Mechanisms of iron–sulfur cluster assembly: the SUF machinery

Biosynthesis of iron-sulfur clusters is a cellular process which depends on complex protein machineries. Escherichia coli contains two such biosynthetic systems, ISC and SUF. In this review article we specifically make a presentation of the various Suf proteins and discuss the molecular mechanisms by which these proteins work together to assemble Fe and S atoms within a scaffold and to transfer the resulting cluster to target proteins. An erratum to this article can be found at     

Mycobacterium tuberculosis requires SufT for Fe-S cluster maturation,metabolism, and survival in vivo

Iron-sulfur (Fe-S) cluster proteins carry out essential cellular functions in diverse organisms, including the human pathogen Mycobacterium tuberculosis (Mtb). The mechanisms underlying Fe-S cluster biogenesis are poorly defined in Mtb. Here, we show that Mtb SufT (Rv1466), a DUF59 domain-containing essential protein, is required for the Fe-S cluster maturation. Mtb SufT homodimerizes and interacts with Fe-S cluster biogenesis proteins; SufS and SufU. SufT also interacts with the 4Fe-4S cluster containing proteins; aconitase and SufR. Importantly, a hyperactive cysteine in the DUF59 domain mediates interaction of SufT with SufS, SufU, aconitase, and SufR. We efficiently repressed the expression of SufT to generate a SufT knock-down strain in Mtb (SufT-KD) using CRISPR interference. Depleting SufT reduces aconitase’s enzymatic activity under standard growth conditions and in response to oxidative stress and iron limitation. The SufT-KD strain exhibited defective growth and an altered pool of tricarboxylic acid cycle intermediates, amino acids, and sulfur metabolites. Using Seahorse Extracellular Flux analyzer, we demonstrated that SufT depletion diminishes glycolytic rate and oxidative phosphorylation in Mtb. The SufT-KD strain showed defective survival upon exposure to oxidative stress and nitric oxide. Lastly, SufT depletion reduced the survival of Mtb in macrophages and attenuated the ability of Mtb to persist in mice. Altogether, SufT assists in Fe-S cluster maturation and couples this process to bioenergetics of Mtb for survival under low and high demand for Fe-S clusters.     

Protein splicing of SufB is crucial for the functionality of the Mycobacterium tuberculosis SUF machinery

The SufBCD complex is an essential component of the SUF machinery of [Fe-S] cluster biogenesis in many organisms. We show here that in Mycobacterium tuberculosis the formation of this complex is dependent on the protein splicing of SufB, suggesting that this process is a potential new target for antituberculous drugs.     

Identification of a Mycobacterium tuberculosis gene that enhances mycobacterial survival in macrophages

Intracellular survival plays a central role in the pathogenesis of Mycobacterium tuberculosis. To identify M. tuberculosis genes required for intracellular survival within macrophages, an M. tuberculosis H37Rv plasmid library was constructed by using the shuttle vector pOLYG. This plasmid library was electroporated into Mycobacterium smegmatis 1-2c, and the transformants were used to infect the human macrophage-like cell line U-937. Because M. smegmatis does not readily survive within macrophages, any increased intracellular survival is likely due to cloned M. tuberculosis H37Rv DNA. After six sequential passages of M. smegmatis transformants through U-937 cells, one clone (p69) was enriched more than 70% as determined by both restriction enzyme and PCR analyses. p69 demonstrated significantly enhanced survival compared to that of the vector control, ranging from 2.4- to 5.3-fold at both 24 and 48 h after infection. DNA sequence analysis revealed three open reading frames (ORFs) in the insert of p69. ORF2 (1.2 kb) was the only one which contained a putative promoter region and a ribosome-binding site. Deletion analysis of the p69 insert DNA showed that disruption of ORF2 resulted in complete loss of the enhanced intracellular survival phenotype. This gene was named the enhanced intracellular survival (eis) gene. By using an internal region of eis as a probe for Southern analysis, eis was found in the genomic DNA of various M. tuberculosis strains and of Mycobacterium bovis BCG but not in that of M. smegmatis or 10 other nonpathogenic mycobacterial species. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis showed that all M. smegmatis eis-containing constructs expressed a unique protein of 42 kDa, the predicted size of Eis. The expression of this 42-kDa protein directly correlated to the enhanced survival of M. smegmatis p69 in U-937 cells. These results suggest a possible role for eis and its protein product in the intracellular survival of M. tuberculosis.     

SufA/IscA: reactivity studies of a class of scaffold proteins involved in [Fe-S] cluster assembly

IscA/SufA proteins belong to complex protein machineries which are involved in iron-sulfur cluster biosynthesis. They are defined as scaffold proteins from which preassembled clusters are transferred to target apoproteins. The experiments described here demonstrate that the transfer reaction proceeds in two observable steps: a first fast one leading to a protein–protein complex between the cluster donor (SufA/IscA) and the acceptor (biotin synthase), and a slow one consisting of cluster transfer leading to the apoform of the scaffold protein and the holoform of the target protein. Mutation of cysteines in the acceptor protein specifically inhibits the second step of the reaction, showing that these cysteines are involved in the cluster transfer mechanism but not in complex formation. No cluster transfer from IscA to IscU, another scaffold of the isc operon, could be observed, whereas IscU was shown to be an efficient cluster source for cluster assembly in IscA. Implications of these results are discussed.Abbreviations AdoMet S-adenosylmethionine - APS adenosine-5-phosphosulfate - BioB biotin synthase - DAF deazaflavin - DTB dethiobiotin - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - _hisIscU/A six histidine residues at the N-terminus of IscU/A - PCR polymerase chain reaction - PLP pyridoxal 5-phosphate - SufA_his six histidine residues at the C-terminus of SufA     

Siderophore-controlled iron assimilation in the enterobacterium Erwinia chrysanthemi: evidence for the involvement of bacterioferritin and the Suf iron-sulfur cluster assembly machinery

The intracellular fate of iron acquired by bacteria during siderophore-mediated assimilation is poorly understood. We investigated this question in the pathogenic enterobacterium Erwinia chrysanthemi. This bacterium produces two siderophores, chrysobactin and achromobactin, during plant infection. We analyzed the distribution of iron into cytosolic proteins in bacterial cells supplied with 59Fe-chrysobactin using native gel electrophoresis. A parental strain and mutants deficient in bacterioferritin (bfr), miniferritin (dps), ferritin (ftnA), bacterioferredoxin (bfd), or iron-sulfur cluster assembly machinery (sufABCDSE) were studied. In the parental strain, we observed two rapidly 59Fe-labeled protein signals identified as bacterioferritin and an iron pool associated to the protein chain-elongation process. In the presence of increased 59Fe-chrysobactin concentrations, we detected mini-ferritin-bound iron. Iron incorporation into bacterioferritin was severely reduced in nonpolar sufA, sufB, sufD, sufS, and sufE mutants but not in a sufC background. Iron recycling from bacterioferritin did not occur in bfd and sufC mutants. Iron depletion caused a loss of aconitase activity, whereas ferric chrysobactin supplementation stimulated the production of active aconitase in parental cells and in bfr and bfd mutants. Aconitase activity in sufA, sufB, sufD, sufS, and sufE mutant strains was 10 times lower than that in parental cells. In the sufC mutant, it was twice as low as that in the parental strain. Defects observed in the mutants were not caused by altered ferric chrysobactin transport. Our data demonstrate a functional link between bacterioferritin, bacterioferredoxin, and the Suf protein machinery resulting in optimal bacterial growth and a balanced distribution of iron between essential metalloproteins.     

Identification and development of 2-methylimidazo[1,2-a]pyridine-3-carboxamides as Mycobacterium tuberculosis pantothenate synthetase inhibitors

In the present study, we used crystal structure of mycobacterial pantothenate synthetase (PS) bound with 2-(2-(benzofuran-2-ylsulfonylcarbamoyl)-5-methoxy-1H-indol-1-yl) acetic acid inhibitor for virtual screening of antitubercular compound database to identify new scaffolds. One of the identified lead was modified synthetically to obtain thirty novel analogues. These synthesized compounds were evaluated for Mycobacterium tuberculosis (MTB) PS inhibition study, in vitro antimycobacterial activities and cytotoxicity against RAW 264.7 cell line. Among the compounds tested, N′-(1-naphthoyl)-2-methylimidazo[1,2-a]pyridine-3-carbohydrazide (5b) was found to be the most active compound with IC₅₀ of 1.90 ± 0.12 μM against MTB PS, MIC of 4.53 μM against MTB with no cytotoxicity at 50 μM. The binding affinity of the most potent inhibitor 5b was further confirmed biophysically through differential scanning fluorimetry.     

Protein-protein interactions within the Fatty Acid Synthase-II system of Mycobacterium tuberculosis are essential for mycobacterial viability

Despite the existence of efficient chemotherapy, tuberculosis remains a leading cause of mortality worldwide. New drugs are urgently needed to reduce the potential impact of the emergence of multidrug-resistant strains of the causative agent Mycobacterium tuberculosis (Mtb). The front-line antibiotic isoniazid (INH), and several other drugs, target the biosynthesis of mycolic acids and especially the Fatty Acid Synthase-II (FAS-II) elongation system. This biosynthetic pathway is essential and specific for mycobacteria and still represents a valuable system for the search of new anti-tuberculous agents. Several data, in the literature, suggest the existence of protein-protein interactions within the FAS-II system. These interactions themselves might serve as targets for a new generation of drugs directed against Mtb. By using an extensive in vivo yeast two-hybrid approach and in vitro co-immunoprecipitation, we have demonstrated the existence of both homotypic and heterotypic interactions between the known components of FAS-II. The condensing enzymes KasA, KasB and mtFabH interact with each other and with the reductases MabA and InhA. Furthermore, we have designed and constructed point mutations of the FAS-II reductase MabA, able to disrupt its homotypic interactions and perturb the interaction pattern of this protein within FAS-II. Finally, we showed by a transdominant genetic approach that these mutants are dominant negative in both non-pathogenic and pathogenic mycobacteria. These data allowed us to draw a dynamic model of the organization of FAS-II. They also represent an important step towards the design of a new generation of anti-tuberculous agents, as being inhibitors of essential protein-protein interactions.     

Spectroscopic studies on the [4Fe-4S] cluster in adenosine 5'-phosphosulfate reductase from Mycobacterium tuberculosis

Mycobacterium tuberculosis adenosine 5'-phosphosulfate reductase (MtAPR) is an iron-sulfur protein and a validated target to develop new antitubercular agents, particularly for the treatment of latent infection. The enzyme harbors a [4Fe-4S](2+) cluster that is coordinated by four cysteinyl ligands, two of which are adjacent in the amino acid sequence. The iron-sulfur cluster is essential for catalysis; however, the precise role of the [4Fe-4S] cluster in APR remains unknown. Progress in this area has been hampered by the failure to generate a paramagnetic state of the [4Fe-4S] cluster that can be studied by electron paramagnetic resonance spectroscopy. Herein, we overcome this limitation and report the EPR spectra of MtAPR in the [4Fe-4S](+) state. The EPR signal is rhombic and consists of two overlapping S = ½ species. Substrate binding to MtAPR led to a marked increase in the intensity and resolution of the EPR signal and to minor shifts in principle g values that were not observed among a panel of substrate analogs, including adenosine 5'-diphosphate. Using site-directed mutagenesis, in conjunction with kinetic and EPR studies, we have also identified an essential role for the active site residue Lys-144, whose side chain interacts with both the iron-sulfur cluster and the sulfate group of adenosine 5'-phosphosulfate. The implications of these findings are discussed with respect to the role of the iron-sulfur cluster in the catalytic mechanism of APR.     

IscU as a scaffold for iron-sulfur cluster biosynthesis: sequential assembly of [2Fe-2S] and [4Fe-4S] clusters in IscU

Iron-sulfur cluster biosynthesis in both prokaryotic and eukaryotic cells is known to be mediated by two highly conserved proteins, termed IscS and IscU in prokaryotes. The homodimeric IscS protein has been shown to be a cysteine desulfurase that catalyzes the reductive conversion of cysteine to alanine and sulfide. In this work, the time course of IscS-mediated Fe-S cluster assembly in IscU was monitored via anaerobic anion exchange chromatography. The nature and properties of the clusters assembled in discrete fractions were assessed via analytical studies together with absorption, resonance Raman, and M?ssbauer investigations. The results show sequential cluster assembly with the initial IscU product containing one [2Fe-2S](2+) cluster per dimer converting first to a form containing two [2Fe-2S](2+) clusters per dimer and finally to a form that contains one [4Fe-4S](2+) cluster per dimer. Both the [2Fe-2S](2+) and [4Fe-4S](2+) clusters in IscU are reductively labile and are degraded within minutes upon being exposed to air. On the basis of sequence considerations and spectroscopic studies, the [2Fe-2S](2+) clusters in IscU are shown to have incomplete cysteinyl ligation. In addition, the resonance Raman spectrum of the [4Fe-4S](2+) cluster in IscU is best interpreted in terms of noncysteinyl ligation at a unique Fe site. The ability to assemble both [2Fe-2S](2+) and [4Fe-4S](2+) clusters in IscU supports the proposal that this ubiquitous protein provides a scaffold for IscS-mediated assembly of clusters that are subsequently used for maturation of apo Fe-S proteins.     

Stationary phase-associated protein expression in Mycobacterium tuberculosis: function of the mycobacterial alpha-crystallin homolog.

The majority of active tuberculosis cases arise as a result of reactivation of latent organisms which are quiescent within the host. The ability of mycobacteria to survive extended periods without active replication is a complex process whose details await elucidation. We used two-dimensional gel electrophoresis to examine both steady-state protein composition and time-dependent protein synthetic profiles in aging cultures of virulent Mycobacterium tuberculosis. At least seven proteins were maximally synthesized 1 to 2 weeks following the end of log-phase growth. One of these proteins accumulated to become a predominant stationary-phase protein. N-terminal amino acid sequencing and immunoreactivity identified this protein as the 16-kDa alpha-crystallin-like small heat shock protein. The gene for this protein was shown to be limited to the slowly growing M. tuberculosis complex of organisms as assessed by Southern blotting. Overexpression of this protein in wild-type M. tuberculosis resulted in a slower decline in viability following the end of log-phase growth. Accumulation of this protein was observed in log-phase cultures following a shift to oxygen-limiting conditions but not by other external stimuli. The protein was purified to homogeneity from overexpressing M. smegmatis in two steps and shown to have a significant ability to suppress the thermal denaturation of alcohol dehydrogenase. Collectively, these results suggest that the mycobacterial alpha-crystallin protein may play a role in enhancing long-term protein stability and therefore long-term survival of M. tuberculosis.     

Iron-sulfur cluster biosynthesis: characterization of Escherichia coli CYaY as an iron donor for the assembly of [2Fe-2S] clusters in the scaffold IscU

The biogenesis of iron-sulfur [Fe-S] clusters requires the coordinated delivery of both iron and sulfide. Sulfide is provided by cysteine desulfurases that use L-cysteine as sulfur source. So far, the physiological iron donor has not been clearly identified. CyaY, the bacterial ortholog of frataxin, an iron binding protein thought to be involved in iron-sulfur cluster formation in eukaryotes, is a good candidate because it was shown to bind iron. Nevertheless, no functional in vitro studies showing an involvement of CyaY in [Fe-S] cluster biosynthesis have been reported so far. In this paper we demonstrate for the first time a specific interaction between CyaY and IscS, a cysteine desulfurase participating in iron-sulfur cluster assembly. Analysis of the iron-loaded CyaY protein demonstrated a strong binding of Fe(3+) and a weak binding of Fe(2+) by CyaY. Biochemical analysis showed that the CyaY-Fe(3+) protein corresponds to a mixture of monomer, intermediate forms (dimer-pentamers), and oligomers with the intermediate one corresponding to the only stable and soluble iron-containing form of CyaY. Using spectroscopic methods, this form was further demonstrated to be functional in vitro as an iron donor during [Fe-S] cluster assembly on the scaffold protein IscU in the presence of IscS and cysteine. All of these results point toward a link between CyaY and [Fe-S] cluster biosynthesis, and a possible mechanism for the process is discussed.     

Investigation of the iron-sulfur cluster in Mycobacterium tuberculosis APS reductase: implications for substrate binding and catalysis

The sulfur assimilation pathway is a key metabolic system in prokaryotes that is required for production of cysteine and cofactors such as coenzyme A. In the first step of the pathway, APS reductase catalyzes the reduction of adenosine 5'-phosphosulfate (APS) to adenosine 5'-phosphate (AMP) and sulfite with reducing equivalents from the protein cofactor, thioredoxin. The primary sequence of APS reductase is distinguished by a conserved iron-sulfur cluster motif, -CC-X( approximately )(80)-CXXC-. Of the sequence motifs that are associated with 4Fe-4S centers, the cysteine dyad is atypical and has generated discussion with respect to coordination as well as the cluster's larger functional significance. Herein, we have used biochemical, spectroscopic, and mass spectrometry analysis to investigate the iron-sulfur cluster and its role in the mechanism of Mycobacterium tuberculosis APS reductase. Site-directed mutagenesis of any cysteine residue within the conserved motif led to a loss of cluster with a concomitant loss in catalytic activity, while secondary structure was preserved. Studies of 4Fe-4S cluster stability and cysteine reactivity in the presence and absence of substrates, and in the free enzyme versus the covalent enzyme-intermediate (E-Cys-S-SO(3)(-)), suggest a structural rearrangement that occurs during the catalytic cycle. Taken together, these results demonstrate that the active site functionally communicates with the iron-sulfur cluster and also suggest a functional significance for the cysteine dyad in promoting site differentiation within the 4Fe-4S cluster.     

The SufE protein and the SufBCD complex enhance SufS cysteine desulfurase activity as part of a sulfur transfer pathway for Fe-S cluster assembly in Escherichia coli

The sufABCDSE operon of the Gram-negative bacterium Escherichia coli is induced by oxidative stress and iron deprivation. To examine the biochemical roles of the Suf proteins, we purified all of the proteins and assayed their effect on SufS cysteine desulfurase activity. Here we report that the SufE protein can stimulate the cysteine desulfurase activity of the SufS enzyme up to 8-fold and accepts sulfane sulfur from SufS. This sulfur transfer process from SufS to SufE is sheltered from the environment based on its resistance to added reductants and on the analysis of available crystal structures of the proteins. We also found that the SufB, SufC, and SufD proteins associate in a stable complex and that, in the presence of SufE, the SufBCD complex further stimulates SufS activity up to 32-fold. Thus, the SufE protein and the SufBCD complex act synergistically to modulate the cysteine desulfurase activity of SufS. We propose that this sulfur transfer mechanism may be important for limiting sulfide release during oxidative stress conditions in vivo.     

Structure of the Mycobacterium tuberculosis OmpATb protein: a model of an oligomeric channel in the mycobacterial cell wall

The pore-forming outer membrane protein OmpATb from Mycobacterium tuberculosis is a virulence factor required for acid resistance in host phagosomes. In this study, we determined the 3D structure of OmpATb by NMR in solution. We found that OmpATb is composed of two independent domains separated by a proline-rich hinge region. As expected, the high-resolution structure of the C-terminal domain (OmpATb(198-326)) revealed a module structurally related to other OmpA-like proteins from Gram-negative bacteria. The N-terminal domain of OmpATb (73-204), which is sufficient to form channels in planar lipid bilayers, exhibits a fold, which belongs to the α+β sandwich class fold. Its peculiarity is to be composed of two overlapping subdomains linked via a BON (Bacterial OsmY and Nodulation) domain initially identified in bacterial proteins predicted to interact with phospholipids. Although OmpATb(73-204) is highly water soluble, current-voltage measurements demonstrate that it is able to form conducting pores in model membranes. A HADDOCK modeling of the NMR data gathered on the major monomeric form and on the minor oligomeric populations of OmpATb(73-204) suggest that OmpATb(73-204) can form oligomeric rings able to insert into phospholipid membrane, similar to related proteins from the Type III secretion systems, which form multisubunits membrane-associated rings at the basal body of the secretion machinery.     

Nfu2: a scaffold protein required for [4Fe-4S] and ferredoxin iron-sulphur cluster assembly in Arabidopsis chloroplasts

Nfu proteins are candidates to act as scaffold protein in vivo for iron-sulphur cluster biogenesis. In this work, Nfu2 protein function in the chloroplast was investigated in vivo using T-DNA insertion lines disrupted in AtNfu2 gene. Both alleles characterized presented the same dwarf phenotype due to photosynthetic and metabolic limitations. Nfu2 cDNA expression in nfu2.1 mutant rescued this phenotype. Photosynthesis study of these mutants revealed an altered photosystem I (PSI) activity together with a decrease in PSI amount confirmed by immunodetection experiments, and leading to an over reduction of the plastoquinol pool. Decrease of plastid 4Fe-4S sulphite reductase activity correlates with PSI amount decrease and supports an alteration of 4Fe-4S cluster biogenesis in nfu2 chloroplasts. The decrease of electron flow from the PSI is combined with a decrease in ferredoxin amount in nfu2 mutants. Our results are therefore in favour of a requirement of Nfu2 protein for 4Fe-4S and 2Fe-2S ferredoxin cluster assembly, conferring to this protein an important function for plant growth and photosynthesis as demonstrated by nfu2 mutant phenotype. As glutamate synthase and Rieske Fe-S proteins are not affected in nfu2 mutants, these data indicate that different pathways are involved in Fe-S biogenesis in Arabidopsis chloroplasts.     

Resistome analysis of Mycobacterium tuberculosis: Identification of aminoglycoside 2'-Nacetyltransferase (AAC) as co-target for drug desigining

The emergence of multidrug resistant tuberculosis (MDRTB) highlights the urgent need to understand the mechanisms of resistance to the drugs and to develop a new arena of therapeutics to treat the disease. Ethambutol, isonazid, pyrazinamide, rifampicin are first line of drugs against TB, whereas aminoglycoside, polypeptides, fluoroquinolone, ethionamide are important second line of bactericidal drugs used to treat MDRTB, and resistance to one or both of these drugs are defining characteristic of extensively drug resistant TB. We retrieved 1,221 resistant genes from Antibiotic Resistance Gene Database (ARDB), which are responsible for resistance against first and second line antibiotics used in treatment of Mycobacterium tuberculosis infection. From network analysis of these resistance genes, 53 genes were found to be common. Phylogenetic analysis shows that more than 60% of these genes code for acetyltransferase. Acetyltransferases detoxify antibiotics by acetylation, this mechanism plays central role in antibiotic resistance. Seven acetyltransferase (AT-1 to AT-7) were selected from phylogenetic analysis. Structural alignment shows that these acetyltransferases share common ancestral core, which can be used as a template for structure based drug designing. From STRING analysis it is found that acetyltransferase interact with 10 different proteins and it shows that, all these interaction were specific to M. tuberculosis. These results have important implications in designing new therapeutic strategies with acetyltransferase as lead co-target to combat against MDR as well as Extreme drug resistant (XDR) tuberculosis.

Abbreviations

AA - amino acid, AT - Acetyltransferase, AAC - Aminoglycoside 2''-N-acetyltransferase, XDR - Extreme drug-resistant, MDR - Multidrug-resistant, Mtb - Mycobacterium tuberculosis, TB - Tuberculosis.