Search

Key Message: The molybdenum cofactor sulfurase gene ( AnMCSU ) was cloned from xerophytic desert plant Ammopiptanthus nanus and validated for its function of tolerance toward abiotic stresses by heterologous expression in Arabidopsis thaliana. Molybdenum cofactor sulfurase participates in catalyzing biosynthesis of abscisic acid, which plays a crucial role in the response of plants to abiotic stresses. In this study, we cloned molybdenum cofactor sulfurase gene (AnMCSU) from a super-xerophytic desert plant, Ammopiptanthus nanus, by using rapid amplification of cDNA ends method. This gene has a total length of 2544 bp, with a 5'- and a 3'-untranslated region of 167 and 88 bp, and an open reading frame of 2289 bp, which encodes an 84.85 kDa protein of 762 amino acids. The putative amino acid sequence shares high homology and conserved amino acid residues crucial for the function of molybdenum cofactor sulfurases with other leguminous species. The encoded protein of the AnMCSU gene was located in the cytoplasm by transient expression in Nicotiana benthamiana. The result of real-time quantitative PCR showed that the expression of the AnMCSU gene was induced by heat, dehydration, high salt stresses, and ABA induction, and inhibited by cold stress. The heterologous expression of the AnMCSU gene significantly enhanced the tolerance of Arabidopsis thaliana to high salt, cold, osmotic stresses, and abscisic acid induction. All these results suggest that the AnMCSU gene might play a crucial role in the adaptation of A. nanus to abiotic stress and has potential to be applied to transgenic improvement of commercial crops.

The metallo-β-lactamase VIM-31 differs from VIM-2 by only two Tyr224His and His252Arg substitutions. Located close to the active site, the Tyr224His substitution is also present in VIM-1, VIM-4, VIM-7 and VIM-12. The VIM-31 variant was reported in 2012 from Enterobacter cloacae and kinetically characterized. It exhibits globally lower catalytic efficiencies than VIM-2. In the present study, we report the three-dimensional structures of VIM-31 in its native (reduced) and oxidized forms. The so-called 'flapping-loop' (loop 1) and loop 3 of VIM-31 were not positioned as in VIM-2 but instead were closer to the active site as in VIM-4, resulting in a narrower active site in VIM-31. Also, the presence of His224 in VIM-31 disrupts hydrogen-bonding networks close to the active site. Moreover, a third zinc-binding site, which also exists in VIM-2 structures, could be identified as a structural explanation for the decreased activity of VIM-MBLs at high zinc concentrations., (© 2015 FEBS.)

Over the last decade, small (often noncoding) RNA molecules have been discovered as important regulators influencing myriad aspects of bacterial physiology and virulence. In particular, small RNAs (sRNAs) have been implicated in control of both primary and secondary metabolic pathways in many bacterial species. This chapter describes characteristics of the major classes of sRNA regulators, and highlights what is known regarding their mechanisms of action. Specific examples of sRNAs that regulate metabolism in gram-negative bacteria are discussed, with a focus on those that regulate gene expression by base pairing with mRNA targets to control their translation and stability.

Accurate prediction of protein function in humans is important for understanding biological processes at the molecular level in biomedicine and drug design. Over a third of proteins are commonly held to bind metal, and ∼10% of human proteins, to bind zinc. Therefore, an initial step in protein function prediction frequently involves predicting metal ion binding. In recent years, methods have been developed to predict a set of residues in 3D space forming the metal-ion binding site, often with a high degree of accuracy. Here, using extensions of these methods, we provide an extensive list of human proteins and their putative metal ion binding site residues, using translated gene sequences derived from the complete, resolved human genome. Under conditions of ∼90% selectivity, over 900 new human putative metal ion binding proteins are identified. A statistical analysis of resolved metal ion binding sites in the human metalloproteome is furnished and the importance of remote homology analysis is demonstrated. As an example, a novel metal-ion binding site involving a complex of a botulinum substrate with its inhibitor is presented. On the basis of the location of the predicted site and the interactions of the contacting residues at the complex interface, we postulate that metal ion binding in this region could influence complex formation and, consequently, the functioning of the protein. Thus, this work provides testable hypotheses about novel functions of known proteins., (© 2015 Wiley Periodicals, Inc.)

This review presents recent examples of metal-binding promiscuity in protein scaffolds and highlights the effect of metal variation on catalytic functionality. Naturally evolved binding sites, as well as unnatural amino acids and cofactors can bind a diverse range of metals, including non-biological transition elements. Computational screening and rational design have been successfully used to create promiscuous binding-sites. Incorporation of non-native metals into proteins expands the catalytic range of transformations catalysed by enzymes and enhances their potential for application in chemicals synthesis., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Diamond-Blackfan anaemia is a congenital bone marrow failure syndrome that is characterized by red blood cell aplasia. The disease has been associated with mutations or large deletions in 11 ribosomal protein genes including RPS7, RPS10, RPS17, RPS19, RPS24, RPS26, RPS29, RPL5, RPL11, RPL26 and RPL35A as well as GATA1 in more than 50% of patients. However, the molecular aetiology of many Diamond-Blackfan anaemia cases remains to be uncovered. To identify new mutations responsible for Diamond-Blackfan anaemia, we performed whole-exome sequencing analysis of 48 patients with no documented mutations/deletions involving known Diamond-Blackfan anaemia genes except for RPS7, RPL26, RPS29 and GATA1. Here, we identified a de novo splicing error mutation in RPL27 and frameshift deletion in RPS27 in sporadic patients with Diamond-Blackfan anaemia. In vitro knockdown of gene expression disturbed pre-ribosomal RNA processing. Zebrafish models of rpl27 and rps27 mutations showed impairments of erythrocyte production and tail and/or brain development. Additional novel mutations were found in eight patients, including RPL3L, RPL6, RPL7L1T, RPL8, RPL13, RPL14, RPL18A and RPL31. In conclusion, we identified novel germline mutations of two ribosomal protein genes responsible for Diamond-Blackfan anaemia, further confirming the concept that mutations in ribosomal protein genes lead to Diamond-Blackfan anaemia., (© 2014 John Wiley & Sons Ltd.)

Bacterioferritin is a bacterial iron storage and detoxification protein that is capable of forming a ferric oxyhydroxide mineral core within its central cavity. To do this, iron must traverse the bacterioferritin protein shell, which is expected to occur through one or more of the channels through the shell identified by structural studies. The size and negative electrostatic potential of the 24 B-type channels suggest that they could provide a route for iron into bacterioferritin. Residues at the B-type channel (Asn-34, Glu-66, Asp-132, and Asp-139) of E. coli bacterioferritin were substituted to determine if they are important for iron core formation. A significant decrease in the rates of initial oxidation of Fe(II) at the ferroxidase center and subsequent iron mineralization was observed for the D132F variant. The crystal structure of this variant shows that substitution of residue 132 with phenylalanine caused a steric blockage of the B-type channel and no other material structural perturbation. We conclude that the B-type channel is a major route for iron entry into both the ferroxidase center and the iron storage cavity of bacterioferritin., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Drought stress limits yield severely in most of the crops. Plants utilize complex gene regulation mechanisms to tolerate water deficiency as well as other abiotic stresses. MicroRNAs (miRNAs) are a class of small non-coding RNAs that are progressively recognized as important regulators of gene expression acting at post-transcriptional level. miR408, conserved in terrestrial plants, targets copper related genes. Although, expression level of miR408 is influenced by various environmental factors including drought stress, the biological action of miR408 is still unclear. To examine the miR408 function upon drought stress in chickpea, transgenic lines overexpressing the miR408 were generated. Induced tolerance was observed in the plants with enhanced miR408 expression upon 17-day water deficiency. Expression levels of miR408 target gene together with seven drought responsive genes were measured using qRT-PCR. Here, the involvement of miR408 in drought stress response has been reported. The overexpression leading plantacyanin transcript repression caused regulation of DREB and other drought responsive genes., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Ammonium and methane are inert molecules and dedicated enzymes are required to break up the N-H and C-H bonds. Until recently, only aerobic microorganisms were known to grow by the oxidation of ammonium or methane. Apart from respiration, oxygen was specifically utilized to activate the inert substrates. The presumed obligatory need for oxygen may have resisted the search for microorganisms that are capable of the anaerobic oxidation of ammonium and of methane. However extremely slowly growing, these "impossible" organisms exist and they found other means to tackle ammonium and methane. Anaerobic ammonium-oxidizing (anammox) bacteria use the oxidative power of nitric oxide (NO) by forging this molecule to ammonium, thereby making hydrazine (N2H4). Nitrite-dependent anaerobic methane oxidizers (N-DAMO) again take advantage of NO, but now apparently disproportionating the compound into dinitrogen and dioxygen gas. This intracellularly produced dioxygen enables N-DAMO bacteria to adopt an aerobic mechanism for methane oxidation.Although our understanding is only emerging how hydrazine synthase and the NO dismutase act, it seems clear that reactions fully rely on metal-based catalyses known from other enzymes. Metal-dependent conversions not only hold for these key enzymes, but for most other reactions in the central catabolic pathways, again supported by well-studied enzymes from model organisms, but adapted to own specific needs. Remarkably, those accessory catabolic enzymes are not unique for anammox bacteria and N-DAMO. Close homologs are found in protein databases where those homologs derive from (partly) known, but in most cases unknown species that together comprise an only poorly comprehended microbial world.

The transition element molybdenum (Mo) is of primordial importance for biological systems, because it is required by enzymes catalyzing key reactions in the global carbon, sulfur, and nitrogen metabolism. To gain biological activity, Mo has to be complexed by a special cofactor. With the exception of bacterial nitrogenase, all Mo-dependent enzymes contain a unique pyranopterin-based cofactor coordinating a Mo atom at their catalytic site. Various types of reactions are catalyzed by Mo-enzymes in prokaryotes including oxygen atom transfer, sulfur or proton transfer, hydroxylation, or even nonredox reactions. Mo-enzymes are widespread in prokaryotes and many of them were likely present in the Last Universal Common Ancestor. To date, more than 50--mostly bacterial--Mo-enzymes are described in nature. In a few eubacteria and in many archaea, Mo is replaced by tungsten bound to the same unique pyranopterin. How Mo-cofactor is synthesized in bacteria is reviewed as well as the way until its insertion into apo-Mo-enzymes.

The generation of new enzymatic activities has mainly relied on repurposing the interiors of preexisting protein folds because of the challenge in designing functional, three-dimensional protein structures from first principles. Here we report an artificial metallo-β-lactamase, constructed via the self-assembly of a structurally and functionally unrelated, monomeric redox protein into a tetrameric assembly that possesses catalytic zinc sites in its interfaces. The designed metallo-β-lactamase is functional in the Escherichia coli periplasm and enables the bacteria to survive treatment with ampicillin. In vivo screening of libraries has yielded a variant that displays a catalytic proficiency [(k(cat)/K(m))/k(uncat)] for ampicillin hydrolysis of 2.3 × 10(6) and features the emergence of a highly mobile loop near the active site, a key component of natural β-lactamases to enable substrate interactions., (Copyright © 2014, American Association for the Advancement of Science.)

Type-1 copper proteins participate in redox reactions and biological catalysis. Significant variation exists within the electronic structure of type-1 copper sites, producing both blue and green proteins. Classical, "blue" sites have been extensively studied, but "green" sites have been poorly characterized. We recently discovered a green copper protein, called auracyanin D. Here, we report a series of axial ligand mutations in auracyanin D, and characterize the resulting spectral and redox changes. The resulting mutants appear blue, green, and red and vary in redox potential from +56mV to +786mV. This is the largest change in redox potential to date for any type-1 center. We found that in this green protein, modifications of the axial ligand produce significantly larger changes than similar mutations in blue type-1 copper sites., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background & Aims: Little is known about factors that promote gastric carcinogenesis. We analyzed multiple microarray data sets for messenger RNAs (mRNAs) that were increased significantly in human gastric tumor samples, compared with the adjacent normal gastric tissue. We found expression of tripartite motif 59 (TRIM59), which encodes a putative ubiquitin ligase, to be increased, and investigated its effects in gastric cancer cell lines., Methods: We analyzed microarray data sets from the Oncomine database. We used quantitative polymerase chain reaction and immunoblotting to measure levels of TRIM59 mRNA and protein in 50 human gastric cancer and paired normal tissues, obtained from Renji Hospital and the First Affiliated Hospital of Nanchang University, in China. We also measured protein levels in the gastric epithelial cell line GES-1; the cancer cell lines MKN45, AGS, SGC7901, BGC823, Snu5, N87, and Snu1; and in tissue arrays of 108 human gastric tumors. TRIM59 was knocked down and overexpressed in gastric cancer cell lines, and the effects on proliferation, clone formation, migration, and growth of xenograft tumors in nude mice were assessed. TRIM59-related signaling pathways were examined by immunoblotting and quantitative polymerase chain reaction. We analyzed interactions among TRIM59, P53, and ubiquitin in immunoprecipitation studies., Results: Levels of TRIM59 mRNA and protein were increased significantly in gastric tumors compared with nontumor tissues; increased levels were associated with advanced tumor stage and shorter patient survival times. TRIM59 knockdown reduced proliferation, clone formation, and migration of gastric cancer cell lines, as well as growth of xenograft tumors in nude mice; overexpression of TRIM59 had the opposite effects. TRIM59 interacted physically with P53, increasing its ubiquitination and degradation. Increased levels of TRIM59 in human gastric tumors correlated with reduced expression of P53 target genes., Conclusions: The putative ubiquitin ligase TRIM59 is up-regulated in human gastric tumors compared with nontumor tissues. Levels of TRIM59 correlate with tumor progression and patient survival times. TRIM59 interacts with P53, promoting its ubiquitination and degradation, and TRIM59 might promote gastric carcinogenesis via this mechanism., (Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Mononuclear iron enzymes can tightly bind non-activating metals. How do cells avoid mismetallation? The model bacterium Escherichia coli may control its metal pools so that thermodynamics favor the correct metallation of each enzyme. This system is disrupted, however, by superoxide and hydrogen peroxide. These species oxidize ferrous iron and thereby displace it from many iron-dependent mononuclear enzymes. Ultimately, zinc binds in its place, confers little activity, and imposes metabolic bottlenecks. Data suggest that E. coli compensates by using thiols to extract the zinc and by importing manganese to replace the catalytic iron atom. Manganese resists oxidants and provides substantial activity., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Metal ion assimilation is essential for all forms of life. However, organisms must properly control the availability of these nutrients within the cell to avoid inactivating proteins by mismetallation. To safeguard against an imbalance between supply and demand in eukaryotes, intracellular compartments contain metal transporters that load and unload metals. Although the vacuoles of Saccharomyces cerevisiae and Arabidopsis thaliana are well established locales for the storage of copper, zinc, iron, and manganese, related compartments are emerging as important mediators of metal homeostasis. Here we describe these compartments and review their metal transporter complement., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

More than one third of all proteins are metalloproteins. They catalyze important reactions such as photosynthesis, nitrogen fixation and CO2 reduction. Metalloproteins such as the olfactory receptors also serve as highly elaborate sensors. Here we review recent developments in functional metalloprotein design using the genetic code expansion approach. We show that, through the site-specific incorporation of metal-chelating unnatural amino acids (UAAs), proton and electron transfer mediators, and UAAs bearing bioorthogonal reaction groups, small soluble proteins can recapitulate and expand the important functions of complex metalloproteins. Further developments along this route may result in cell factories and live-cell sensors with unprecedented efficiency and selectivity.

Flavodiiron proteins are known to have crucial and specific roles in photoprotection of photosystems I and II in cyanobacteria. The filamentous, heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 contains, besides the four flavodiiron proteins Flv1A, Flv2, Flv3A, and Flv4 present in vegetative cells, two heterocyst-specific flavodiiron proteins, Flv1B and Flv3B. Here, we demonstrate that Flv3B is responsible for light-induced O2 uptake in heterocysts, and that the absence of the Flv3B protein severely compromises the growth of filaments in oxic, but not in microoxic, conditions. It is further demonstrated that Flv3B-mediated photosynthetic O2 uptake has a distinct role in heterocysts which cannot be substituted by respiratory O2 uptake in the protection of nitrogenase from oxidative damage and, thus, in an efficient provision of nitrogen to filaments. In line with this conclusion, the Δflv3B strain has reduced amounts of nitrogenase NifHDK subunits and shows multiple symptoms of nitrogen deficiency in the filaments. The apparent imbalance of cytosolic redox state in Δflv3B heterocysts also has a pronounced influence on the amounts of different transcripts and proteins. Therefore, an O2-related mechanism for control of gene expression is suggested to take place in heterocysts.

The incidence of melanoma continues to rise globally and is increasing at a rate greater than any other cancer. To systematically search for new genes involved in melanomagenesis, we collated exome sequencing data from independent melanoma cohort datasets, including those in the public domain. We identified recurrent mutations that may drive melanoma growth, survival or metastasis, and which may hold promise for the design of novel therapies to treat melanoma. These included a frequent recurrent (i.e. hotspot) mutation in the 5' untranslated region of RPS27 in ~10% of samples. We show that the mutation expands the 5'TOP element, a motif known to regulate the expression of most of the ribosomal protein family, to which RPS27 belongs, and thus might sensitize the mutated transcript to growth-mediated regulation. This finding highlights not only the important role of non-protein coding genetic aberrations in cancer development but also their potential as novel therapeutic targets.

Molybdenum (Mo) is a trace element that is essential for important cellular processes. To gain biological activity, Mo must be complexed in the molybdenum cofactor (Moco), a pterin derivative of low molecular weight. Moco synthesis is a multi-step pathway that involves a variable number of genes in eukaryotes, which are assigned to four steps of eukaryotic Moco biosynthesis. Moco biosynthesis mutants lack any Moco-dependent enzymatic activities, including assimilation of nitrate (plants and fungi), detoxification of sulfite (humans and plants) and utilization of hypoxanthine as sole N-source (fungi). We report the first comprehensive genetic characterization of the Neurospora crassa (N. crassa) Moco biosynthesis pathway, annotating five genes which encode all pathway enzymes, and compare it with the characterized Aspergillus nidulans pathway. Biochemical characterization of the corresponding knock-out mutants confirms our annotation model, documenting the N. crassa/A. nidulans (fungal) Moco biosynthesis as unique, combining the organizational structure of both plant and human Moco biosynthesis genes., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Mycobacterium tuberculosis, the pathogen that causes tuberculosis, has evolved sophisticated mechanisms for evading assault by the human host. This review focuses on M. tuberculosis regulatory metalloproteins that are sensitive to exogenous stresses attributed to changes in the levels of gaseous molecules (i.e., molecular oxygen, carbon monoxide and nitric oxide) to elicit an intracellular response. In particular, we highlight recent developments on the subfamily of Whi proteins, redox sensing WhiB-like proteins that contain iron-sulfur clusters, sigma factors and their cognate anti-sigma factors of which some are zinc-regulated, and the dormancy survival regulon DosS/DosT-DosR heme sensory system. Mounting experimental evidence suggests that these systems contribute to a highly complex and interrelated regulatory network that controls M. tuberculosis biology. This review concludes with a discussion of strategies that M. tuberculosis has developed to maintain redox homeostasis, including mechanisms to regulate endogenous nitric oxide and carbon monoxide levels., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Zinc oxide nanoparticles (ZnONPs) are used in large quantities by the cosmetic, food and textile industries. Here we exposed Caenorhabditis elegans wild-type and a metal sensitive triple knockout mutant (mtl-1;mtl-2;pcs-1) to ZnONPs (0-50mg/L) to study strain and exposure specific effects on transcription, reactive oxygen species generation, the biomolecular phenotype (measured by Raman microspectroscopy) and key endpoints of the nematode life cycle (growth, reproduction and lifespan). A significant dissolution effect was observed, where dissolved ZnO constituted over 50% of total Zn within a two day exposure to the test medium, suggesting that the nominal exposure to pure ZnONPs represents in vivo, at best, a mixture exposure of ionic zinc and nanoparticles. Nevertheless, the analyses provided evidence that the metallothioneins (mtl-1 and mtl-2), the phytochelatin synthase (pcs-1) and an apoptotic marker (cep-1) were transcriptionally activated. In addition, the DCFH-DA assay provided in vitro evidence of the oxidative potential of ZnONPs in the metal exposure sensitive triple mutant. Raman spectroscopy highlighted that the biomolecular phenotype changes significantly in the mtl-1;mtl-2;pcs-1 triple knockout worm upon ZnONP exposure, suggesting that these metalloproteins are instrumental in the protection against cytotoxic damage. Finally, ZnONP exposure was shown to decrease growth and development, reproductive capacity and lifespan, effects which were amplified in the triple knockout. By combining diverse toxicological strategies, we identified that individuals (genotypes) housing mutations in key metalloproteins and phytochelatin synthase are more susceptible to ZnONP exposure, which underlines their importance to minimize ZnONP induced toxicity., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background: Conceptual parallels exist between bacterial and eukaryotic small-RNA (sRNA) pathways, yet relatively little is known about which protein may recognize and recruit bacterial sRNAs to interact with targets. In eukaryotes, Argonaute (AGO) proteins discharge such functions. The highly conserved bacterial YbeY RNase has structural similarities to the MID domain of AGOs. A limited study had indicated that in Sinorhizobium meliloti the YbeY ortholog regulates the accumulation of sRNAs as well as the target mRNAs, raising the possibility that YbeY may play a previously unrecognized role in bacterial sRNA regulation., Results: We have applied a multipronged approach of loss-of-function studies, genome-wide mRNA and sRNA expression profiling, pathway analysis, target prediction, literature mining and network analysis to unravel YbeY-dependent molecular responses of E. coli exposed to hydroxyurea (HU). Loss of ybeY function, which results in a marked resistance to HU, had global affects on sRNA-mediated gene expression. Of 54 detectable E. coli sRNAs in our microarray analysis, 30 sRNAs showed a differential expression upon HU stress, of which 28 sRNAs displayed a YbeY-dependent change in expression. These included 12 Hfq-dependent and 16 Hfq-independent sRNAs. We successfully identified at least 57 experimentally inferred sRNA-mRNA relationships. Further applying a 'context likelihood of relatedness' algorithm, we reverse engineered the YbeY-dependent Hfq-dependent sRNA-mRNA network as well as YbeY-dependent Hfq-independent sRNA-mRNA network., Conclusion: YbeY extensively modulates Hfq-dependent and independent sRNA-mRNA interactions. YbeY-dependent sRNAs have central roles in modulating cellular response to HU stress.

ATP:co(I)rrinoid adenosyltransferase (ACAT) enzymes convert vitamin B12 to coenzyme B12. EutT is the least understood ACAT. We report the purification of EutT to homogeneity and show that, in vitro, free dihydroflavins drive the adenosylation of cob(II)alamin bound to EutT. Results of chromatography analyses indicate that EutT is dimeric in solution, and unlike other ACATs, EutT catalyzes the reaction with sigmoidal kinetics indicative of positive cooperativity for cob(II)alamin. Maximal EutT activity was obtained after metalation with ferrous ions. EutT/Fe(II) protein lost all activity upon exposure to air and H2O2, consistent with previously reported results indicating that EutT was an oxygen-labile metalloprotein containing a redox-active metal. Results of in vivo and in vitro analyses of single-amino-acid variants affecting a HX11CCXXC(83) motif conserved in EutT proteins showed that residues His67, Cys80, and Cys83 were required for EutT function in vivo, while Cys79 was not. Unlike that of other variants, the activity of the EutT(C80A) variant was undetectable in vitro, suggesting that Cys80 was critical to EutT function. Results of circular dichroism studies indicate that the presence or absence of a metal ion does not affect protein folding. EutT can now be purified in the presence of oxygen and reactivated with ferrous ions for maximal activity.

In this work, we have developed a method that uses hydrogen-deuterium exchange (HDX) of C2-hydrogens of histidines coupled with mass spectrometry (MS) to identify Zn-bound histidines in metalloproteins. This method relies on differences in HDX reaction rates of Zn-bound and Zn-free His residues. Using several model peptides and proteins, we find that all Zn-bound His residues have substantially lower HDX reaction rates in the presence of the metal. The vast majority of non-Zn-binding His residues undergo no significant changes in HDX reaction rates when their reactivity is compared in the presence and absence of Zn. Using this new approach, we then determined the Zn binding site of β-2-microglobulin, a protein associated with metal-induced amyloidosis. Together, these results suggest that HDX-MS of His C2-hydrogens is a promising new method for identifying Zn-bound histidines in metalloproteins.

Given the similar chemical properties of zinc and cadmium, zinc finger domains have been often proposed as mediators of the toxic and carcinogenic effects exerted by this xenobiotic metal. The effects of zinc replacement by cadmium in different eukaryotic zinc fingers have been reported. In the present work, to evaluate the effects of such substitution in the prokaryotic zinc finger, we report a detailed study of its functional and structural consequences on the Ros DNA binding domain (Ros87). We show that this protein, which bears important structural differences with respect to the eukaryotic domains, appears to structurally tolerate the zinc to cadmium substitution and the presence of cadmium does not affect the DNA binding activity of the protein. Moreover, we show for the first time how zinc to cadmium replacement can also take place in a cellular context. Our findings both complement and extend previous results obtained for different eukaryotic zinc fingers, suggesting that metal substitution in zinc fingers may be of relevance to the toxicity and/or carcinogenicity mechanisms of this metal.

Burkholderia thailandensis is closely related to Burkholderia pseudomallei, a bacterial pathogen and the causative agent of melioidosis. B. pseudomallei can survive and persist within a hypoxic environment for up to one year and has been shown to grow anaerobically in the presence of nitrate. Currently, little is known about the role of anaerobic respiration in pathogenesis of melioidosis. Using B. thailandensis as a model, a library of 1344 transposon mutants was created to identify genes required for anaerobic nitrate respiration. One transposon mutant (CA01) was identified with an insertion in BTH_I1704 (moeA), a gene required for the molybdopterin biosynthetic pathway. This pathway is involved in the synthesis of a molybdopterin cofactor required for a variety of molybdoenzymes, including nitrate reductase. Disruption of molybdopterin biosynthesis prevented growth under anaerobic conditions, when using nitrate as the sole terminal electron acceptor. Defects in anaerobic respiration, nitrate reduction, motility and biofilm formation were observed for CA01. Mutant complementation with pDA-17:BTH_I1704 was able to restore anaerobic growth on nitrate, nitrate reductase activity and biofilm formation, but did not restore motility. This study highlights the potential importance of molybdoenzyme-dependent anaerobic respiration in the survival and virulence of B. thailandensis., (Copyright © 2013 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

The crystal structure of formiminoglutamase from Trypanosoma cruzi (TcFIGase) is reported at 1.85 Å resolution. Although the structure of this enzyme was previously determined by the Structural Genomics of Pathogenic Protozoa Consortium (PDB accession code 2A0M), this structure was determined at low pH and lacked bound metal ions; accordingly, the protein was simply annotated as "arginase superfamily protein" with undetermined function. We show that reconstitution of this protein with Mn²⁺ confers maximal catalytic activity in the hydrolysis of formiminoglutamate to yield glutamate and formamide, thereby demonstrating that this protein is a metal-dependent formiminoglutamase. Equilibration of TcFIGase crystals with MnCl₂ at higher pH yields a binuclear manganese cluster similar to that observed in arginase, except that the histidine ligand to the Mn²⁺(A) ion of arginase is an asparagine ligand (N114) to the Mn²⁺(A) ion of TcFIGase. The crystal structure of N114H TcFIGase reveals a binuclear manganese cluster essentially identical to that of arginase, but the mutant exhibits a modest 35% loss of catalytic efficiency (k(cat)/K(M)). Interestingly, when TcFIGase is prepared and crystallized in the absence of reducing agents at low pH, a disulfide linkage forms between C35 and C242 in the active site. When reconstituted with Mn²⁺ at higher pH, this oxidized enzyme exhibits a modest 33% loss of catalytic efficiency. Structure determinations of the metal-free and metal-bound forms of oxidized TcFIGase reveal that although disulfide formation constricts the main entrance to the active site, other structural changes open alternative channels to the active site that may help sustain catalytic activity.

Molybdoenzymes contain a molybdenum cofactor in their active site to catalyze various redox reactions in all domains of life. To decipher crucial steps during their biogenesis, the TorA molybdoenzyme of Escherichia coli had played a major role to understand molybdoenzyme maturation process driven by specific chaperones. TorD, the specific chaperone of TorA, is also involved in TorA protection. Here, we show that immature TorA (apoTorA) is degraded in vivo and in vitro by the Lon protease. Lon interacts with apoTorA but not with holoTorA. Lon and TorD compete for apoTorA binding but TorD binding protects apoTorA against degradation. Lon is the first protease shown to eliminate an immature or misfolded molybdoenzyme probably by targeting its inactive catalytic site., (© 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Metaproteomics enables the investigation of the protein repertoire expressed by complex microbial communities. However, to unleash its full potential, refinements in bioinformatic approaches for data analysis are still needed. In this context, sequence databases selection represents a major challenge. This work assessed the impact of different databases in metaproteomic investigations by using a mock microbial mixture including nine diverse bacterial and eukaryotic species, which was subjected to shotgun metaproteomic analysis. Then, both the microbial mixture and the single microorganisms were subjected to next generation sequencing to obtain experimental metagenomic- and genomic-derived databases, which were used along with public databases (namely, NCBI, UniProtKB/SwissProt and UniProtKB/TrEMBL, parsed at different taxonomic levels) to analyze the metaproteomic dataset. First, a quantitative comparison in terms of number and overlap of peptide identifications was carried out among all databases. As a result, only 35% of peptides were common to all database classes; moreover, genus/species-specific databases provided up to 17% more identifications compared to databases with generic taxonomy, while the metagenomic database enabled a slight increment in respect to public databases. Then, database behavior in terms of false discovery rate and peptide degeneracy was critically evaluated. Public databases with generic taxonomy exhibited a markedly different trend compared to the counterparts. Finally, the reliability of taxonomic attribution according to the lowest common ancestor approach (using MEGAN and Unipept software) was assessed. The level of misassignments varied among the different databases, and specific thresholds based on the number of taxon-specific peptides were established to minimize false positives. This study confirms that database selection has a significant impact in metaproteomics, and provides critical indications for improving depth and reliability of metaproteomic results. Specifically, the use of iterative searches and of suitable filters for taxonomic assignments is proposed with the aim of increasing coverage and trustworthiness of metaproteomic data.

MPS-1 (metallopanstimulin-1), also known as ribosomal protein S27, was overexpressed in gastric cancer cells. However, how MPS-1 contributes to gastric carcinogenesis has not been well characterized. Here, we show that high expression of MPS-1 was observed in gastric cancer tissues and associated with gastric cancer cell metastasis. Alteration of MPS-1 expression regulates invasion and migration of gastric cancer cells both in vitro and in vivo. Furthermore, by using Signal-Net and cluster analyses of microarray data we identified integrin β4 (ITGB4) as a downstream target of MPS-1 that mediates its effects on cell metastasis. Knockdown of MPS-1 expression in gastric cancer cells led to significant reduction of ITGB4 expression at both the RNA and protein levels. Mechanically, we found that overexpression of ITGB4 in MPS-1 knockdown cells largely recovers the ability of invasion and migration. Conversely, knockdown of ITGB4 partially reduced cell invading/migrating ability induced by MPS-1 overexpression. Moreover, MPS-1 and ITGB4 expressions are positively correlated in gastric cancer cell lines and tissues. Finally, the survival analyses show that the expression of MPS-1 and ITGB4 is associated with poor outcomes in gastric cancer patients. Collectively, our findings suggest that MPS-1 regulates cell invasiveness and migration partially through ITGB4 and that MPS-1/ITGB4 signaling axis may serve as therapeutic targets in the treatment of gastric cancer.

The metal sites of electron transfer proteins are tuned for function. The type 1 copper site is one of the most utilized metal sites in electron transfer reactions. This site can be tuned by the protein environment from +80 mV to +680 mV in typical type 1 sites. Accompanying this huge variation in midpoint potentials are large changes in electronic structure, resulting in proteins that are blue, green, or even red. Here, we report a family of blue copper proteins, the auracyanins, from the filamentous anoxygenic phototroph Chloroflexus aurantiacus that display the entire known spectral and redox variations known in the type 1 copper site. C. aurantiacus encodes four auracyanins, labeled A-D. The midpoint potentials vary from +83 mV (auracyanin D) to +423 mV (auracyanin C). The electronic structures vary from classical blue copper UV-vis absorption spectra (auracyanin B) to highly perturbed spectra (auracyanins C and D). The spectrum of auracyanin C is temperature-dependent. The expansion and divergent nature of the auracyanins is a previously unseen phenomenon.

Alternative Complex III (ACIII) is a multisubunit integral membrane protein electron transfer complex that is proposed to be an energy-conserving functional replacement for the bacterial cytochrome bc1 or b6f complexes. Clues to the structure and function of this novel complex come from its relation to other bacterial enzyme families. The ACIII complex has menaquinone: electron acceptor oxidoreductase activity and contains protein subunits with multiple Fe-S centers and c-type hemes. ACIII is found in a diverse group of bacteria, including both phototrophic and nonphototrophic taxa. In the phototrophic filamentous anoxygenic phototrophs, the electron acceptor is the small blue copper protein auracyanin instead of a soluble cytochrome. Recent work on ACIII and the copper protein auracyanin is reviewed with focus on the photosynthetic systems and potential electron transfer pathways and mechanisms. Taken together, the ACIII complexes constitute a unique system for photosynthetic electron transfer and energy conservation. This article is part of a Special Issue entitled: Respiratory Complex III and related bc complexes., (Copyright © 2013 Elsevier B.V. All rights reserved.)

LOS5/ABA3 gene encoding molybdenum cofactor sulphurase is involved in aldehyde oxidase (AO) activity in Arabidopsis, which indirectly regulates ABA biosynthesis and increased stress tolerance. Here, we used a constitutive super promoter to drive LOS5/ABA3 overexpression in soybean (Glycine max L.) to enhance drought tolerance in growth chamber and field conditions. Expression of LOS5/ABA3 was up-regulated by drought stress, which led to increasing AO activity and then a notable increase in ABA accumulation. Transgenic soybean under drought stress had reduced water loss by decreased stomatal aperture size and transpiration rate, which alleviated leaf wilting and maintained higher relative water content. Exposed to drought stress, transgenic soybean exhibited reduced cell membrane damage by reducing electrolyte leakage and production of malondialdehyde and promoting proline accumulation and antioxidant enzyme activities. Also, overexpression of LOS5/ABA3 enhanced expression of stress-up-regulated genes. Furthermore, the seed yield of transgenic plants is at least 21% higher than that of wide-type plants under drought stress conditions in the field. These data suggest that overexpression of LOS5/ABA3 could improve drought tolerance in transgenic soybean via enhanced ABA accumulation, which could activate expression of stress-up-regulated genes and cause a series of physiological and biochemical resistant responses., (© 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.)

Background: The development and progression of colorectal cancer (CRC) involve a complex process of multiple genetic changes. Tumor suppressor p53 is capable of determining the fate of CRC cells. However, the role of a p53-inducible modulator, ribosomal protein S27-like (RPS27L), in CRC is unknown., Methods: Here, the differential expression of RPS27L was examined in the feces and colonic tissues of CRC patients, to explore its possible correlation with patient survival and to investigate the cellular mechanisms underlying their clinical outcomes. Eighty intermediate-stage CRC patients (42 at stage II and 38 at stage III) were divided into two groups according to their fecal RPS27L mRNA levels. The survival probabilities of the groups were estimated using the Kaplan-Meier method. The RPS27L protein in the colonic tissues of stage III patients with different prognoses was further examined immunohistochemically. RPS27L expression in LoVo cells was manipulated to examine the possible cellular responses in vitro., Results: Elevated RPS27L expression, in either feces or tissues, was related to a better prognosis. In vitro, RPS27L-expressing LoVo cells ceased DNA synthesis and apoptotic activity while the expression of their DNA repair molecules was upregulated., Conclusions: Elevated RPS27L may improve the prognoses of certain CRC patients by enhancing the DNA repair capacity of their colonic cells, and can be determined in feces. By integrating clinical, molecular, and cellular data, our study demonstrates that fecal RPS27L may be a useful index for predicting prognoses and guiding personalized therapeutic strategies, especially in patients with intermediate-stage CRC.

The continued interest in protein engineering has led to intense efforts in developing novel stable enzymes, which could not only give boost to industrial and biomedical applications, but also enhance our understanding of the structure-function relationships of proteins. We present here the generation of three hybrid proteins of cytochrome c (cyt c) and peroxidase via structure-based rational mutagenesis of cyt c. Several residues (positions 67, 70, 71 and 80) in the distal heme region of cyt c were mutated to the highly conserved amino acids in the heme pocket of peroxidases. The multiple mutants were found to exhibit high peroxidase activity and conserve the impressive stability of cyt c. We expect that this strategy could be extended to other cases of metalloprotein engineering, and lead to the development of stable and active biocatalysts for industrial uses. Besides, this study also provides insight into the structure-function relationships of hemoproteins.

The green Cu-NirK from Haloferax mediterranei (Cu-NirK) has been expressed, refolded and retrieved as a trimeric enzyme using an expression method developed for halophilic Archaea. This method utilizes Haloferax volcanii as a halophilic host and an expression vector with a constitutive and strong promoter. The enzymatic activity of recombinant Cu-NirK was detected in both cellular fractions (cytoplasmic fraction and membranes) and in the culture media. The characterization of the enzyme isolated from the cytoplasmic fraction as well as the culture media revealed important differences in the primary structure of both forms indicating that Hfx. mediterranei could carry out a maturation and exportation process within the cell before the protein is exported to the S-layer. Several conserved signals found in Cu-NirK from Hfx. mediterranei sequence indicate that these processes are closely related to the Tat system. Furthermore, the N-terminal sequence of the two Cu-NirK subunits constituting different isoforms revealed that translation of this protein could begin at two different points, identifying two possible start codons. The hypothesis proposed in this work for halophilic Cu-NirK processing and exportation via the Tat system represents the first approximation of this mechanism in the Halobacteriaceae family and in Prokarya in general., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Arachnomelia syndrome (AS) is a recessive inherited disease in cattle. Although the arachnomelia phenotypes are virtually identical in Brown Swiss and Simmental cattle, the causative mutation are different, which are a 1 bp insertion c.363-364insG in the sulfite oxidase (SUOX) gene and a 2 bp deletion c.1224_1225delCA in the molybdenum cofactor syn-thesis step 1 (MOCS1) gene, respectively. In the current study, combining fluorescence PCR with capillary electrophoresis technology, an automatic fluorescence method was established, which could detect the two causative loci rapidly and cor-rectly with a single reaction. Samples from 51 Simmental bulls, 80 cows mated artificially using semen of Simmental bulls and their resulted 106 progeny, together with 55 Xinjiang Brown were collected and used for validation of the newly de-signed methods. Our results have laid a foundation for screening AS disease causing mutations in Chinese cattle.

The genetic identity and cofactor composition of the bacterial tellurate reductase are currently unknown. In this study, we examined the requirement of molybdopterin biosynthesis and molybdate transporter genes for tellurate reduction in Escherichia coli K-12. The results show that mutants deleted of the moaA, moaB, moaE, or mog gene in the molybdopterin biosynthesis pathway lost the ability to reduce tellurate. Deletion of the modB or modC gene in the molybdate transport pathway also resulted in complete loss of tellurate reduction activity. Genetic complementation by the wild-type sequences restored tellurate reduction activity in the mutant strains. These findings provide genetic evidence that tellurate reduction in E. coli involves a molybdoenzyme.

Legionella pneumophila is a water-borne bacterium that causes pneumonia in humans. PlcA and PlcB are two previously defined L. pneumophila proteins with homology to the phosphatidylcholine-specific phospholipase C (PC-PLC) of Pseudomonas fluorescens. Additionally, we found that Lpg0012 shows similarity to PLCs and has been shown to be a Dot/Icm-injected effector, CegC1, which is designated here as PlcC. It remained unclear, however, whether these L. pneumophila proteins exhibit PLC activity. PlcC expressed in Escherichia coli hydrolyzed a broad phospholipid spectrum, including PC, phosphatidylglycerol (PG), and phosphatidylinositol. The addition of Zn(2+) ions activated, whereas EDTA inhibited, PlcC-derived PLC activity. Protein homology search revealed that the three Legionella enzymes and P. fluorescens PC-PLC share conserved domains also present in uncharacterized fungal proteins. Fifteen conserved amino acids were essential for enzyme activity as identified via PlcC mutagenesis. Analysis of defined L. pneumophila knock-out mutants indicated Lsp-dependent export of PG-hydrolyzing PLC activity. PlcA and PlcB exhibited PG-specific activity and contain a predicted Sec signal sequence. In line with the reported requirement of host cell contact for Dot/Icm-dependent effector translocation, PlcC showed cell-associated PC-specific PLC activity after bacterial growth in broth. A PLC triple mutant, but not single or double mutants, exhibited reduced host killing in a Galleria mellonella infection model, highlighting the importance of the three PLCs in pathogenesis. In summary, we describe here a novel Zn(2+)-dependent PLC family present in Legionella, Pseudomonas, and fungi with broad substrate preference and function in virulence.

Metalloproteins have long been recognized as key determinants of endogenous contrast in magnetic resonance imaging (MRI) of biological subjects. More recently, both natural and engineered metalloproteins have been harnessed as biotechnological tools to probe gene expression, enzyme activity, and analyte concentrations by MRI. Metalloprotein MRI probes are paramagnetic and function by analogous mechanisms to conventional gadolinium or iron oxide-based MRI contrast agents. Compared with synthetic agents, metalloproteins typically offer worse sensitivity, but the possibilities of using protein engineering and targeted gene expression approaches in conjunction with metalloprotein contrast agents are powerful and sometimes definitive strengths. This review summarizes theoretical and practical aspects of metalloprotein-based contrast agents, and discusses progress in the exploitation of these proteins for molecular imaging applications., (Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Although largely deemed as structurally conserved, catalytic metal ion sites can rearrange, thereby contributing to enzyme evolvability. Here, we show that in paraoxonase-1, a lipo-lactonase, catalytic promiscuity and divergence into an organophosphate hydrolase are correlated with an alternative mode of the catalytic Ca(2+). We describe the crystal structures of active-site mutants bearing mutations at position 115. The histidine at this position acts as a base to activate the lactone-hydrolyzing water molecule. Mutations to Trp or Gln indeed diminish paraoxonase-1's lactonase activity; however, the promiscuous organophosphate hydrolase activity is enhanced. The structures reveal a 1.8-Å upward displacement towards the enzyme's surface of the catalytic Ca(2+) in the His115 mutants and configurational changes in the ligating side chains and water molecules, relative to the wild-type enzyme. Biochemical analysis and molecular dynamics simulations suggest that this alternative, upward metal mode mediates the promiscuous hydrolysis of organophosphates. The upward Ca(2+) mode observed in the His115 mutants also appears to mediate the wild type's paraoxonase activity. However, whereas the upward mode dominates in the Trp115 mutant, it is scarcely populated in wild type. Thus, the plasticity of active-site metal ions may permit alternative, latent, promiscuous activities and also provide the basis for the divergence of new enzymatic functions., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

During rotavirus infection, replication and packaging of the viral genome occur in viral factories, termed viroplasms. The viral nonstructural protein NSP5 is a major building block of viroplasms; it recruits the viral polymerase VP1, the core protein VP2, and the ATPase NSP2 inside the viroplasm to form the viral replication complex. Here we report that NSP5 is a unique viral metalloprotein that coordinates a [2Fe-2S] iron-sulfur cluster as demonstrated by the metal and labile sulfide contents, UV-visible light absorption, and electron paramagnetic resonance. Point mutations in NSP5 allowed us to identify C171 and C174, arranged in a CXC motif, as essential residues for cluster coordination. When coexpressed with NSP2, an NSP5 mutant devoid of the iron-sulfur cluster still forms viroplasm-like structures. The cluster is therefore neither involved in the interaction with NSP2 nor in the formation of viroplasm-like structures and thus presumably in viroplasm formation. Finally, we show using microscale thermophoresis that the iron-sulfur cluster modulates the affinity of NSP5 for single-stranded RNA. Because the cluster is near the binding sites of both the polymerase VP1 and the ATPase NSP2, we anticipate that this cluster is crucial for NSP5 functions, in either packaging or replication of the viral genome.

Sut1 is a transcriptional regulator of the Zn(II)(2)Cys(6) family in the budding yeast Saccharomyces cerevisiae. The only function that has been attributed to Sut1 is sterol uptake under anaerobic conditions. Here, we show that Sut1 is also expressed in the presence of oxygen, and we identify a novel function for Sut1. SUT1 overexpression blocks filamentous growth, a response to nutrient limitation, in both haploid and diploid cells. This inhibition by Sut1 is independent of its function in sterol uptake. Sut1 downregulates the expression of GAT2, HAP4, MGA1, MSN4, NCE102, PRR2, RHO3, and RHO5. Several of these Sut1 targets (GAT2, HAP4, MGA1, RHO3, and RHO5) are essential for filamentation in haploids and/or diploids. Furthermore, the expression of the Sut1 target genes, with the exception of MGA1, is induced during filamentous growth. We also show that SUT1 expression is autoregulated and inhibited by Ste12, a key transcriptional regulator of filamentation. We propose that Sut1 partially represses the expression of GAT2, HAP4, MGA1, MSN4, NCE102, PRR2, RHO3, and RHO5 when nutrients are plentiful. Filamentation-inducing conditions relieve this repression by Sut1, and the increased expression of Sut1 targets triggers filamentous growth.

Metalloproteins (MPs) comprise one-third of all known protein structures. This diverse set of proteins contain a plethora of unique inorganic moieties capable of performing chemistry that would otherwise be impossible using only the amino acids found in nature. Most of the well-studied MPs are generally viewed as being very rigid in structure, and it is widely thought that the properties of the metal centers are primarily determined by the small fraction of amino acids that make up the local environment. Here we examine both theoretically and experimentally whether distal regions can influence the metal center in the diabetes drug target mitoNEET. We demonstrate that a loop (L2) 20 Å away from the metal center exerts allosteric control over the cluster binding domain and regulates multiple properties of the metal center. Mutagenesis of L2 results in significant shifts in the redox potential of the [2Fe-2S] cluster and orders of magnitude effects on the rate of [2Fe-2S] cluster transfer to an apo-acceptor protein. These surprising effects occur in the absence of any structural changes. An examination of the native basin dynamics of the protein using all-atom simulations shows that twisting in L2 controls scissoring in the cluster binding domain and results in perturbations to one of the cluster-coordinating histidines. These allosteric effects are in agreement with previous folding simulations that predicted L2 could communicate with residues surrounding the metal center. Our findings suggest that long-range dynamical changes in the protein backbone can have a significant effect on the functional properties of MPs.

Chlamydomonas reinhardtii is a eukaryotic, unicellular, biflagellate green alga. In Chlamydomonas nitrate assimilation depends on the activity of the enzyme nitrate reductase that requires the molybdenum cofactor (Moco). We have characterized the Chlamydomonas strains 102 (nit5nit6), 104 (nit4nit5) and 106 (nit6). These mutations lead to the exchanges V171A (nit4) and G183D (nit6) in the CNX1E protein of Moco biosynthesis that result in Moco deficiency and hence inability to grow on nitrate. CNX1E inserts molybdenum into molybdopterin (MPT) to yield Moco through the intermediate MPT-AMP. The CNX1E variant G183D is unable to bind MPT-AMP. However, the CNX1E variant V171A is able to bind MPT-AMP but is not able to hydrolyze it and insert the molybdenum to obtain Moco. We show that intermolecular complementation between the CNX1E variants V171A and G183D both in vivo and in vitro restored its activity. The molecular characterization of strain 106 led us to discover the presence of wild type and nit6 CNX1E alleles in this strain (nit6/+). Then, we found out that 106 is indeed a diploid strain. It is proposed that 102, the strain from which 106 derived, may contain a genetic alteration that produces an aberrant meiotic division of the mature zygotes., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

Sequencing of entire bacterial genomes has led to the identification of many membrane-associated transporters, including several multidrug resistance transport proteins, in recent years. However, the regulators and signaling pathways involved in the expression of these genes remain largely unknown. In this study, we have identified Ms2173, a GntR/FadR family transcription factor, as a novel global regulator in Mycobacterium smegmatis. Ms2173 was found to specifically recognize a 15-bp palindromic motif and to broadly regulate expression of 292 genes, including 37 genes that encode membrane-associated transport proteins. Copper ions induced Ms2173 to form inactive proteins lacking DNA-binding activity. Ms2173 was shown to function as a repressor of its target genes. Interestingly, we found that the function of Ms2173 was linked to mycobacterial drug resistance. Compared with the substantially enhanced drug resistance in the Ms2173-deleted mutant strain, the strains overexpressing Ms2173 were more sensitive to anti-tuberculosis drugs than the wild-type strain. Additionally, copper ions could partially counteract the in vivo function of Ms2173. We have thus characterized the first mycobacterial GntR/Fad-like transcription factor that functions as a copper ion-responsive global repressor that we have renamed GfcR. These findings further enhance our understanding of membrane-associated transporter regulation and drug resistance in mycobacteria.

We amplified and analyzed the complete cDNA of metalloprotein II (MP II) from the somatic muscle of the polychaete Perinereis aibuhitensis, the full length cDNA is 904 bp encoding 119 amino acids. The MP II cDNA sequence was subjected to BLAST searching in NCBI and was found to share high homology with hemerythrin of other worms. MP II expression of P. aibuhitensis exposed to single and combined metals (Cu(II), Cd(II)) was analyzed using real time-PCR. MP II mRNA expression increased at the start of Cu(II) exposure, then decreased and finally return to the normal level. Expression pattern of MP II under Cd(II) exposure was time- and dose-dependent. MP II expression induced by a combination of Cd(II) and Cu(II) was similar to that induced by Cd(II) alone., (Copyright © 2012 Elsevier B.V. All rights reserved.)

[FeFe]-hydrogenases are iron-sulfur proteins characterized by a complex active site, the H-cluster, whose assembly requires three conserved maturases. HydE and HydG are radical S-adenosylmethionine enzymes that chemically modify a H-cluster precursor on HydF, a GTPase with a dual role of scaffold on which this precursor is synthesized, and carrier to transfer it to the hydrogenase. Coordinate structural and functional relationships between HydF and the two other maturases are crucial for the H-cluster assembly. However, to date only qualitative analysis of this protein network have been provided. In this work we showed that the interactions of HydE and HydG with HydF are distinct events, likely occurring in a precise functional order driven by different kinetic properties, independently of the HydF GTPase activity, which is instead involved in the dissociation of the maturases from the scaffold. We also found that HydF is able to interact with the hydrogenase only when co-expressed with the two other maturases, indicating that under these conditions it harbors per se all the structural elements needed to transfer the H-cluster precursor, thus completing the maturation process. These results open new working perspectives aimed at improving the knowledge of how these complex metalloenzymes are biosynthesized.

The thio-modification of tRNA that occurs in virtually all organisms affects the accuracy and efficiency of protein translation and is therefore biologically important. However, the molecular mechanism responsible for this tRNA modification in plants is largely unclear. We demonstrate here that Arabidopsis sulfurtransferase Cnx5, a ubiquitin-activating enzyme-like (UBA) protein involved in molybdopterin (MPT) biosynthesis, is strictly required for the thio-modification of cytosolic tRNAs in vivo. A previously uncharacterized ubiquitin-like (Ubl) protein Urm11 is also essential for tRNA thio-modification in Arabidopsis. When expressed in Saccharomyces cerevisiae, Cnx5 and Urm11 can substitute for the corresponding yeast orthologs ScUba4 and ScUrm1, respectively, in the thio-modification of yeast cytosolic tRNAs. However, another Ubl protein, Cnx7 of Arabidopsis, which is involved in MPT biosynthesis in conjunction with Cnx5, cannot replace yeast ScUrm1. Interestingly, the expression of a mutant form of Cnx7 in which the carboxyl-terminal six amino acids are substituted by those of Urm11 can significantly restore the thio-modification of tRNAs in the yeast urm1Δ mutant. These findings suggest that in Arabidopsis the common UBA protein Cnx5 collaborates with two functionally differentiated Ubl proteins, Urm11 and Cnx7, in the thio-modification of tRNA and MPT biosynthesis, respectively. Phylogenetic analysis revealed that although most eukaryotes contained a Cnx5-Urm11 ortholog pair and the tRNA thio-modification some fungi, including S. cerevisiae, had lost the Cnx7 ortholog and the ability to synthesize the molybdenum cofactor.

The twin-arginine translocation (Tat) pathway is dedicated to the transport of fully folded proteins across the cytoplasmic membranes of many bacteria and the chloroplast thylakoidal membrane. Accordingly, Tat-dependently translocated proteins are known to be delivered to the periplasm of Gram-negative bacteria, the growth medium of Gram-positive bacteria, and the thylakoid lumen. Here, we present the first example of a protein, YkuE of Bacillus subtilis, that is specifically targeted by the Tat pathway to the cell wall of a Gram-positive bacterium. The cell wall binding of YkuE is facilitated by electrostatic interactions. Interestingly, under particular conditions, YkuE can also be targeted to the cell wall in a Tat-independent manner. The biological function of YkuE was so far unknown. Our present studies show that YkuE is a metal-dependent phosphoesterase that preferentially binds manganese and zinc.