Fe-S biogenesis by SMS and SUF pathways: A focus on the assembly step.

FeS clusters are prosthetic groups present in all organisms. Proteins with FeS centers are involved in most cellular processes. ISC and SUF are machineries necessary for the formation and insertion of FeS in proteins.

Structural and Biochemical Characterization of Zinc SufU-SufS Complex.

Iron-sulfur (Fe-S) clusters are inorganic prosthetic groups in proteins composed exclusively of iron and inorganic sulfide. These cofactors are required in a wide range of critical cellular pathways. Iron-sulfur clusters do not form spontaneously in vivo; several proteins are required to mobilize sulfur and iron, assemble and traffic-nascent clusters.

An early origin of iron-sulfur cluster biosynthesis machineries before Earth oxygenation.

Iron-sulfur (Fe-S) clusters are ubiquitous cofactors essential for life. It is largely thought that the emergence of oxygenic photosynthesis and progressive oxygenation of the atmosphere led to the origin of multiprotein machineries (ISC, NIF and SUF) assisting Fe-S cluster synthesis in the presence of oxidative stress and shortage of bioavailable iron. However, previous analyses have left unclear the origin and evolution of these systems.

Cellular assays identify barriers impeding iron-sulfur enzyme activity in a non-native prokaryotic host.

Iron-sulfur (Fe-S) clusters are ancient and ubiquitous protein cofactors and play irreplaceable roles in many metabolic and regulatory processes. Fe-S clusters are built and distributed to Fe-S enzymes by dedicated protein networks. The core components of these networks are widely conserved and highly versatile.

Transient Formation of a Second Active Site Cavity during Quinolinic Acid Synthesis by NadA.

Quinolinate synthase, also called NadA, is a [4Fe-4S]-containing enzyme that uses what is probably the oldest pathway to generate quinolinic acid (QA), the universal precursor of the biologically essential cofactor nicotinamide adenine dinucleotide (NAD). Its synthesis comprises the condensation of dihydroxyacetone phosphate (DHAP) and iminoaspartate (IA), which involves dephosphorylation, isomerization, cyclization, and two dehydration steps. The convergence of the three homologous domains of NadA defines a narrow active site that contains a catalytically essential [4Fe-4S] cluster.

Design of specific inhibitors of quinolinate synthase based on [4Fe-4S] cluster coordination.

Quinolinate synthase (NadA) is a [4Fe-4S] cluster-containing enzyme involved in the formation of quinolinic acid, the precursor of the essential NAD coenzyme. Here, we report the synthesis and activity of derivatives of the first inhibitor of NadA. Using multidisciplinary approaches we have investigated their action mechanism and discovered additional specific inhibitors of this enzyme.

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

The article "Iron-sulfur clusters biogenesis by the SUF machinery: close to the molecular mechanism understanding", written by J. Pérard, Sandrine Ollagnier de Choudens was originally published electronically on the publisher's internet portal (currently SpringerLink) 26 December, 2017 without open access.

Crystallographic Trapping of Reaction Intermediates in Quinolinic Acid Synthesis by NadA.

NadA is a multifunctional enzyme that condenses dihydroxyacetone phosphate (DHAP) with iminoaspartate (IA) to generate quinolinic acid (QA), the universal precursor of the nicotinamide adenine dinucleotide (NAD(P)) cofactor. Using X-ray crystallography, we have (i) characterized two of the reaction intermediates of QA synthesis using a "pH-shift" approach and a slowly reacting Thermotoga maritima NadA variant and (ii) observed the QA product, resulting from the degradation of an intermediate analogue, bound close to the entrance of a long tunnel leading to the solvent medium. We have also used molecular docking to propose a condensation mechanism between DHAP and IA based on two previously published Pyrococcus horikoshi NadA structures.

The ErpA/NfuA complex builds an oxidation-resistant Fe-S cluster delivery pathway.

Fe-S cluster-containing proteins occur in most organisms, wherein they assist in myriad processes from metabolism to DNA repair via gene expression and bioenergetic processes. Here, we used both and methods to investigate the capacity of the four Fe-S carriers, NfuA, SufA, ErpA, and IscA, to fulfill their targeting role under oxidative stress. Likewise, Fe-S clusters exhibited varying half-lives, depending on the carriers they were bound to; an NfuA-bound Fe-S cluster was more stable ( = 100 min) than those bound to SufA ( = 55 min), ErpA ( = 54 min), or IscA ( = 45 min).

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

Iron-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.

Genetic, Biochemical, and Biophysical Methods for Studying FeS Proteins and Their Assembly.

FeS clusters containing proteins are structurally and functionally diverse and present in most organisms. Our understanding of FeS cluster production and insertion into polypeptides has benefited from collaborative efforts between in vitro and in vivo studies. The former allows a detailed description of FeS-containing protein and a deep understanding of the molecular mechanisms catalyzing FeS cluster assembly.

ISCA1 is essential for mitochondrial FeS biogenesis in vivo.

Mammalian A-type proteins, ISCA1 and ISCA2, are evolutionarily conserved proteins involved in iron-sulfur cluster (Fe-S) biogenesis. Recently, it was shown that ISCA1 and ISCA2 form a heterocomplex that is implicated in the maturation of mitochondrial FeS proteins. Here we report that mouse ISCA1 and ISCA2 are FeS-containing proteins that combine all features of Fe-S carrier proteins.

Crystal Structures of Quinolinate Synthase in Complex with a Substrate Analogue, the Condensation Intermediate, and Substrate-Derived Product.

The enzyme NadA catalyzes the synthesis of quinolinic acid (QA), the precursor of the universal nicotinamide adenine dinucleotide (NAD) cofactor. Here, we report the crystal structures of complexes between the Thermotoga maritima (Tm) NadA K219R/Y107F variant and (i) the first intermediate (W) resulting from the condensation of dihydroxyacetone phosphate (DHAP) with iminoaspartate and (ii) the DHAP analogue and triose-phosphate isomerase inhibitor phosphoglycolohydroxamate (PGH). In addition, using the TmNadA K219R/Y21F variant, we have reacted substrates and obtained a crystalline complex between this protein and the QA product.

Structural insights into the Escherichia coli lysine decarboxylases and molecular determinants of interaction with the AAA+ ATPase RavA.

The inducible lysine decarboxylase LdcI is an important enterobacterial acid stress response enzyme whereas LdcC is its close paralogue thought to play mainly a metabolic role. A unique macromolecular cage formed by two decamers of the Escherichia coli LdcI and five hexamers of the AAA+ ATPase RavA was shown to counteract acid stress under starvation. Previously, we proposed a pseudoatomic model of the LdcI-RavA cage based on its cryo-electron microscopy map and crystal structures of an inactive LdcI decamer and a RavA monomer.

Dual activity of quinolinate synthase: triose phosphate isomerase and dehydration activities play together to form quinolinate.

Quinolinate synthase (NadA) is an Fe4S4 cluster-containing dehydrating enzyme involved in the synthesis of quinolinic acid (QA), the universal precursor of the essential coenzyme nicotinamide adenine dinucleotide. The reaction catalyzed by NadA is not well understood, and two mechanisms have been proposed in the literature that differ in the nature of the molecule (DHAP or G-3P) that condenses with iminoaspartate (IA) to form QA. In this article, using biochemical approaches, we demonstrate that DHAP is the triose that condenses with IA to form QA.

Turning Escherichia coli into a Frataxin-Dependent Organism.

Fe-S bound proteins are ubiquitous and contribute to most basic cellular processes. A defect in the ISC components catalyzing Fe-S cluster biogenesis leads to drastic phenotypes in both eukaryotes and prokaryotes. In this context, the Frataxin protein (FXN) stands out as an exception.

Molecular investigation of iron-sulfur cluster assembly scaffolds under stress.

Fe/S biosynthesis is controlled in Escherichia coli by two machineries, the housekeeping ISC machinery and the SUF system that is functional under stress conditions. Despite many in vivo studies showing that SUF is more adapted for Fe/S assembly under stress, no molecular data supporting this concept have been provided so far. This work focuses on molecular studies of key actors in Fe/S assembly, the SufB and IscU scaffolds under oxidative stress and iron limitation.

An integrative computational model for large-scale identification of metalloproteins in microbial genomes: a focus on iron-sulfur cluster proteins.

Metalloproteins represent a ubiquitous group of molecules which are crucial to the survival of all living organisms. While several metal-binding motifs have been defined, it remains challenging to confidently identify metalloproteins from primary protein sequences using computational approaches alone. Here, we describe a comprehensive strategy based on a machine learning approach to design and assess a penalized generalized linear model.

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

Quinolinate 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.

Insights into the function of YciM, a heat shock membrane protein required to maintain envelope integrity in Escherichia coli.

The cell envelope of Gram-negative bacteria is an essential organelle that is important for cell shape and protection from toxic compounds. Proteins involved in envelope biogenesis are therefore attractive targets for the design of new antibacterial agents. In a search for new envelope assembly factors, we screened a collection of Escherichia coli deletion mutants for sensitivity to detergents and hydrophobic antibiotics, a phenotype indicative of defects in the cell envelope.

In vivo [Fe-S] cluster acquisition by IscR and NsrR, two stress regulators in Escherichia coli.

The multi-proteins Isc and Suf systems catalyse the biogenesis of [Fe-S] proteins. Here we investigate how NsrR and IscR, transcriptional regulators that sense NO and [Fe-S] homeostasis, acquire their [Fe-S] clusters under both normal and iron limitation conditions. Clusters directed at the apo-NsrR and apo-IscR proteins are built on either of the two scaffolds, IscU or SufB.

Mammalian frataxin controls sulfur production and iron entry during de novo Fe4S4 cluster assembly.

Iron-sulfur (Fe-S) cluster-containing proteins are essential components of cells. In eukaryotes, Fe-S clusters are synthesized by the mitochondrial iron-sulfur cluster (ISC) machinery and the cytosolic iron-sulfur assembly (CIA) system. In the mammalian ISC machinery, preassembly of the Fe-S cluster on the scaffold protein (ISCU) involves a cysteine desulfurase complex (NFS1/ISD11) and frataxin (FXN), the protein deficient in Friedreich's ataxia.

Molecular organization, biochemical function, cellular role and evolution of NfuA, an atypical Fe-S carrier.

Biosynthesis of iron-sulphur (Fe-S) proteins is catalysed by multi-protein systems, ISC and SUF. However, 'non-ISC, non-SUF' Fe-S biosynthesis factors have been described, both in prokaryotes and eukaryotes. Here we report in vitro and in vivo investigations of such a 'non-ISC, non SUF' component, the Nfu proteins.

Studies of inhibitor binding to the [4Fe-4S] cluster of quinolinate synthase.

Stop for NadA! A [4Fe-4S] enzyme, NadA, catalyzes the formation of quinolinic acid in de novo nicotinamide adenine dinucleotide (NAD) biosynthesis. A structural analogue of an intermediate, 4,5-dithiohydroxyphthalic acid (DTHPA), has an in vivo NAD biosynthesis inhibiting activity in E. coli.