Biosynthesis of iron-sulphur clusters is a complex and highly conserved process

Iron-sulphur ([Fe-S]) clusters are simple inorganic prosthetic groups that are contained in a variety of proteins having functions related to electron transfer, gene regulation, environmental sensing and substrate activation. In spite of their simple structures, biological [Fe-S] clusters are not formed spontaneously. Rather, a consortium of highly conserved proteins is required for both the formation of [Fe-S] clusters and their insertion into various protein partners. Among the [Fe-S] cluster biosynthetic proteins are included a pyridoxal phosphate-dependent enzyme (NifS) that is involved in the activation of sulphur from l-cysteine, and a molecular scaffold protein (NifU) upon which [Fe-S] cluster precursors are formed. The formation or transfer of [Fe-S] clusters appears to require an electron-transfer step. Another complexity is that molecular chaperones homologous to DnaJ and DnaK are involved in some aspect of the maturation of [Fe-S]-cluster-containing proteins. It appears that the basic biochemical features of [Fe-S] cluster formation are strongly conserved in Nature, since organisms from all three life Kingdoms contain the same consortium of homologous proteins required for [Fe-S] cluster formation that were discovered in the eubacteria.     

Computational structural and functional analysis of hypothetical proteins of Staphylococcus aureus

Genome sequencing projects has led to an explosion of large amount of gene products in which many are of hypothetical proteins with unknown function. Analyzing and annotating the functions of hypothetical proteins is important in Staphylococcus aureus which is a pathogenic bacterium that cause multiple types of diseases by infecting various sites in humans and animals. In this study, ten hypothetical proteins of Staphylococcus aureus were retrieved from NCBI and analyzed for their structural and functional characteristics by using various bioinformatics tools and databases. The analysis revealed that some of them possessed functionally important domains and families and protein-protein interacting partners which were ABC transporter ATP-binding protein, Multiple Antibiotic Resistance (MAR) family, export proteins, Helix-Turn-helix domains, arsenate reductase, elongation factor, ribosomal proteins, Cysteine protease precursor, Type-I restriction endonuclease enzyme and plasmid recombination enzyme which might have the same functions in hypothetical proteins. The structural prediction of those proteins and binding sites prediction have been done which would be useful in docking studies for aiding in the drug discovery.     

Identification and physical organization of the gene cluster involved in the biosynthesis of Burkholderia cepacia complex exopolysaccharide

Bacteria belonging to the Burkholderia cepacia complex (BCC) are important opportunistic pathogens in patients with cystic fibrosis (CF). Since approximately 80% of the CF isolates examined produce exopolysaccharide (EPS), it was hypothesized that this EPS may play a role in the colonization and persistence of these bacteria in the CF lung. The present study describes the identification and physical organization of the EPS biosynthetic gene cluster. This bce gene cluster was identified following the isolation of three EPS-defective mutants from the highly mucoid CF isolate IST408, belonging to BCC genomovar I, based on random plasposon insertion mutagenesis and comparison of the nucleotide sequence of the interrupted genes with the available genome of Burkholderia cenocepacia J2315. This 16.2 kb cluster includes 12 genes and is located on chromosome 2. Database searches for homologous proteins and secondary structure analysis for the deduced Bce amino acid sequences revealed genes predicted to encode enzymes required for the formation of nucleotide sugar precursors, glycosyltransferases involved in the repeat-unit assembly, and other proteins involved in polymerization and export of bacterial surface polysaccharides.     

An approach for cloning biosynthetic genes of 2-deoxystreptamine-containing aminocyclitol antibiotics: isolation of a biosynthetic gene cluster of tobramycin from Streptomyces tenebrarius

Genes homologous to 2-deoxystreptamine (DOS) biosynthetic genes were isolated from aminoglycoside producers, Micromonospora and Streptomyces spp., using PCR primers based on the core sequences of 2-deoxy-scyllo-inosose (DOI) synthase and L-glutamine: scyllo-inosose aminotransferase (GIA). Identities of 40-45% were observed for DOI synthases, and 65-75% were observed for GIAs. The gene cluster of tobramycin biosynthesis was isolated from the genomic library of Streptomyces tenebrarius using DOI synthase as a probe. Sequencing of 33.9 kb revealed 24 putative open reading frames including the tobramycin biosynthetic gene cluster (13.8 kb) and a transport protein. This cluster encodes proteins homologous to 2-deoxystreptamine biosynthetic enzymes, glycosyltransferase and other aminocyclitols biosynthetic enzymes. Sequence analysis revealed the evolution of DOI synthases from 3-dehydroquinate (DHQ) synthases in actinomycetes. DOI synthases and GIA are therefore useful for cloning biosynthetic genes of DOS-containing aminocyclitol antibiotics or for screening such metabolites producers.     

Insights into an Unusual Nonribosomal Peptide Synthetase Biosynthesis: IDENTIFICATION AND CHARACTERIZATION OF THE GE81112 BIOSYNTHETIC GENE CLUSTER*

The GE81112 tetrapeptides (1–3) represent a structurally unique class of antibiotics, acting as specific inhibitors of prokaryotic protein synthesis. Here we report the cloning and sequencing of the GE81112 biosynthetic gene cluster from Streptomyces sp. L-49973 and the development of a genetic manipulation system for Streptomyces sp. L-49973. The biosynthetic gene cluster for the tetrapeptide antibiotic GE81112 (getA-N) was identified within a 61.7-kb region comprising 29 open reading frames (open reading frames), 14 of which were assigned to the biosynthetic gene cluster. Sequence analysis revealed the GE81112 cluster to consist of six nonribosomal peptide synthetase (NRPS) genes encoding incomplete di-domain NRPS modules and a single free standing NRPS domain as well as genes encoding other biosynthetic and modifying proteins. The involvement of the cloned gene cluster in GE81112 biosynthesis was confirmed by inactivating the NRPS gene getE resulting in a GE81112 production abolished mutant. In addition, we characterized the NRPS A-domains from the pathway by expression in Escherichia coli and in vitro enzymatic assays. The previously unknown stereochemistry of most chiral centers in GE81112 was established from a combined chemical and biosynthetic approach. Taken together, these findings have allowed us to propose a rational model for GE81112 biosynthesis. The results further open the door to developing new derivatives of these promising antibiotic compounds by genetic engineering.     

Identification of potential apicoplast associated therapeutic targets in human and animal pathogen Toxoplasma gondii ME49

Toxoplasma gondii ME49 is an obligatory intracellular apicomplexa parasite that causes toxoplasmosis in humans, domesticated and wild animals. Waterborne outbreaks of acute toxoplasmosis worldwide reinforce the transmission of Toxoplasma gondii ME49 to humans through contaminated water and may have a greater epidemiological impact than previously believed. In the quest for drug and vaccine target identification subtractive genomics involving subtraction between the host and pathogen genome has been implemented for enlisting essential pathogen specific proteins. Using this approach, our analysis on both human and Toxoplasma gondii ME49 reveals that out of 7987 protein coding sequences of the pathogen, 950 represent essential non human-homologous proteins. Subcellular localization prediction & comparative-biochemical pathway analysis of these essential proteins gives a list of apicoplast-associated proteins having unique pathogen-specific metabolic pathway. These apicoplast-associated enzymes involved in fatty acid biosynthesis pathway of Toxoplasma gondii ME49, may be used as potential drug targets, as the pathway is vital for the protozoan's survival. Structure prediction of drug target proteins was done using fold based recognition method. Screening of the functional inhibitors against these novel targets may result in discovery of novel therapeutic compounds that can be effective against Toxoplasma gondii ME49. ABBREVIATIONS: DEG - Database of Essential Gene, KEGG - Kyoto Encyclopaedia of Genes and Genomes, KAAS - KEGG Automated Annotation Server, PFP - Protein Function Prediction, COG - Cluster of Orthologous Genes.     

Proteome analysis of<Emphasis Type="Italic"> Aspergillus nidulans</Emphasis> reveals proteins associated with the response to the antibiotic concanamycin A,produced by<Emphasis Type="Italic"> Streptomyces</Emphasis> species

Competition between microbes is common to all ecosystems, but the exact nature of the competition is in most cases unknown. We have previously studied the antagonism between Streptomyces halstedii and several fungi at both the organismal and gene expression levels. Here we analysed the effect of an antibiotic produced by Streptomyces, concanamycin A, on protein levels in the filamentous fungus Aspergillus nidulans. Two-dimensional gel electrophoresis revealed that 20 proteins either increased or decreased in abundance upon treatment of the fungus with the antibiotic. Five of the most prominent proteins which changed in abundance were identified based on peptide analysis by mass spectrometry. Two of these correspond to proteins previously described in A. nidulans, and three others are homologous to proteins found in other organisms. Of these, one down-regulated protein was identified as glyceraldehyde dehydrogenase, a protein involved in general metabolic pathways. A second down-regulated protein, CpcB, affects the initiation of sexual development. Among the proteins not previously described in A. nidulans, all of them up-regulated by concanamycin A, we found two proteins with described homologues in other fungal species. The first is homologous to a cadmium-induced protein in Candida sp. The second protein is homologous to LovC, an enoyl transferase involved in the biosynthesis of lovastatin, a secondary metabolite identified in A. terreus. A third protein has a homologue in A. niger, which is of unknown function. This study indicates that proteome analysis may be a useful method for studying effects on gene expression during competitive interactions between bacteria and filamentous fungi.     

The actin binding protein, fesselin, is a member of the synaptopodin family

Fesselin is a natively unfolded protein that is abundant in avian smooth muscle. Like many natively unfolded proteins, fesselin has multiple binding partners including actin, myosin, calmodulin and α-actinin. Fesselin accelerates actin polymerization and bundles actin. These and other observations suggest that fesselin is a component of the cytoskeleton. We have now cloned fesselin and have determined the cDNA derived amino acid sequence. We verified parts of the sequence by Edman analysis and by mass spectroscopy. Our results confirmed fesselin is homologous to human synaptopodin 2 and belongs to the synaptopodin family of proteins.     

Functional assignment to maize group 1 LEA protein EMB564 within the cell nucleus using computational analysis

Maize protein EMB564 is a member of group 1 LEA (late embryogenesis abundant) proteins. Currently, the molecular functions of group 1 LEA proteins remain largely unclear. We here report on the functional assignment to EMB564 by computational analysis. EMB564 is predicted as nuclear localization by five different predictors including CELLO, Plant-mPLoc, WoLF PSORT, Predotar and TargetP. EMB564 is found to be remote homologous with DNA/RNA helicases and single-stranded DNA-binding proteins, and their sequences contains similar DNA/RNA binding sites. Furthermore, the three-dimensional (3D) model of EMB564 structurally resembles a variety of nuclear and DNA/RNA-binding proteins, especially those involving in the regulation of cell division, chromosomal replication and DNA unwinding or repairing. Our results reveal that EMB564 protein is most likely to function within the cell nucleus.     

Crystal structures and proposed structural/functional classification of three protozoan proteins from the isochorismatase superfamily

We have determined the crystal structures of three homologous proteins from the pathogenic protozoans Leishmania donovani, Leishmania major, and Trypanosoma cruzi. We propose that these proteins represent a new subfamily within the isochorismatase superfamily (CDD classification cd004310). Their overall fold and key active site residues are structurally homologous both to the biochemically well-characterized N-carbamoylsarcosine-amidohydrolase, a cysteine hydrolase, and to the phenazine biosynthesis protein PHZD (isochorismase), an aspartyl hydrolase. All three proteins are annotated as mitochondrial-associated ribonuclease Mar1, based on a previous characterization of the homologous protein from L. tarentolae. This would constitute a new enzymatic activity for this structural superfamily, but this is not strongly supported by the observed structures. In these protozoan proteins, the extended active site is formed by inter-subunit association within a tetramer, which implies a distinct evolutionary history and substrate specificity from the previously characterized members of the isochorismatase superfamily. The characterization of the active site is supported crystallographically by the presence of an unidentified ligand bound at the active site cysteine of the T. cruzi structure.     

SUMO and SUMOylation in plants

The traditional focus on the central dogma of molecular biology, from gene through RNA to protein, has now been replaced by the recognition of an additional mechanism. The new regulatory mechanism, post-translational modifications to proteins, can actively alter protein function or activity introducing additional levels of functional complexity by altering cellular and sub-cellular location, protein interactions and the outcome of biochemical reaction chains. Modifications by ubiquitin (Ub) and ubiquitin-like modifiers systems are conserved in all eukaryotic organisms. One of them, small ubiquitin-like modifier (SUMO) is present in plants. The SUMO mechanism includes several isoforms of proteins that are involved in reactions of sumoylation and de-sumoylation. Sumoylation affects several important processes in plants. Outstanding among those are responses to environmental stresses. These may be abiotic stresses, such as phosphate deficiency, heat, low temperature, and drought, or biotic stressses, as well including defense reactions to pathogen infection. Also, the regulations of flowering time, cell growth and development, and nitrogen assimilation have recently been added to this list. Identification of SUMO targets is material to characterize the function of sumoylation or desumoylation. Affinity purification and mass spectrometric identification have been done lately in plants. Further SUMO noncovalent binding appears to have function in other model organisms and SUMO interacting proteins in plants will be of interest to plant biologists who dissect the dynamic function of SUMO. This review will discuss results of recent insights into the role of sumoylation in plants.     

The crystal structure of a thermophilic glucose binding protein reveals adaptations that interconvert mono and di-saccharide binding sites

Periplasmic binding proteins (PBPs) comprise a protein superfamily that is involved in prokaryotic solute transport and chemotaxis. These proteins have been used to engineer reagentless biosensors to detect natural or non-natural ligands. There is considerable interest in obtaining very stable members of this superfamily from thermophilic bacteria to use as robust engineerable parts in biosensor development. Analysis of the recently determined genome sequence of Thermus thermophilus revealed the presence of more than 30 putative PBPs in this thermophile. One of these is annotated as a glucose binding protein (GBP) based on its genetic linkage to genes that are homologous to an ATP-binding cassette glucose transport system, although the PBP sequence is homologous to periplasmic maltose binding proteins (MBPs). Here we present the cloning, over-expression, characterization of cognate ligands, and determination of the X-ray crystal structure of this gene product. We find that it is a very stable (apo-protein Tm value is 100(+/- 2) degrees C; complexes 106(+/- 3) degrees C and 111(+/- 1) degrees C for glucose and galactose, respectively) glucose (Kd value is 0.08(+/- 0.03) microM) and galactose (Kd value is 0.94(+/- 0.04) microM) binding protein. Determination of the X-ray crystal structure revealed that this T. thermophilus glucose binding protein (ttGBP) is structurally homologous to MBPs rather than other GBPs. The di or tri-saccharide ligands in MBPs are accommodated in long relatively shallow grooves. In the ttGBP binding site, this groove is partially filled by two loops and an alpha-helix, which create a buried binding site that allows binding of only monosaccharides. Comparison of ttGBP and MBP provides a clear example of structural adaptations by which the size of ligand binding sites can be controlled in the PBP super family.     

Crystal structures of the bacterial solute receptor AcbH displaying an exclusive substrate preference for β-D-galactopyranose

Solute receptors (binding proteins) are indispensable components of canonical ATP-binding cassette importers in prokaryotes. Here, we report on the characterization and crystal structures in the closed and open conformations of AcbH, the solute receptor of the putative carbohydrate transporter AcbFG which is encoded in the acarbose (acarviosyl-1,4-maltose) biosynthetic gene cluster from Actinoplanes sp. SE50/110. Binding assays identified AcbH as a high-affinity monosaccharide-binding protein with a dissociation constant (K_d) for β-d-galactopyranose of 9.8 ± 1.0 nM. Neither galactose-containing di- and trisaccharides, such as lactose and raffinose, nor monosaccharides including d-galacturonic acid, l-arabinose, d-xylose and l-rhamnose competed with [¹⁴C]galactose for binding to AcbH. Moreover, AcbH does not bind d-glucose, which is a common property of all but one d-galactose-binding proteins characterized to date. Strikingly, determination of the X-ray structure revealed that AcbH is structurally homologous to maltose-binding proteins rather than to glucose-binding proteins. Two helices are inserted in the substrate-binding pocket, which reduces the cavity size and allows the exclusive binding of monosaccharides, specifically β-d-galactopyranose, in the ⁴C₁ conformation. Site-directed mutagenesis of three residues from the binding pocket (Arg82, Asp361 and Arg362) that interact with the axially oriented O4-H hydroxyl of the bound galactopyranose and subsequent functional analysis indicated that these residues are crucial for galactose binding. To our knowledge, this is the first report of the tertiary structure of a solute receptor with exclusive affinity for β-d-galactopyranose. The putative role of a galactose import system in the context of acarbose metabolism in Actinoplanes sp. is discussed.     

Molecular cloning and characterization of an ML-236B (compactin) biosynthetic gene cluster in Penicillium citrinum

Cloning of genes encoding polyketide synthases (PKSs) has allowed us to identify a gene cluster for ML-236B biosynthesis in Penicillium citrinum. Like lovastatin, which is produced by Aspergillus terreus, ML-236B (compactin) inhibits the enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. Genomic sequencing and Northern analysis showed that nine predicted genes for ML-236B biosynthesis were located within a 38-kb region and were transcribed when ML-236B was produced. The predicted amino acid sequences encoded by these nine genes, designated mlcA- mlcH and mlcR, were similar to those encoded by the genes for lovastatin synthesis, and were therefore assumed to be involved either directly or indirectly in ML-236B biosynthesis. Targeted disruption experiments provided evidence that two PKS genes in the cluster, mlcA and mlcB, are required for the biosynthesis of the nonaketide and the diketide moieties, respectively, of ML-236B, suggesting that the gene cluster as a whole is responsible for ML-236B biosynthesis in P. citrinum. Bioconversion of some of the predicted intermediates by an mlcA-disrupted mutant was also investigated in order to analyze the ML-236B biosynthetic pathway. The molecular organization of the gene cluster and proposed functions for the ML-236B biosynthetic genes in P. citrinum are described.     

Nondisjunctional Segregations in Drosophila Female Meiosis I Are Preceded by Homolog Malorientation at Metaphase Arrest

The essential outer membrane β-barrel protein BamA forms a complex with four lipoprotein partners BamBCDE that assembles β-barrel proteins into the outer membrane of Escherichia coli. Detailed genetic studies have shown that BamA cycles through multiple conformations during substrate assembly, suggesting that a complex network of residues may be involved in coordinating conformational changes and lipoprotein partner function. While genetic analysis of BamA has been informative, it has also been slow in the absence of a straightforward selection for mutants. Here we take a bioinformatic approach to identify candidate residues for mutagenesis using direct coupling analysis. Starting with the BamA paralog FhaC, we show that direct coupling analysis works well for large β-barrel proteins, identifying pairs of residues in close proximity in tertiary structure with a true positive rate of 0.64 over the top 50 predictions. To reduce the effects of noise, we designed and incorporated a novel structured prior into the empirical correlation matrix, dramatically increasing the FhaC true positive rate from 0.64 to 0.88 over the top 50 predictions. Our direct coupling analysis of BamA implicates residues R661 and D740 in a functional interaction. We find that the substitutions R661G and D740G each confer OM permeability defects and destabilize the BamA β-barrel. We also identify synthetic phenotypes and cross-suppressors that suggest R661 and D740 function in a similar process and may interact directly. We expect that the direct coupling analysis approach to informed mutagenesis will be particularly useful in systems lacking adequate selections and for dynamic proteins with multiple conformations.     

Assembly of the yeast vacuolar H+-ATPase and ATP hydrolysis occurs in the absence of subunit c''

The V-ATPases are ubiquitous enzymes of eukaryotes. They are involved in many cellular processes via their ability to pump protons across biological membranes. They are two domain enzymes comprising an ATP hydrolysing sector and a proton translocating sector. Both sectors are functionally coupled. The proton tanslocating sector, V0, is comprised of five polypeptides in an as yet undetermined stoichiometry. In V0 three homologous proteins, subunit c, c' and c' have previously been reported to be essential for assembly of the enzyme. However, we report that subunit c' is not essential for assembly but is for functional coupling of the enzyme.     

Production of recombinant proteins by filamentous fungi

The initial focus of recombinant protein production by filamentous fungi related to exploiting the extraordinary extracellular enzyme synthesis and secretion machinery of industrial strains, including Aspergillus, Trichoderma, Penicillium and Rhizopus species, was to produce single recombinant protein products. An early recognized disadvantage of filamentous fungi as hosts of recombinant proteins was their common ability to produce homologous proteases which could degrade the heterologous protein product and strategies to prevent proteolysis have met with some limited success. It was also recognized that the protein glycosylation patterns in filamentous fungi and in mammals were quite different, such that filamentous fungi are likely not to be the most suitable microbial hosts for production of recombinant human glycoproteins for therapeutic use. By combining the experience gained from production of single recombinant proteins with new scientific information being generated through genomics and proteomics research, biotechnologists are now poised to extend the biomanufacturing capabilities of recombinant filamentous fungi by enabling them to express genes encoding multiple proteins, including, for example, new biosynthetic pathways for production of new primary or secondary metabolites. It is recognized that filamentous fungi, most species of which have not yet been isolated, represent an enormously diverse source of novel biosynthetic pathways, and that the natural fungal host harboring a valuable biosynthesis pathway may often not be the most suitable organism for biomanufacture purposes. Hence it is expected that substantial effort will be directed to transforming other fungal hosts, non-fungal microbial hosts and indeed non microbial hosts to express some of these novel biosynthetic pathways. But future applications of recombinant expression of proteins will not be confined to biomanufacturing. Opportunities to exploit recombinant technology to unravel the causes of the deleterious impacts of fungi, for example as human, mammalian and plant pathogens, and then to bring forward solutions, is expected to represent a very important future focus of fungal recombinant protein technology.