Jeverson Frazzon

Formation of iron–sulfur clusters in bacteria: an emerging field in bioinorganic chemistry

Biological iron-sulfur clusters are chemically versatile inorganic structures that are attached to many proteins. These clusters are intimately involved in the functions of their partner proteins and they are required to sustain life on earth. Recent work has demonstrated that, in spite of their simple structures, the assembly and insertion of iron-sulfur clusters into their protein partners is a complex biological process. This complexity is probably related to the cellular toxicity of iron and sulfur in their free forms.

Chloroplast HCF101 is a scaffold protein for [4Fe-4S] cluster assembly

Oxygen-evolving chloroplasts possess their own iron-sulfur cluster assembly proteins including members of the SUF (sulfur mobilization) and the NFU family. Recently, the chloroplast protein HCF101 (high chlorophyll fluorescence 101) has been shown to be essential for the accumulation of the membrane complex Photosystem I and the soluble ferredoxin-thioredoxin reductases, both containing [4Fe-4S] clusters. The protein belongs to the FSC-NTPase ([4Fe-4S]-cluster-containing P-loop NTPase) superfamily, several members of which play a crucial role in Fe/S cluster biosynthesis. Although the C-terminal ISC-binding site, conserved in other members of the FSC-NTPase family, is not present in chloroplast HCF101 homologues using Mössbauer and EPR spectroscopy, we provide evidence that HCF101 binds a [4Fe-4S] cluster. 55Fe incorporation studies of mitochondrially targeted HCF101 in Saccharomyces cerevisiae confirmed the assembly of an Fe/S cluster in HCF101 in an Nfs1-dependent manner. Site-directed mutagenesis identified three HCF101-specific cysteine residues required for assembly and/or stability of the cluster. We further demonstrate that the reconstituted cluster is transiently bound and can be transferred from HCF101 to a [4Fe-4S] apoprotein. Together, our findings suggest that HCF101 may serve as a chloroplast scaffold protein that specifically assembles [4Fe-4S] clusters and transfers them to the chloroplast membrane and soluble target proteins.

Reference Genes for the Normalization of Gene Expression in Eucalyptus Species

Abstract Gene expression analysis is increasingly important in biological research, with reverse transcription–quantitative PCR (RT–qPCR) becoming the method of choice for high-throughput and accurate expression profiling of selected genes. Considering the increased sensitivity, reproducibility and large dynamic range of this method, the requirements for proper internal reference gene(s) for relative expression normalization have become much more stringent. Given the increasing interest in the functional genomics of Eucalyptus, we sought to identify and experimentally verify suitable reference genes for the normalization of gene expression associated with the flower, leaf and xylem of six species of the genus. We selected 50 genes that exhibited the least variation in microarrays of E. grandis leaves and xylem, and E. globulus xylem. We further performed the experimental analysis using RT–qPCR for six Eucalyptus species and three different organs/tissues. Employing algorithms geNorm and NormFinder, we assessed the gene expression stability of eight candidate new reference genes. Classic housekeeping genes were also included in the analysis. The stability profiles of candidate genes were in very good agreement. PCR results proved that the expression of novel Eucons04, Eucons08 and Eucons21 genes was the most stable in all Eucalyptus organs/tissues and species studied. We showed that the combination of these genes as references when measuring the expression of a test gene results in more reliable patterns of expression than traditional housekeeping genes. Hence, novel Eucons04, Eucons08 and Eucons21 genes are the best suitable references for the normalization of expression studies in the Eucalyptus genus.

Functional Characterization by Genetic Complementation of aroB-Encoded Dehydroquinate Synthase from Mycobacterium tuberculosis H37Rv and Its Heterologous Expression and Purification

The recent recrudescence of Mycobacterium tuberculosis infection and the emergence of multidrug-resistant strains have created an urgent need for new therapeutics against tuberculosis. The enzymes of the shikimate pathway are attractive drug targets because this route is absent in mammals and, in M. tuberculosis, it is essential for pathogen viability. This pathway leads to the biosynthesis of aromatic compounds, including aromatic amino acids, and it is found in plants, fungi, bacteria, and apicomplexan parasites. The aroB-encoded enzyme dehydroquinate synthase is the second enzyme of this pathway, and it catalyzes the cyclization of 3-deoxy-D-arabino-heptulosonate-7-phosphate in 3-dehydroquinate. Here we describe the PCR amplification and cloning of the aroB gene and the overexpression and purification of its product, dehydroquinate synthase, to homogeneity. In order to probe where the recombinant dehydroquinate synthase was active, genetic complementation studies were performed. The Escherichia coli AB2847 mutant was used to demonstrate that the plasmid construction was able to repair the mutants, allowing them to grow in minimal medium devoid of aromatic compound supplementation. In addition, homogeneous recombinant M. tuberculosis dehydroquinate synthase was active in the absence of other enzymes, showing that it is homomeric. These results will support the structural studies with M. tuberculosis dehydroquinate synthase that are essential for the rational design of antimycobacterial agents.

Structural studies of the Enterococcus faecalis SufU [Fe-S] cluster protein

BackgroundIron-sulfur clusters are ubiquitous and evolutionarily ancient inorganic prosthetic groups, the biosynthesis of which depends on complex protein machineries. Three distinct assembly systems involved in the maturation of cellular Fe-S proteins have been determined, designated the NIF, ISC and SUF systems. Although well described in several organisms, these machineries are poorly understood in Gram-positive bacteria. Within the Firmicutes phylum, the Enterococcus spp. genus have recently assumed importance in clinical microbiology being considered as emerging pathogens for humans, wherein Enterococcus faecalis represents the major species associated with nosocomial infections. The aim of this study was to carry out a phylogenetic analysis in Enterococcus faecalis V583 and a structural and conformational characterisation of it SufU protein.ResultsBLAST searches of the Enterococcus genome revealed a series of genes with sequence similarity to the Escherichia coli SUF machinery of [Fe-S] cluster biosynthesis, namely sufB, sufC, sufD and SufS. In addition, the E. coli IscU ortholog SufU was found to be the scaffold protein of Enterococcus spp., containing all features considered essential for its biological activity, including conserved amino acid residues involved in substrate and/or co-factor binding (Cys50,76,138 and Asp52) and, phylogenetic analyses showed a close relationship with orthologues from other Gram-positive bacteria. Molecular dynamics for structural determinations and molecular modeling using E. faecalis SufU primary sequence protein over the PDB:1su0 crystallographic model from Streptococcus pyogenes were carried out with a subsequent 50 ns molecular dynamic trajectory. This presented a stable model, showing secondary structure modifications near the active site and conserved cysteine residues. Molecular modeling using Haemophilus influenzae IscU primary sequence over the PDB:1su0 crystal followed by a MD trajectory was performed to analyse differences in the C-terminus region of Gram-positive SufU and Gram-negative orthologous proteins, in which several modifications in secondary structure were observed.ConclusionThe data describe the identification of the SUF machinery for [Fe-S] cluster biosynthesis present in the Firmicutes genome, showing conserved sufB, sufC, sufD and sufS genes and the presence of the sufU gene coding for scaffold protein, instead of sufA; neither sufE nor sufR are present. Primary sequences and structural analysis of the SufU protein demonstrated its structural-like pattern to the scaffold protein IscU nearby on the ISC machinery. E. faecalis SufU molecular modeling showed high flexibility over the active site regions, and demonstrated the existence of a specific region in Firmicutes denoting the G ram p ositive r egion (GPR), suggested as a possible candidate for interaction with other factors and/or regulators.

Antimicrobial resistance profile of Enterococcus spp isolated from food in Southern Brazil

Fifty-six Enterococcus spp. strains were isolated from foods in Southern Brazil, confirmed by PCR and classified as Enterococcus faecalis (27), Enterococcus faecium (23) and Enterococcus spp (6). Antimicrobial susceptibility tests showed resistance phenotypes to a range of antibiotics widely administrated in humans such as gentamycin, streptomycin, ampicillin and vancomycin.

Functional analysis of Arabidopsis genes involved in mitochondrial iron–sulfur cluster assembly

Machinery for the assembly of the iron–sulfur ([Fe–S]) clusters that function as cofactors in a wide variety of proteins has been identified in microbes, insects, and animals. Homologs of the genes involved in [Fe–S] cluster biogenesis have recently been found in plants, as well, and point to the existence of two distinct systems in these organisms, one located in plastids and one in mitochondria. Here we present the first biochemical confirmation of the activity of two components of the mitochondrial machinery in Arabidopsis, AtNFS1 and AtISU1. Analysis of the expression patterns of the corresponding genes, as well as AtISU2 and AtISU3, and the phenotypes of plants in which these genes are up or down-regulated are consistent with a role for the mitochondrial [Fe–S] assembly system in the maturation of proteins required for normal plant development.

Role of conserved cysteines in mediating sulfur transfer from IscS to IscU.

The role of the three conserved cysteine residues on Azotobacter vinelandii IscU in accepting sulfane sulfur and forming a covalent complex with IscS has been evaluated using electrospray‐ionization mass spectrometry studies of variants involving individual cysteine‐to‐alanine substitutions. The results reveal that IscS can transfer sulfur to each of the three alanine‐substituted forms of IscU to yield persulfide or polysulfide species, and formation of a heterodisulfide covalent complex between IscS and Cys37 on IscU. It is concluded that S transfer from IscS to IscU does not involve a specific cysteine on IscU or the formation of an IscS–IscU heterodisulfide complex.

Feedback regulation of iron-sulfur cluster biosynthesis

Iron-sulfur clusters ([Fe-S] clusters) represent one of natures simplest, functionally versatile, and perhaps most ancient prosthetic groups (1). Among the familiar types of [Fe-S] clusters are [2Fe-2S] and [4Fe-4S] clusters, which usually are attached to their protein partners by four cysteine thiol ligands (Fig. 1). Proteins that contain one or more [Fe-S] clusters are commonly called [Fe-S] proteins, and they represent a large class of structurally and functionally diverse proteins that participate in many metabolic processes. For example, [Fe-S] proteins are essential players in the life-sustaining processes of respiration, nitrogen fixation, and photosynthesis with [Fe-S] clusters participating as agents of electron transfer, substrate activation, catalysis, and environmental sensing. Given the structural simplicity of [Fe-S] clusters and the participation of [Fe-S] proteins in so many metabolic processes it may be surprising that the pathway for biological formation of [Fe-S] clusters is only now beginning to emerge. Pioneering work in the laboratory of Berg and Holm (2) established that the chemical synthesis of structural analogs to many biological [Fe-S] clusters is achieved when Fe3+/2+ and S2− are combined under controlled conditions and in the presence of the appropriate thiolate donors. Along these same lines, Malkin and Rabinowitz (3) showed that purified [Fe-S] proteins, for which the cluster has been removed, are often reconstituted with the correct [Fe-S] cluster species by simple treatment with Fe2+ and S2− under reducing conditions. Such in vitro “spontaneous” assembly, however, cannot represent the complete mechanism of biological [Fe-S] cluster formation because free Fe2+ and S2− are metabolic poisons. Rather, it is now known that a group of highly conserved proteins are responsible for directing the controlled assembly of [Fe-S] clusters and the maturation of [Fe-S] proteins (4).

Molecular analysis of the iap gene of Listeria monocytogenes isolated from cheeses in Rio Grande do Sul, Brazil

The polymorphic region sequences in the iap gene were analyzed in 25 strains of Listeria monocytogenes isolated from cheeses in the state of Rio Grande do Sul, and compared with reference strains. This investigation distinguished two clusters of L. monocytogenes: I (20 strains) and II (5 strains).