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copper starvation-inducible protein for cytochrome oxidase biogenesis in bradyrhizobium japonicum.microarray analysis of bradyrhizobium japonicum grown under copper limitation uncovered five genes named pcuabcde, which are co-transcribed and co-regulated as an operon. the predicted gene products are periplasmic proteins (pcua, pcuc, and pcud), a tonb-dependent outer membrane receptor (pcub), and a cytoplasmic membrane-integral protein (pcue). homologs of pcuc and pcue had been discovered in other bacteria, namely pcu(a)c and ycnj, where they play a role in cytochrome oxidase biogenesis and c ...201223012364
spectroscopic and kinetic investigation of the fully reduced and mixed valence states of ba3-cytochrome c oxidase from thermus thermophilus: a fourier transform infrared (ftir) and time-resolved step-scan ftir study.the complete understanding of a molecular mechanism of action requires the thermodynamic and kinetic characterization of different states and intermediates. cytochrome c oxidase reduces o(2) to h(2)o, a reaction coupled to proton translocation across the membrane. therefore, it is necessary to undertake a thorough characterization of the reduced form of the enzyme and the determination of the electron transfer processes and pathways between the redox-active centers. in this study fourier transfo ...201222927441
a three-dimensional topology of complex i inferred from evolutionary correlations.the quaternary structure of eukaryotic nadh:ubiquinone oxidoreductase (complex i), the largest complex of the oxidative phosphorylation, is still mostly unresolved. furthermore, it is unknown where transiently bound assembly factors interact with complex i. we therefore asked whether the evolution of complex i contains information about its 3d topology and the binding positions of its assembly factors. we approached these questions by correlating the evolutionary rates of eukaryotic complex i su ...201222857522
structural determinants of the β-selectivity of a bacterial aminotransferase.chiral β-amino acids occur as constituents of various natural and synthetic compounds with potentially useful bioactivities. the pyridoxal 5'-phosphate (plp)-dependent s-selective transaminase from mesorhizobium sp. strain luk (mesat) is a fold type i aminotransferase that can be used for the preparation of enantiopure β-phe and derivatives thereof. using x-ray crystallography, we solved structures of mesat in complex with (s)-β-phe, (r)-3-amino-5-methylhexanoic acid, 2-oxoglutarate, and the inh ...201222745123
from static structure to living protein: computational analysis of cytochrome c oxidase main-chain flexibility.crystallographic structure and deuterium accessibility comparisons of cco in different redox states have suggested conformational changes of mechanistic significance. to predict the intrinsic flexibility and low energy motions in cco, this work has analyzed available high-resolution crystallographic structures with proflex and elnémo computational methods. the results identify flexible regions and potential conformational changes in cco that correlate well with published structural and biochemic ...201222824280
structure, function, and assembly of heme centers in mitochondrial respiratory complexes.the sequential flow of electrons in the respiratory chain, from a low reduction potential substrate to o(2), is mediated by protein-bound redox cofactors. in mitochondria, hemes-together with flavin, iron-sulfur, and copper cofactors-mediate this multi-electron transfer. hemes, in three different forms, are used as a protein-bound prosthetic group in succinate dehydrogenase (complex ii), in bc(1) complex (complex iii) and in cytochrome c oxidase (complex iv). the exact function of heme b in comp ...201222554985
mechanistic stoichiometry of proton translocation by cytochrome cbb3.cytochrome cbb(3) belongs to the superfamily of respiratory heme-copper oxidases that couple the reduction of molecular oxygen to proton translocation across the bacterial or mitochondrial membrane. the cbb(3)-type enzymes are found only in bacteria, and are both structurally and functionally the most distant from their mitochondrial counterparts. the mechanistic h(+)/e(-) stoichiometry of proton translocation in these cbb(3)-type cytochrome c oxidases has remained controversial. a stoichiometri ...201222529361
product-controlled steady-state kinetics between cytochrome aa(3) from rhodobacter sphaeroides and equine ferrocytochrome c analyzed by a novel spectrophotometric approach.cytochrome c oxidase (cco) catalyzes the reduction of molecular oxygen to water using ferrocytochrome c (cyt c(2+)) as the electron donor. in this study, the oxidation of horse cyt c(2+) by cco from rhodobacter sphaeroides, was monitored using stopped-flow spectrophotometry. a novel analytic procedure was applied in which the spectra were deconvoluted into the reduced and oxidized forms of cyt c by a least-squares fitting method, yielding the reaction rates at various concentrations of cyt c(2+) ...201222516686
electron transfer in subunit nuoi (tyky) of escherichia coli nadh:quinone oxidoreductase (ndh-1).bacterial proton-translocating nadh:quinone oxidoreductase (ndh-1) consists of a peripheral and a membrane domain. the peripheral domain catalyzes the electron transfer from nadh to quinone through a chain of seven iron-sulfur (fe/s) clusters. subunit nuoi in the peripheral domain contains two [4fe-4s] clusters (n6a and n6b) and plays a role in bridging the electron transfer from cluster n5 to the terminal cluster n2. we constructed mutants for eight individual cys-coordinating fe/s clusters. wi ...201222474289
exploring the proton pump and exit pathway for pumped protons in cytochrome ba3 from thermus thermophilus.the heme-copper oxygen reductases are redox-driven proton pumps. in the current work, the effects of mutations in a proposed exit pathway for pumped protons are examined in the ba(3)-type oxygen reductase from thermus thermophilus, leading from the propionates of heme a(3) to the interface between subunits i and ii. recent studies have proposed important roles for his376 and asp372, both of which are hydrogen-bonded to propionate-a of heme a(3), and for glu126(ii) (subunit ii), which is hydrogen ...201222431640
comparative analysis of two phenotypically-similar but genomically-distinct burkholderia cenocepacia-specific bacteriophages.genomic analysis of bacteriophages infecting the burkholderia cepacia complex (bcc) is an important preliminary step in the development of a phage therapy protocol for these opportunistic pathogens. the objective of this study was to characterize kl1 (vb_bces_kl1) and ah2 (vb_bces_ah2), two novel burkholderia cenocepacia-specific siphoviruses isolated from environmental samples.201222676492
genome signature difference between deinococcus radiodurans and thermus thermophilus.the extremely radioresistant bacteria of the genus deinococcus and the extremely thermophilic bacteria of the genus thermus belong to a common taxonomic group. considering the distinct living environments of deinococcus and thermus, different genes would have been acquired through horizontal gene transfer after their divergence from a common ancestor. their guanine-cytosine (gc) contents are similar; however, we hypothesized that their genomic signatures would be different. our findings indicate ...201222500246
phylogenomics of prokaryotic ribosomal proteins.archaeal and bacterial ribosomes contain more than 50 proteins, including 34 that are universally conserved in the three domains of cellular life (bacteria, archaea, and eukaryotes). despite the high sequence conservation, annotation of ribosomal (r-) protein genes is often difficult because of their short lengths and biased sequence composition. we developed an automated computational pipeline for identification of r-protein genes and applied it to 995 completely sequenced bacterial and 87 arch ...201222615861
bioinformatic characterization of the 4-toluene sulfonate uptake permease (tsup) family of transmembrane proteins.the ubiquitous sequence diverse 4-toluene sulfonate uptake permease (tsup) family contains few characterized members and is believed to catalyze the transport of several sulfur-based compounds. prokaryotic members of the tsup family outnumber the eukaryotic members substantially, and in prokaryotes, but not eukaryotes, extensive lateral gene transfer occurred during family evolution. despite unequal representation, homologues from the three taxonomic domains of life share well-conserved motifs. ...201222192777
bioinformatic characterization of the 4-toluene sulfonate uptake permease (tsup) family of transmembrane proteins.the ubiquitous sequence diverse 4-toluene sulfonate uptake permease (tsup) family contains few characterized members and is believed to catalyze the transport of several sulfur-based compounds. prokaryotic members of the tsup family outnumber the eukaryotic members substantially, and in prokaryotes, but not eukaryotes, extensive lateral gene transfer occurred during family evolution. despite unequal representation, homologues from the three taxonomic domains of life share well-conserved motifs. ...201222192777
structural and biochemical characterization of hp0315 from helicobacter pylori as a vapd protein with an endoribonuclease activity.vapd-like virulence-associated proteins have been found in many organisms, but little is known about this protein family including the 3d structure of these proteins. recently, a relationship between the cas2 family of ribonucleases associated with the crispr system of microbial immunity and vapd was suggested. here, we show for the first time the structure of a member of the vapd family and present a relationship of vapd with cas2 family and toxin-antitoxin (ta) systems. the crystal structure o ...201222241770
characterization of the alkaline laccase ssl1 from streptomyces sviceus with unusual properties discovered by genome mining.fungal laccases are well investigated enzymes with high potential in diverse applications like bleaching of waste waters and textiles, cellulose delignification, and organic synthesis. however, they are limited to acidic reaction conditions and require eukaryotic expression systems. this raises a demand for novel laccases without these constraints. we have taken advantage of the laccase engineering database lcced derived from genome mining to identify and clone the laccase ssl1 from streptomyces ...201223285009
engineering the substrate specificity of a thermophilic penicillin acylase from thermus thermophilus.a homologue of the escherichia coli penicillin acylase is encoded in the genomes of several thermophiles, including in different thermus thermophilus strains. although the natural substrate of this enzyme is not known, this acylase shows a marked preference for penicillin k over penicillin g. three-dimensional models were created in which the catalytic residues and the substrate binding pocket were identified. through rational redesign, residues were replaced to mimic the aromatic binding site o ...201223263966
simultaneous quantification of mitochondrial dna damage and copy number in circulating blood: a sensitive approach to systemic oxidative stress.systemic oxidative stress is associated with a wide range of pathological conditions. oxidative dna damage is frequently measured in circulating lymphocytes. mitochondrial dna (mtdna) is known to be more sensitive to oxidative damage than nuclear dna but is rarely used for direct measurement of dna damage in clinical studies. based on the supercoiling-sensitive real-time pcr method, we propose a new approach for the noninvasive monitoring of systemic oxidative stress by quantifying the mtdna str ...201223484085
simultaneous quantification of mitochondrial dna damage and copy number in circulating blood: a sensitive approach to systemic oxidative stress.systemic oxidative stress is associated with a wide range of pathological conditions. oxidative dna damage is frequently measured in circulating lymphocytes. mitochondrial dna (mtdna) is known to be more sensitive to oxidative damage than nuclear dna but is rarely used for direct measurement of dna damage in clinical studies. based on the supercoiling-sensitive real-time pcr method, we propose a new approach for the noninvasive monitoring of systemic oxidative stress by quantifying the mtdna str ...201223484085
the aeropath project targeting pseudomonas aeruginosa: crystallographic studies for assessment of potential targets in early-stage drug discovery.bacterial infections are increasingly difficult to treat owing to the spread of antibiotic resistance. a major concern is gram-negative bacteria, for which the discovery of new antimicrobial drugs has been particularly scarce. in an effort to accelerate early steps in drug discovery, the eu-funded aeropath project aims to identify novel targets in the opportunistic pathogen pseudomonas aeruginosa by applying a multidisciplinary approach encompassing target validation, structural characterization ...201223295481
the aeropath project targeting pseudomonas aeruginosa: crystallographic studies for assessment of potential targets in early-stage drug discovery.bacterial infections are increasingly difficult to treat owing to the spread of antibiotic resistance. a major concern is gram-negative bacteria, for which the discovery of new antimicrobial drugs has been particularly scarce. in an effort to accelerate early steps in drug discovery, the eu-funded aeropath project aims to identify novel targets in the opportunistic pathogen pseudomonas aeruginosa by applying a multidisciplinary approach encompassing target validation, structural characterization ...201223295481
a multicenter blinded analysis indicates no association between chronic fatigue syndrome/myalgic encephalomyelitis and either xenotropic murine leukemia virus-related virus or polytropic murine leukemia virus.the disabling disorder known as chronic fatigue syndrome or myalgic encephalomyelitis (cfs/me) has been linked in two independent studies to infection with xenotropic murine leukemia virus-related virus (xmrv) and polytropic murine leukemia virus (pmlv). although the associations were not confirmed in subsequent studies by other investigators, patients continue to question the consensus of the scientific community in rejecting the validity of the association. here we report blinded analysis of p ...201222991430
comparing the similarity of different groups of bacteria to the human proteome.numerous aspects of the relationship between bacteria and human have been investigated. one aspect that has recently received attention is sequence overlap at the proteomic level. however, there has not yet been a study that comprehensively characterizes the level of sequence overlap between bacteria and human, especially as it relates to bacterial characteristics like pathogenicity, g-c content, and proteome size. in this study, we began by performing a general characterization of the range of ...201222558081
bactquant: an enhanced broad-coverage bacterial quantitative real-time pcr assay.bacterial load quantification is a critical component of bacterial community analysis, but a culture-independent method capable of detecting and quantifying diverse bacteria is needed. based on our analysis of a diverse collection of 16 s rrna gene sequences, we designed a broad-coverage quantitative real-time pcr (qpcr) assay--bactquant--for quantifying 16 s rrna gene copy number and estimating bacterial load. we further utilized in silico evaluation to complement laboratory-based qpcr characte ...201222510143
a blueprint for a mutationist theory of replicative strand asymmetries formation.in the present review, we summarized current knowledge on replicative strand asymmetries in prokaryotic genomes. a cornerstone for the creation of a theory of their formation has been overviewed. according to our recent works, the probability of nonsense mutation caused by replication-associated mutational pressure is higher for genes from lagging strands than for genes from leading strands of both bacterial and archaeal genomes. lower density of open reading frames in lagging strands can be exp ...201222942675
acyl carrier protein structural classification and normal mode analysis.all acyl carrier protein primary and tertiary structures were gathered into the thyme database. they are classified into 16 families by amino acid sequence similarity, with members of the different families having sequences with statistically highly significant differences. these classifications are supported by tertiary structure superposition analysis. tertiary structures from a number of families are very similar, suggesting that these families may come from a single distant ancestor. normal ...201222374859
stable isotope peptide mass spectrometry to decipher amino acid metabolism in dehalococcoides strain cbdb1.dehalococcoides species are key players in the anaerobic transformation of halogenated solvents at contaminated sites. here, we analyze isotopologue distributions in amino acid pools from peptides of dehalococcoides strain cbdb1 after incubation with (13)c-labeled acetate or bicarbonate as a carbon source. the resulting data were interpreted with regard to genome annotations to identify amino acid biosynthesis pathways. in addition to using gas chromatography-mass spectrometry (gc-ms) for analyz ...201222661690
structure, function and inhibition of the two- and three-domain 4fe-4s ispg proteins.ispg is a 4fe4s protein involved in isoprenoid biosynthesis. most bacterial ispgs contain two domains: a tim barrel (a) and a 4fe4s domain (b), but in plants and malaria parasites, there is a large insert domain (a*) whose structure and function are unknown. we show that bacterial ispgs function in solution as (ab)(2) dimers and that mutations in either both a or both b domains block activity. chimeras harboring an a-mutation in one chain and a b-mutation in the other have 50% of the activity se ...201222586085
in silico analysis of a therapeutic target in leishmania infantum: the guanosine-diphospho-d-mannose pyrophosphorylase.leishmaniases are tropical and sub-tropical diseases for which classical drugs (i.e. antimonials) exhibit toxicity and drug resistance. such a situation requires to find new chemical series with antileishmanial activity. this work consists in analyzing the structure of a validated target in leishmania: the gdp-mannose pyrophosphorylase (gdp-mp), an enzyme involved in glycosylation and essential for amastigote survival. by comparing both human and l. infantum gdp-mp 3d homology models, we identif ...201222314241
the genome of pelobacter carbinolicus reveals surprising metabolic capabilities and physiological features.the bacterium pelobacter carbinolicus is able to grow by fermentation, syntrophic hydrogen/formate transfer, or electron transfer to sulfur from short-chain alcohols, hydrogen or formate; it does not oxidize acetate and is not known to ferment any sugars or grow autotrophically. the genome of p. carbinolicus was sequenced in order to understand its metabolic capabilities and physiological features in comparison with its relatives, acetate-oxidizing geobacter species.201223227809
oxygen and hydrogen peroxide in the early evolution of life on earth: in silico comparative analysis of biochemical pathways.in the universe, oxygen is the third most widespread element, while on earth it is the most abundant one. moreover, oxygen is a major constituent of all biopolymers fundamental to living organisms. besides o(2), reactive oxygen species (ros), among them hydrogen peroxide (h(2)o(2)), are also important reactants in the present aerobic metabolism. according to a widely accepted hypothesis, aerobic metabolism and many other reactions/pathways involving o(2) appeared after the evolution of oxygenic ...201222970865
characterization of the genome, proteome, and structure of yersiniophage ϕr1-37.the bacteriophage vb_yecm-ϕr1-37 (ϕr1-37) is a lytic yersiniophage that can propagate naturally in different yersinia species carrying the correct lipopolysaccharide receptor. this large-tailed phage has deoxyuridine (du) instead of thymidine in its dna. in this study, we determined the genomic sequence of phage ϕr1-37, mapped parts of the phage transcriptome, characterized the phage particle proteome, and characterized the virion structure by cryo-electron microscopy and image reconstruction. t ...201222973030
mathematical modeling and comparison of protein size distribution in different plant, animal, fungal and microbial species reveals a negative correlation between protein size and protein number, thus providing insight into the evolution of proteomes.the sizes of proteins are relevant to their biochemical structure and for their biological function. the statistical distribution of protein lengths across a diverse set of taxa can provide hints about the evolution of proteomes.201222296664
diversity in genetic in vivo methods for protein-protein interaction studies: from the yeast two-hybrid system to the mammalian split-luciferase system.the yeast two-hybrid system pioneered the field of in vivo protein-protein interaction methods and undisputedly gave rise to a palette of ingenious techniques that are constantly pushing further the limits of the original method. sensitivity and selectivity have improved because of various technical tricks and experimental designs. here we present an exhaustive overview of the genetic approaches available to study in vivo binary protein interactions, based on two-hybrid and protein fragment comp ...201222688816
cellular function and molecular structure of ecto-nucleotidases.ecto-nucleotidases play a pivotal role in purinergic signal transmission. they hydrolyze extracellular nucleotides and thus can control their availability at purinergic p2 receptors. they generate extracellular nucleosides for cellular reuptake and salvage via nucleoside transporters of the plasma membrane. the extracellular adenosine formed acts as an agonist of purinergic p1 receptors. they also can produce and hydrolyze extracellular inorganic pyrophosphate that is of major relevance in the c ...201222555564
on the molecular mechanism of gc content variation among eubacterial genomes.as a key parameter of genome sequence variation, the gc content of bacterial genomes has been investigated for over half a century, and many hypotheses have been put forward to explain this gc content variation and its relationship to other fundamental processes. previously, we classified eubacteria into dnae-based groups (the dimeric combination of dna polymerase iii alpha subunits), according to a hypothesis where gc content variation is essentially governed by genome replication and dna repai ...201222230424
accommodation of profound sequence differences at the interfaces of eubacterial rna polymerase multi-protein assembly.evolutionarily divergent proteins have been shown to change their interacting partners. rna polymerase assembly is one of the rare cases which retain its component proteins in the course of evolution. this ubiquitous molecular assembly, involved in transcription, consists of four core subunits (alpha, beta, betaprime, and omega), which assemble to form the core enzyme. remarkably, the orientation of the four subunits in the complex is conserved from prokaryotes to eukaryotes although their seque ...201222359428
a predictive model of intein insertion site for use in the engineering of molecular switches.inteins are intervening protein domains with self-splicing ability that can be used as molecular switches to control activity of their host protein. successfully engineering an intein into a host protein requires identifying an insertion site that permits intein insertion and splicing while allowing for proper folding of the mature protein post-splicing. by analyzing sequence and structure based properties of native intein insertion sites we have identified four features that showed significant ...201222649521
structural, bioinformatic, and in vivo analyses of two treponema pallidum lipoproteins reveal a unique trap transporter.treponema pallidum, the bacterial agent of syphilis, is predicted to encode one tripartite atp-independent periplasmic transporter (trap-t). trap-ts typically employ a periplasmic substrate-binding protein (sbp) to deliver the cognate ligand to the transmembrane symporter. herein, we demonstrate that the genes encoding the putative trap-t components from t. pallidum, tp0957 (the sbp), and tp0958 (the symporter), are in an operon with an uncharacterized third gene, tp0956. we determined the cryst ...201222306465
capreomycin susceptibility is increased by tlya-directed 2'-o-methylation on both ribosomal subunits.the binding site of the cyclic peptide antibiotics capreomycin and viomycin is located on the ribosomal subunit interface close to nucleotides c1409 in 16s rrna and c1920 in 23s rrna. in mycobacterium tuberculosis, the 2'-hydroxyls of both nucleotides are methylated by the enzyme tlya. loss of these methylations through inactivation of tlya confers resistance to capreomycin and viomycin. we report here that tlya orthologues occur in diverse bacteria and fall into two distinct groups. one group, ...201222779429
Alanyl-tRNA synthetase genes of Vanderwaltozyma polyspora arose from duplication of a dual-functional predecessor of mitochondrial origin.In eukaryotes, the cytoplasmic and mitochondrial forms of a given aminoacyl-tRNA synthetase (aaRS) are typically encoded by two orthologous nuclear genes, one of eukaryotic origin and the other of mitochondrial origin. We herein report a novel scenario of aaRS evolution in yeast. While all other yeast species studied possess a single nuclear gene encoding both forms of alanyl-tRNA synthetase (AlaRS), Vanderwaltozyma polyspora, a yeast species descended from the same whole-genome duplication even ...201221908394
Alanyl-tRNA synthetase genes of Vanderwaltozyma polyspora arose from duplication of a dual-functional predecessor of mitochondrial origin.In eukaryotes, the cytoplasmic and mitochondrial forms of a given aminoacyl-tRNA synthetase (aaRS) are typically encoded by two orthologous nuclear genes, one of eukaryotic origin and the other of mitochondrial origin. We herein report a novel scenario of aaRS evolution in yeast. While all other yeast species studied possess a single nuclear gene encoding both forms of alanyl-tRNA synthetase (AlaRS), Vanderwaltozyma polyspora, a yeast species descended from the same whole-genome duplication even ...201221908394
recent progress in bacillus subtilis sporulation.the gram-positive bacterium bacillus subtilis can initiate the process of sporulation under conditions of nutrient limitation. here, we review some of the last 5 years of work in this area, with a particular focus on the decision to initiate sporulation, dna translocation, cell-cell communication, protein localization and spore morphogenesis. the progress we describe has implications not only just for the study of sporulation but also for other biological systems where homologs of sporulation-sp ...201222091839
recent progress in bacillus subtilis sporulation.the gram-positive bacterium bacillus subtilis can initiate the process of sporulation under conditions of nutrient limitation. here, we review some of the last 5 years of work in this area, with a particular focus on the decision to initiate sporulation, dna translocation, cell-cell communication, protein localization and spore morphogenesis. the progress we describe has implications not only just for the study of sporulation but also for other biological systems where homologs of sporulation-sp ...201222091839
Loss of elongation factor p disrupts bacterial outer membrane integrity.Elongation factor P (EF-P) is posttranslationally modified at a conserved lysyl residue by the coordinated action of two enzymes, PoxA and YjeK. We have previously established the importance of this modification in Salmonella stress resistance. Here we report that, like poxA and yjeK mutants, Salmonella strains lacking EF-P display increased susceptibility to hypoosmotic conditions, antibiotics, and detergents and enhanced resistance to the compound S-nitrosoglutathione. The susceptibility pheno ...201222081389
Unique structural features and sequence motifs of proline utilization A (PutA).Proline utilization A proteins (PutAs) are bifunctional enzymes that catalyze the oxidation of proline to glutamate using spatially separated proline dehydrogenase and pyrroline-5-carboxylate dehydrogenase active sites. Here we use the crystal structure of the minimalist PutA from Bradyrhizobium japonicum (BjPutA) along with sequence analysis to identify unique structural features of PutAs. This analysis shows that PutAs have secondary structural elements and domains not found in the related mon ...201222201760
expression and cellular localization of inducible nitric oxide synthase in lipopolysaccharide-treated rat kidneys. 201222260992
physiology of resistant deinococcus geothermalis bacterium aerobically cultivated in low manganese medium.this dynamic proteome study describes the physiology of growth and survival of deinococcus geothermalis, in conditions simulating paper machine waters being aerobic, warm, and low in carbon and manganese. industrial environment of this species differ from its natural habitats, geothermal springs and deep ocean subsurfaces, by being highly exposed to oxygen. quantitative proteome analysis using two-dimensional gel electrophoresis and bioinformatic tools showed expression change for 165 proteins f ...201222228732
construction and transformation of a thermotoga-e. coli shuttle vector.abstract: background: thermotoga spp. are attractive candidates for producing biohydrogen, green chemicals, and thermostable enzymes. they may also serve as model systems for understanding life sustainability under hyperthermophilic conditions. a lack of genetic tools has hampered the investigation and application of these organisms. this study aims to develop a genetic transfer system for thermotoga spp.. results: methods for preparing and handling thermotoga solid cultures under aerobic condi ...201222225774
key role of two ftsa terminal domains in its bidirectional polymerization.the effect of two different truncations involving either the 1c domain or the simultaneous absence of the s12-13 β-strands of the ftsa protein from streptococcus pneumoniae, located at opposite terminal sides in the molecular structure, suggests that they are essential for atp-dependent polymerization. these two truncated proteins are not able to polymerize themselves but can be incorporated to some extent into the ftsa+ polymers during the assembling process. consequently, they block the growth ...201222247552
structure of n-formylglycinamide ribonucleotide amidotransferase ii (purl) from thermus thermophilus hb8.the crystal structure of purl from thermus thermophilus hb8 (ttpurl; ttha1519) was determined in complex with an adenine nucleotide, po(4)(3-) and mg(2+) at 2.35 å resolution. ttpurl consists of 29 α-helices and 28 β-strands, and one loop is disordered. ttpurl consists of four domains, a1, a2, b1 and b2, and the structures of the a1-b1 and a2-b2 domains were almost identical to each other. although the sequence identity between ttpurl and purl from thermotoga maritima (tmpurl) is higher than tha ...201222232163
an unusual n-terminal ααβαββα fold of pilq from t. thermophilus mediates ring formation and is essential for piliation.dna translocators of natural transformation systems are complex systems critical for the uptake of free dna and provide a powerful mechanism for adaptation to changing environmental conditions. in natural transformation machineries, outer membrane secretins are suggested to form a multimeric pore for the uptake of external dna. recently, we reported on a novel structure of the dna translocator secretin complex, pilq, in thermus thermophilus hb27 comprising a stable cone and cup structure and six ...201222253437
mycobacterium tuberculosis rv2419c, the missing glucosyl-3-phosphoglycerate phosphatase for the second step in methylglucose lipopolysaccharide biosynthesis.mycobacteria synthesize intracellular methylglucose lipopolysaccharides (mglp) proposed to regulate fatty acid synthesis. although their structures have been elucidated, the identity of most biosynthetic genes remains unknown. the first step in mglp biosynthesis is catalyzed by a glucosyl-3-phosphoglycerate synthase (gpgs, rv1208 in mycobacterium tuberculosis h37rv). however, a typical glucosyl-3-phosphoglycerate phosphatase (gpgp, ec3.1.3.70) for dephosphorylation of glucosyl-3-phosphoglycerate ...201122355692
crystallization and preliminary neutron diffraction studies of adp-ribose pyrophosphatase-i from thermus thermophilus hb8.adp-ribose pyrophosphatase-i from thermus thermophilus hb8 (ttadprase-i) prevents the intracellular accumulation of adp-ribose by hydrolyzing it to amp and ribose 5'-phosphate. to understand the catalytic mechanism of ttadprase-i, it is necessary to investigate the role of glutamates and metal ions as well as the coordination of water molecules located at the active site. a macroseeding method was developed in order to obtain a large ttadprase-i crystal which was suitable for a neutron diffracti ...201122232170
structural changes that occur upon photolysis of the fe(ii)(a3)-co complex in the cytochrome ba(3)-oxidase of thermus thermophilus: a combined x-ray crystallographic and infrared spectral study demonstrates co binding to cu(b).the purpose of the work was to provide a crystallographic demonstration of the venerable idea that co photolyzed from ferrous heme-a(3) moves to the nearby cuprous ion in the cytochrome c oxidases. crystal structures of co-bound cytochrome ba(3)-oxidase from thermus thermophilus, determined at ~2.8-3.2å resolution, reveal a fe-c distance of ~2.0å, a cu-o distance of 2.4å and a fe-c-o angle of ~126°. upon photodissociation at 100k, x-ray structures indicate loss of fe(a3)-co and appearance of cu( ...201122226917
in vivo, in vitro, and x-ray crystallographic analyses suggest the involvement of an uncharacterized triose-phosphate isomerase (tim) barrel protein in protection against oxidative stress.accumulating genome sequences have revealed the existence of a large number of conserved hypothetical proteins. characterization of these proteins is considered essential in the elucidation of intracellular biological pathways. our previous transcriptomic analysis suggested that, in thermus thermophilus hb8, loss of an oxidized dna-repairing activity leads to the up-regulation of a function-unknown gene, tthb071, which is conserved in a wide range of bacteria. interestingly, the tthb071 gene pro ...201121984829
structural and mutational studies of a hyperthermophilic intein from dna polymerase ii of pyrococcus abyssi.protein splicing is a precise self-catalyzed process in which an intein excises itself from a precursor with the concomitant ligation of the flanking polypeptides (exteins). protein splicing proceeds through a four-step reaction but the catalytic mechanism is not fully understood at the atomic level. we report the solution nmr structures of the hyperthermophilic pyrococcus abyssi polii intein, which has a noncanonical c-terminal glutamine instead of an asparagine. the nmr structures were determi ...201121914805
Tilt-pair analysis of images from a range of different specimens in single-particle electron cryomicroscopy.The comparison of a pair of electron microscope images recorded at different specimen tilt angles provides a powerful approach for evaluating the quality of images, image-processing procedures, or three-dimensional structures. Here, we analyze tilt-pair images recorded from a range of specimens with different symmetries and molecular masses and show how the analysis can produce valuable information not easily obtained otherwise. We show that the accuracy of orientation determination of individua ...201121939668
isolation and characterization of the prochlorococcus carboxysome reveal the presence of the novel shell protein csos1d.cyanobacteria, including members of the genus prochlorococcus, contain icosahedral protein microcompartments known as carboxysomes that encapsulate multiple copies of the co(2)-fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco) in a thin protein shell that enhances the catalytic performance of the enzyme in part through the action of a shell-associated carbonic anhydrase. however, the exact mechanism by which compartmentation provides a catalytic advantage to the enzyme is n ...201122155772
bacteriophage t4 mota activator and the β-flap tip of rna polymerase target the same set of σ70 carboxyl-terminal residues.sigma factors, the specificity subunits of rna polymerase, are involved in interactions with promoter dna, the core subunits of rna polymerase, and transcription factors. the bacteriophage t4-encoded activator, mota, is one such factor, which engages the c terminus of the escherichia coli housekeeping sigma factor, σ(70). mota functions in concert with a phage-encoded co-activator, asia, as a molecular switch. this process, termed sigma appropriation, inhibits host transcription while activating ...201121911499
crystal structure of the bacteriophage t4 late-transcription coactivator gp33 with the β-subunit flap domain of escherichia coli rna polymerase.activated transcription of the bacteriophage t4 late genes, which is coupled to concurrent dna replication, is accomplished by an initiation complex containing the host rna polymerase associated with two phage-encoded proteins, gp55 (the basal promoter specificity factor) and gp33 (the coactivator), as well as the dna-mounted sliding-clamp processivity factor of the phage t4 replisome (gp45, the activator). we have determined the 3.0 å-resolution x-ray crystal structure of gp33 complexed with it ...201122135460
An ensemble of structures of Burkholderia pseudomallei 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase.Burkholderia pseudomallei is a soil-dwelling bacterium endemic to Southeast Asia and Northern Australia. Burkholderia is responsible for melioidosis, a serious infection of the skin. The enzyme 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase (PGAM) catalyzes the interconversion of 3-phosphoglycerate and 2-phosphoglycerate, a key step in the glycolytic pathway. As such it is an extensively studied enzyme and X-ray crystal structures of PGAM enzymes from multiple species have been elucid ...201121904048
identification of human fumarylacetoacetate hydrolase domain-containing protein 1 (fahd1) as a novel mitochondrial acylpyruvase.the human fumarylacetoacetate hydrolase (fah) domain-containing protein 1 (fahd1) is part of the fah protein superfamily, but its enzymatic function is unknown. in the quest for a putative enzymatic function of fahd1, we found that fahd1 exhibits acylpyruvase activity, demonstrated by the hydrolysis of acetylpyruvate and fumarylpyruvate in vitro, whereas several structurally related compounds were not hydrolyzed as efficiently. conserved amino acids asp-102 and arg-106 of fahd1 were found import ...201121878618
pseudomonas aeruginosa 4-amino-4-deoxychorismate lyase: spatial conservation of an active site tyrosine and classification of two types of enzyme.4-amino-4-deoxychorismate lyase (pabc) catalyzes the formation of 4-aminobenzoate, and release of pyruvate, during folate biosynthesis. this is an essential activity for the growth of gram-negative bacteria, including important pathogens such as pseudomonas aeruginosa. a high-resolution (1.75 å) crystal structure of pabc from p. aeruginosa has been determined, and sequence-structure comparisons with orthologous structures are reported. residues around the pyridoxal 5'-phosphate cofactor are high ...201121935381
Crystal structures of an archaeal class II DNA photolyase and its complex with UV-damaged duplex DNA.Class II photolyases ubiquitously occur in plants, animals, prokaryotes and some viruses. Like the distantly related microbial class I photolyases, these enzymes repair UV-induced cyclobutane pyrimidine dimer (CPD) lesions within duplex DNA using blue/near-UV light. Methanosarcina mazei Mm0852 is a class II photolyase of the archaeal order of Methanosarcinales, and is closely related to plant and metazoan counterparts. Mm0852 catalyses light-driven DNA repair and photoreduction, but in contrast ...201121892138
structures of phosphopantetheine adenylyltransferase from burkholderia pseudomallei.phosphopantetheine adenylyltransferase (ppat) catalyzes the fourth of five steps in the coenzyme a biosynthetic pathway, reversibly transferring an adenylyl group from atp onto 4'-phosphopantetheine to yield dephospho-coenzyme a and pyrophosphate. burkholderia pseudomallei is a soil- and water-borne pathogenic bacterium and the etiologic agent of melioidosis, a potentially fatal systemic disease present in southeast asia. two crystal structures are presented of the ppat from b. pseudomallei with ...201121904046
Overexpression, crystallization and preliminary X-ray crystallographic analysis of shikimate dehydrogenase from Archaeoglobus fulgidus.Shikimate dehydrogenase (SDH), which catalyses the NADPH-dependent reduction of 3-dehydroshikimate to shikimate in the shikimate pathway, is an attractive target for the development of herbicides and antimicrobial agents. Previous structural studies have shown that SDH exists in two conformations, an open and a closed form, and it is believed that the conformational state is crucial to understanding its catalytic mechanism. In order to facilitate further structural comparisons among SDHs, includ ...201122139165
Systematic chromosomal deletion of bacterial ribosomal protein genes.Detailed studies of ribosomal proteins (RPs), essential components of the protein biosynthetic machinery, have been hampered by the lack of readily accessible chromosomal deletions of the corresponding genes. Here, we report the systematic genomic deletion of 41 individual RP genes in Escherichia coli, which are not included in the Keio collection. Chromosomal copies of these genes were replaced by an antibiotic resistance gene in the presence of an inducible, easy-to-exchange plasmid-born allel ...201121945294
antimutator variants of dna polymerases.evolution balances dna replication speed and accuracy to optimize replicative fitness and genetic stability. there is no selective pressure to improve dna replication fidelity beyond the background mutation rate from other sources, such as dna damage. however, dna polymerases remain amenable to amino acid substitutions that lower intrinsic error rates. here, we review these 'antimutagenic' changes in dna polymerases and discuss what they reveal about mechanisms of replication fidelity. pioneerin ...201121977975
Activity and regulation of an archaeal DNA-alkyltransferase: a conserved protein involved in repair of DNA alkylation damage.Agents that form methylation adducts in DNA are highly mutagenic and carcinogenic, and organisms have evolved specialized cellular pathways devoted to their repair, including DNA-alkyltransferases. These are proteins conserved in Eucarya, Bacteria and Archaea, acting by a unique reaction mechanism, which leads to direct repair of DNA alkylation damage and irreversible protein alkylation. The alkylated form of DNA-alkyltransferases is inactive and in eukaryotes is rapidly directed to degradation. ...201122167184
Activity and regulation of an archaeal DNA-alkyltransferase: a conserved protein involved in repair of DNA alkylation damage.Agents that form methylation adducts in DNA are highly mutagenic and carcinogenic, and organisms have evolved specialized cellular pathways devoted to their repair, including DNA-alkyltransferases. These are proteins conserved in Eucarya, Bacteria and Archaea, acting by a unique reaction mechanism, which leads to direct repair of DNA alkylation damage and irreversible protein alkylation. The alkylated form of DNA-alkyltransferases is inactive and in eukaryotes is rapidly directed to degradation. ...201122167184
The role of E. coli Nus-factors in transcription regulation and transcription:translation coupling: From structure to mechanism.Bacterial transcription mediated by RNA polymerase (RNAP) is a highly regulated process and RNAP action is modulated during the different phases of initiation, elongation and termination by proteins such as the Escherichia coli Nus transcription-factors. Here we discuss the structural interplay and the mechanistic role of the Nus-factors that are directly involved in the processivity of elongation, transcription:translation coupling and termination, as well as the varying effects of these protei ...201121922055
neutrons, magnets, and photons: a career in structural biology.the purpose of reflections articles, it seems, is to give elderly scientists a chance to write about the "good old days," when everyone walked to school in the snow. they enjoy this activity so much that your editor, martha fedor, must have known that i would accept her invitation to write such an article, no matter how much i demurred at first. as everyone knows, flattery will get you everywhere. it may comfort the apprehensive reader to learn that there is not going to be much walking to schoo ...201122086921
neutrons, magnets, and photons: a career in structural biology.the purpose of reflections articles, it seems, is to give elderly scientists a chance to write about the "good old days," when everyone walked to school in the snow. they enjoy this activity so much that your editor, martha fedor, must have known that i would accept her invitation to write such an article, no matter how much i demurred at first. as everyone knows, flattery will get you everywhere. it may comfort the apprehensive reader to learn that there is not going to be much walking to schoo ...201122086921
a photolyase-like protein from agrobacterium tumefaciens with an iron-sulfur cluster.photolyases and cryptochromes are evolutionarily related flavoproteins with distinct functions. while photolyases can repair uv-induced dna lesions in a light-dependent manner, cryptochromes regulate growth, development and the circadian clock in plants and animals. here we report about two photolyase-related proteins, named phra and phrb, found in the phytopathogen agrobacterium tumefaciens. phra belongs to the class iii cyclobutane pyrimidine dimer (cpd) photolyases, the sister class of plant ...201122066008
Functional characterization of the RuvB homologs from Mycoplasma pneumoniae and Mycoplasma genitalium.Homologous recombination between repeated DNA elements in the genomes of Mycoplasma species has been hypothesized to be a crucial causal factor in sequence variation of antigenic proteins at the bacterial surface. To investigate this notion, studies were initiated to identify and characterize the proteins that form part of the homologous DNA recombination machinery in Mycoplasma pneumoniae as well as Mycoplasma genitalium. Among the most likely participants of this machinery are homologs of the ...201121949077
Biosynthesis of the Pseudomonas aeruginosa Extracellular Polysaccharides, Alginate, Pel, and Psl.Pseudomonas aeruginosa thrives in many aqueous environments and is an opportunistic pathogen that can cause both acute and chronic infections. Environmental conditions and host defenses cause differing stresses on the bacteria, and to survive in vastly different environments, P. aeruginosa must be able to adapt to its surroundings. One strategy for bacterial adaptation is to self-encapsulate with matrix material, primarily composed of secreted extracellular polysaccharides. P. aeruginosa has the ...201121991261
Probing the mechanistic role of the long a-helix in subunit L of respiratory Complex I from Escherichia coli by site-directed mutagenesis.The C-terminus of the NuoL subunit of Complex I includes a long amphipathic a-helix positioned parallel to the membrane, which has been considered to function as a piston in the proton pumping machinery. Here, we have introduced three types of mutations into the nuoL gene to test the piston-like function. First, NuoL was truncated at its C- and N-termini, which resulted in low production of a fragile Complex I with negligible activity. Second, we mutated three partially conserved residues of the ...201122060017
Identification, tissue distribution, and molecular modeling of novel human isoforms of the key enzyme in sialic acid synthesis, UDP-GlcNAc 2-epimerase/ManNAc kinase.UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE) catalyzes the first two committed steps in sialic acid synthesis. In addition to the three previously described human GNE isoforms (hGNE1-hGNE3), our database and polymerase chain reaction analysis yielded five additional human isoforms (hGNE4-hGNE8). hGNE1 is the ubiquitously expressed major isoform, while the hGNE2-hGNE8 isoforms are differentially expressed and may act as tissue-specific regulators of sialylation. hGNE2 and hGNE7 display a 31-residue ...201121910480
Is the sequence-specific binding of aminoacyl-tRNAs by EF-Tu universal among bacteria?Three base pairs in the T-stem are primarily responsible for the sequence-specific interaction of tRNA with Escherichia coli and Thermus thermophilus EF-Tu. While the amino acids on the surface of EF-Tu that contact aminoacyl-tRNA (aa-tRNA) are highly conserved among bacteria, the T-stem sequences of individual tRNA are variable, making it unclear whether or not this protein-nucleic acid interaction is also sequence specific in other bacteria. We propose and validate a thermodynamic model that p ...201121893586
insights into folate/fad-dependent trna methyltransferase mechanism: role of two highly conserved cysteines in catalysis.the flavoprotein trmfo methylates specifically the c5 carbon of the highly conserved uridine 54 in trnas. contrary to most methyltransferases, the 1-carbon unit transferred by trmfo derives from 5,10-methylenetetrahydrofolate and not from s-adenosyl-l-methionine. the enzyme also employs the fad hydroquinone as a reducing agent of the c5 methylene u54-trna intermediate in vitro. by analogy with the catalytic mechanism of thymidylate synthase thya, a conserved cysteine located near the fad isoallo ...201121846722
crystallization and preliminary x-ray diffraction of the first periplasmic domain of secdf, a translocon-associated membrane protein, from thermus thermophilus.a membrane-integrated sec component, secdf, associates with the secyeg protein-conducting channel and facilitates protein secretion and membrane-protein integration. secdf contains 12 transmembrane helices and two periplasmic domains. the first periplasmic domain (p1) plays an important role in protein translocation. here, the overexpression, purification and crystallization of the p1 domain of thermus thermophilus secdf are reported. the crystals diffracted x-rays to 2.3 å resolution and belong ...201122102233
initiation factor eif2γ promotes eif2-gtp-met-trnai(met) ternary complex binding to the 40s ribosome.in contrast to prokaryotic elongation factor ef-tu, which delivers aminoacyl-trnas to the ribosomal a-site, eukaryotic initiation factor eif2 binds methionyl initiator transfer rna (met-trna(i)(met)) to the p-site of the 40s ribosomal subunit. the results of directed hydroxyl radical probing experiments to map binding of saccharomyces cerevisiae eif2 on the ribosome and on met-trna(i)(met) revealed that eif2γ primarily contacts the acceptor stem of met-trna(i)(met) and identified a key binding i ...201122002225
transcriptional regulation of central carbon and energy metabolism in bacteria by redox responsive repressor rex.redox-sensing repressor rex was previously implicated in the control of anaerobic respiration in response to the cellular nadh/nad(+) levels in gram-positive bacteria. we utilized the comparative genomics approach to infer candidate rex-binding dna motifs and assess the rex regulon content in 119 genomes from 11 taxonomic groups. both dna-binding and nad-sensing domains are broadly conserved in rex orthologs identified in the phyla firmicutes, thermotogales, actinobacteria, chloroflexi, deinococ ...201122210771
Mimivirus reveals Mre11/Rad50 fusion proteins with a sporadic distribution in eukaryotes, bacteria, viruses and plasmids.The Mre11/Rad50 complex and the homologous SbcD/SbcC complex in bacteria play crucial roles in the metabolism of DNA double-strand breaks, including DNA repair, genome replication, homologous recombination and non-homologous end-joining in cellular life forms and viruses. Here we investigated the amino acid sequence of the Mimivirus R555 gene product, originally annotated as a Rad50 homolog, and later shown to have close homologs in marine microbial metagenomes.201121899737
Post-translational modification by ß-lysylation is required for the activity of E. coli Elongation Factor P (EF-P).Bacterial elongation factor P (EF-P) is the ortholog of archaeal and eukaryotic initiation factor 5A (aIF5A and eIF5A). EF-P shares sequence homology and crystal structure with eIF5A, but unlike eIF5A, EF-P does not undergo hypusine modification. Recently, two bacterial genes, yjeA and yjeK, encoding truncated homologs of class II lysyl-tRNA synthetase and of lysine-2,3-aminomutase, respectively, have been implicated in the modification of EF-P to convert a specific lysine to a hypothetical ß- ...201122128152
Post-translational modification by ß-lysylation is required for the activity of E. coli Elongation Factor P (EF-P).Bacterial elongation factor P (EF-P) is the ortholog of archaeal and eukaryotic initiation factor 5A (aIF5A and eIF5A). EF-P shares sequence homology and crystal structure with eIF5A, but unlike eIF5A, EF-P does not undergo hypusine modification. Recently, two bacterial genes, yjeA and yjeK, encoding truncated homologs of class II lysyl-tRNA synthetase and of lysine-2,3-aminomutase, respectively, have been implicated in the modification of EF-P to convert a specific lysine to a hypothetical ß- ...201122128152
genomics of bacterial and archaeal viruses: dynamics within the prokaryotic virosphere.prokaryotes, bacteria and archaea, are the most abundant cellular organisms among those sharing the planet earth with human beings (among others). however, numerous ecological studies have revealed that it is actually prokaryotic viruses that predominate on our planet and outnumber their hosts by at least an order of magnitude. an understanding of how this viral domain is organized and what are the mechanisms governing its evolution is therefore of great interest and importance. the vast majorit ...201122126996
estimation of absolute protein quantities of unlabeled samples by selected reaction monitoring mass spectrometry.for many research questions in modern molecular and systems biology information about absolute protein quantities is imperative. these include, for example, kinetic modeling of processes, protein turnover determinations, stoichiometric investigations of protein complexes or quantitative comparisons of different proteins within one or across samples. to date, the vast majority of proteomic studies are limited to providing relative quantitative comparisons of protein levels between limited numbers ...201122101334
estimation of absolute protein quantities of unlabeled samples by selected reaction monitoring mass spectrometry.for many research questions in modern molecular and systems biology information about absolute protein quantities is imperative. these include, for example, kinetic modeling of processes, protein turnover determinations, stoichiometric investigations of protein complexes or quantitative comparisons of different proteins within one or across samples. to date, the vast majority of proteomic studies are limited to providing relative quantitative comparisons of protein levels between limited numbers ...201122101334
Biochemical and structural studies of the uncharacterized protein PA0743 from Pseudomonas aeruginosa revealed a NAD+-dependent L-serine dehydrogenase.The ß-hydroxyacid dehydrogenases form a large family of ubiquitous enzymes that catalyze oxidation of various ß-hydroxyacid substrates to corresponding semialdehydes. Several known enzymes include ß-hydroxyisobutyrate dehydrogenase, 6-phosphogluconate dehydrogenase, 2-(hydroxymethyl)glutarate dehydrogenase, and phenylserine dehydrogenase, but the vast majority of ß-hydroxyacid dehydrogenases remain uncharacterized. Here, we demonstrate that the predicted ß-hydroxyisobutyrate dehydrogenase PA0743 ...201122128181
Biochemical and structural studies of the uncharacterized protein PA0743 from Pseudomonas aeruginosa revealed a NAD+-dependent L-serine dehydrogenase.The ß-hydroxyacid dehydrogenases form a large family of ubiquitous enzymes that catalyze oxidation of various ß-hydroxyacid substrates to corresponding semialdehydes. Several known enzymes include ß-hydroxyisobutyrate dehydrogenase, 6-phosphogluconate dehydrogenase, 2-(hydroxymethyl)glutarate dehydrogenase, and phenylserine dehydrogenase, but the vast majority of ß-hydroxyacid dehydrogenases remain uncharacterized. Here, we demonstrate that the predicted ß-hydroxyisobutyrate dehydrogenase PA0743 ...201122128181
mass spectrometry of intact v-type atpases reveals bound lipids and the effects of nucleotide binding.the ability of electrospray to propel large viruses into a mass spectrometer is established and is rationalized by analogy to the atmospheric transmission of the common cold. much less clear is the fate of membrane-embedded molecular machines in the gas phase. here we show that rotary adenosine triphosphatases (atpases)/synthases from thermus thermophilus and enterococcus hirae can be maintained intact with membrane and soluble subunit interactions preserved in vacuum. mass spectra reveal subuni ...201122021858
translational diffusion of macromolecular assemblies measured using transverse-relaxation-optimized pulsed field gradient nmr.in structural biology, pulsed field gradient (pfg) nmr spectroscopy for the characterization of size and hydrodynamic parameters of macromolecular solutes has the advantage over other techniques that the measurements can be recorded with identical solution conditions as used for nmr structure determination or for crystallization trials. this paper describes two transverse-relaxation-optimized (tro) (15)n-filtered pfg stimulated-echo (ste) experiments for studies of macromolecular translational d ...201121919531
Comparative analyses of transport proteins encoded within the genomes of Mycobacterium tuberculosis and Mycobacterium leprae.The co-emergence of multidrug resistant pathogenic bacterial strains and the HIV pandemic has made tuberculosis a leading public health threat. The causative agent is Mycobacterium tuberculosis (Mtu), a facultative intracellular parasite. Mycobacterium leprae (Mle), a related organism that causes leprosy, is an obligate intracellular parasite. Given that different transporters are required for bacterial growth and persistence under a variety of growth conditions, we conducted comparative analyse ...201122179038
Comparative analyses of transport proteins encoded within the genomes of Mycobacterium tuberculosis and Mycobacterium leprae.The co-emergence of multidrug resistant pathogenic bacterial strains and the HIV pandemic has made tuberculosis a leading public health threat. The causative agent is Mycobacterium tuberculosis (Mtu), a facultative intracellular parasite. Mycobacterium leprae (Mle), a related organism that causes leprosy, is an obligate intracellular parasite. Given that different transporters are required for bacterial growth and persistence under a variety of growth conditions, we conducted comparative analyse ...201122179038
Structure and Function of the Small MutS-Related Domain.MutS family proteins are widely distributed in almost all organisms from bacteria to human and play central roles in various DNA transactions such as DNA mismatch repair and recombinational events. The small MutS-related (Smr) domain was originally found in the C-terminal domain of an antirecombination protein, MutS2, a member of the MutS family. MutS2 is thought to suppress homologous recombination by endonucleolytic resolution of early intermediates in the process. The endonuclease activity of ...201122091410
Probing conformational states of glutaryl-CoA dehydrogenase by fragment screening.Glutaric acidemia type 1 is an inherited metabolic disorder which can cause macrocephaly, muscular rigidity, spastic paralysis and other progressive movement disorders in humans. The defects in glutaryl-CoA dehydrogenase (GCDH) associated with this disease are thought to increase holoenzyme instability and reduce cofactor binding. Here, the first structural analysis of a GCDH enzyme in the absence of the cofactor flavin adenine dinucleotide (FAD) is reported. The apo structure of GCDH from Burkh ...201121904051
structural basis of free reduced flavin generation by flavin reductase from thermus thermophilus hb8.free reduced flavins are involved in a variety of biological functions. they are generated from nad(p)h by flavin reductase via co-factor flavin bound to the enzyme. although recent findings on the structure and function of flavin reductase provide new information about co-factor fad and substrate nad, there have been no reports on the substrate flavin binding site. here we report the structure of ttha0420 from thermus thermophilus hb8, which belongs to flavin reductase, and describe the dual bi ...201122052907
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