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

Adenosyl Radical: Reagent and Catalyst in Enzyme Reactions

Adenosine is undoubtedly an ancient biological molecule that is a component of many enzyme cofactors: ATP, FADH, NAD(P)H, and coenzyme A, to name but a few, and, of course, of RNA. Here we present an overview of the role of adenosine in its most reactive form: as an organic radical formed either by homolytic cleavage of adenosylcobalamin (coenzyme B 12 , AdoCbl) or by single-electron reduction of S -adenosylmethionine (AdoMet) complexed to an iron–sulfur cluster. Although many of the enzymes we discuss are newly discovered, adenosine's role as a radical cofactor most likely arose very early in evolution, before the advent of photosynthesis and the production of molecular oxygen, which rapidly inactivates many radical enzymes. AdoCbl-dependent enzymes appear to be confined to a rather narrow repertoire of rearrangement reactions involving 1,2-hydrogen atom migrations; nevertheless, mechanistic insights gained from studying these enzymes have proved extremely valuable in understanding how enzymes generate and control highly reactive free radical intermediates. In contrast, there has been a recent explosion in the number of radical-AdoMet enzymes discovered that catalyze a remarkably wide range of chemically challenging reactions; here there is much still to learn about their mechanisms. Although all the radical-AdoMet enzymes so far characterized come from anaerobically growing microbes and are very oxygen sensitive, there is tantalizing evidence that some of these enzymes might be active in aerobic organisms including humans.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/69165/1/604_ftp.pd

The apicomplexan plastid and its evolution

Protistan species belonging to the phylum Apicomplexa have a non-photosynthetic secondary plastid—the apicoplast. Although its tiny genome and even the entire nuclear genome has been sequenced for several organisms bearing the organelle, the reason for its existence remains largely obscure. Some of the functions of the apicoplast, including housekeeping ones, are significantly different from those of other plastids, possibly due to the organelle’s unique symbiotic origin

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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