protein contains 3-3.5 iron and 3-3.5 sulfide/ polypeptide chain. Mössbauer spectra of the 57Fe-protein revealed that the majority of the iron is in the form of a (4Fe-4S)2+ cluster. An enzymatic assay for quinolinate synthetase activity was set up and allowed to demonstrate that the cluster is absolutely required for NadA activity. Exposure to air leads to degradation of the cluster and inactivate enzyme

Direct repair of a synthetic 5S-configured spore photoproduct by a spore photoproduct lyase.

International audienceThe spore photoproduct lyase is a Fe-S/AdoMet DNA repair enzyme, which directly repairs spore lesions, induced by UV irradiation of spores, using an unknown radical mechanism. The air sensitive radical SAM enzyme was for the first time challenged with synthetically pure substrates. It was found that the enzyme recognizes a synthetic 5S-configured spore lesion without the central phosphodiester bond. The 5R-configured lesion is in contrast to current belief not a substrate

The crystal structure of Fe₄S₄ quinolinate synthase unravels an enzymatic dehydration mechanism that uses tyrosine and a hydrolase-type triad.

International audienceQuinolinate synthase (NadA) is a Fe4S4 cluster-containing dehydrating enzyme involved in the synthesis of quinolinic acid (QA), the universal precursor of the essential nicotinamide adenine dinucleotide (NAD) coenzyme. A previously determined apo NadA crystal structure revealed the binding of one substrate analog, providing partial mechanistic information. Here, we report on the holo X-ray structure of NadA. The presence of the Fe4S4 cluster generates an internal tunnel and a cavity in which we have docked the last precursor to be dehydrated to form QA. We find that the only suitably placed residue to initiate this process is the conserved Tyr21. Furthermore, Tyr21 is close to a conserved Thr-His-Glu triad reminiscent of those found in proteases and other hydrolases. Our mutagenesis data show that all of these residues are essential for activity and strongly suggest that Tyr21 deprotonation, to form the reactive nucleophilic phenoxide anion, is mediated by the triad. NadA displays a dehydration mechanism significantly different from the one found in archetypical dehydratases such as aconitase, which use a serine residue deprotonated by an oxyanion hole. The X-ray structure of NadA will help us unveil its catalytic mechanism, the last step in the understanding of NAD biosynthesis

The lipoate synthase from Escherichia coli is an iron-sulfur protein

AbstractLipoate synthase catalyzes the last step of the biosynthesis of lipoic acid in microorganisms and plants. The protein isolated from an overexpressing Escherichia coli strain was purified from inclusion bodies. Spectroscopic (UV-visible and electron paramagnetic resonance) properties of the reconstituted protein demonstrate the presence of a (2Fe-2S) center per protein. As observed in biotin synthase, these clusters are converted to (4Fe-4S) centers during reduction under anaerobic conditions. The possible involvement of the cluster in the insertion of sulfur atoms into the octanoic acid backbone is discussed

Iron sulfur Cluster in Bacteria: Mechanism of assembly and transfer

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Iron-Sulfur Clusters toward Stresses: Implication for Understanding and Fighting Tuberculosis

International audienceTuberculosis (TB) remains the leading cause of death due to a single pathogen, accounting for 1.5 million deaths annually on the global level. Mycobacterium tuberculosis, the causative agent of TB, is persistently exposed to stresses such as reactive oxygen species (ROS), reactive nitrogen species (RNS), acidic conditions, starvation, and hypoxic conditions, all contributing toward inhibiting bacterial proliferation and survival. Iron-sulfur (Fe-S) clusters, which are among the most ancient protein prosthetic groups, are good targets for ROS and RNS, and are susceptible to Fe starvation. Mtb holds Fe-S containing proteins involved in essential biological process for Mtb. Fe-S cluster assembly is achieved via complex protein machineries. Many organisms contain several Fe-S assembly systems, while the SUF system is the only one in some pathogens such as Mtb. The essentiality of the SUF machinery and its functionality under the stress conditions encountered by Mtb underlines how it constitutes an attractive target for the development of novel anti-TB

Iron-sulfur clusters biogenesis by the SUF machinery: close to the molecular mechanism understanding.

International audienceIron-sulfur clusters (Fe-S) are amongst the most ancient and versatile inorganic cofactors in nature which are used by proteins for fundamental biological processes. Multiprotein machineries (NIF, ISC, SUF) exist for Fe-S cluster biogenesis which are mainly conserved from bacteria to human. SUF system (sufABCDSE operon) plays a general role in many bacteria under conditions of iron limitation or oxidative stress. In this mini-review, we will summarize the current understanding of the molecular mechanism of Fe-S biogenesis by SUF. The advances in our understanding of the molecular aspects of SUF originate from biochemical, biophysical and recent structural studies. Combined with recent in vivo experiments, the understanding of the Fe-S biogenesis mechanism considerably moved forward