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structural basis for the rescue of stalled ribosomes: structure of yaej bound to the ribosome.in bacteria, the hybrid transfer-messenger rna (tmrna) rescues ribosomes stalled on defective messenger rnas (mrnas). however, certain gram-negative bacteria have evolved proteins that are capable of rescuing stalled ribosomes in a tmrna-independent manner. here, we report a 3.2 angstrom-resolution crystal structure of the rescue factor yaej bound to the thermus thermophilus 70s ribosome in complex with the initiator trna(i)(fmet) and a short mrna. the structure reveals that the c-terminal tail ...201222422986
saccharomyces cerevisiae possesses a stress-inducible glycyl-trna synthetase gene.aminoacyl-trna synthetases are a large family of housekeeping enzymes that are pivotal in protein translation and other vital cellular processes. saccharomyces cerevisiae possesses two distinct nuclear glycyl-trna synthetase (glyrs) genes, grs1 and grs2. grs1 encodes both cytoplasmic and mitochondrial activities, while grs2 is essentially silent and dispensable under normal conditions. we herein present evidence that expression of grs2 was drastically induced upon heat shock, ethanol or hydrogen ...201222438917
simulation of the opening and closing of hsp70 chaperones by coarse-grained molecular dynamics.heat-shock proteins 70 (hsp70s) are key molecular chaperones which assist in the folding and refolding/disaggregation of proteins. hsp70s, which consist of a nucleotide-binding domain (nbd, consisting of nbd-i and nbd-ii subdomains) and a substrate-binding domain [sbd, further split into the β-sheet (sbd-β) and α-helical (sbd-α) subdomains], occur in two major conformations having (a) a closed sbd, in which the sbd and nbd domains do not interact, (b) an open sbd, in which sbd-α interacts with n ...201222737044
when ribonucleases come into play in pathogens: a survey of gram-positive bacteria.it is widely acknowledged that rna stability plays critical roles in bacterial adaptation and survival in different environments like those encountered when bacteria infect a host. bacterial ribonucleases acting alone or in concert with regulatory rnas or rna binding proteins are the mediators of the regulatory outcome on rna stability. we will give a current update of what is known about ribonucleases in the model gram-positive organism bacillus subtilis and will describe their established role ...201222550495
pseudoscorpion mitochondria show rearranged genes and genome-wide reductions of rna gene sizes and inferred structures, yet typical nucleotide composition bias.pseudoscorpions are chelicerates and have historically been viewed as being most closely related to solifuges, harvestmen, and scorpions. no mitochondrial genomes of pseudoscorpions have been published, but the mitochondrial genomes of some lineages of chelicerata possess unusual features, including short rrna genes and trna genes that lack sequence to encode arms of the canonical cloverleaf-shaped trna. additionally, some chelicerates possess an atypical guanine-thymine nucleotide bias on the m ...201222409411
structural and enzymatic characterization of bacd, an l-amino acid dipeptide ligase from bacillus subtilis.bacd is an atp-dependent dipeptide ligase responsible for the biosynthesis of l-alanyl-l-anticapsin, a precursor of an antibiotic produced by bacillus spp. in contrast to the well-studied and phylogenetically related d-alanine: d-alanine ligase (ddl), bacd synthesizes dipeptides using l-amino acids as substrates and has a low substrate specificity in vitro. the enzyme is of great interest because of its potential application in industrial protein engineering for the environmentally friendly biol ...201222407814
2011 william allan award: development and evolution. 201222405084
complete genome sequence of the aerobic, heterotroph marinithermus hydrothermalis type strain (t1(t)) from a deep-sea hydrothermal vent chimney.marinithermus hydrothermalis sako et al. 2003 is the type species of the monotypic genus marinithermus. m. hydrothermalis t1(t) was the first isolate within the phylum "thermus-deinococcus" to exhibit optimal growth under a salinity equivalent to that of sea water and to have an absolute requirement for nacl for growth. m. hydrothermalis t1(t) is of interest because it may provide a new insight into the ecological significance of the aerobic, thermophilic decomposers in the circulation of organi ...201222675595
structural insights into initial and intermediate steps of the ribosome-recycling process.the ribosome-recycling factor (rrf) and elongation factor-g (ef-g) disassemble the 70s post-termination complex (potc) into mrna, trna, and two ribosomal subunits. we have determined cryo-electron microscopic structures of the potc·rrf complex, with and without ef-g. we find that domain ii of rrf initially interacts with universally conserved residues of the 23s rrna helices 43 and 95, and protein l11 within the 50s ribosomal subunit. upon ef-g binding, both rrf and trna are driven towards the t ...201222388519
structure and function of fusb: an elongation factor g-binding fusidic acid resistance protein active in ribosomal translocation and recycling.fusidic acid (fa) is a bacteriostatic antibiotic that locks elongation factor g (ef-g) to the ribosome after gtp hydrolysis during elongation and ribosome recycling. the plasmid pub101-encoded protein fusb causes fa resistance in clinical isolates of staphylococcus aureus through an interaction with ef-g. here, we report 1.6 and 2.3 å crystal structures of fusb. we show that fusb is a two-domain protein lacking homology to known structures, where the n-terminal domain is a four-helix bundle and ...201222645663
response to klyuyev and vassylyev: on the mechanism of tagetitoxin inhibition of transcription.in their commentary, klyuyev and vassylyev dispute a model of transcription inhibition by tagetitoxin (tgt) proposed by us based on biochemical analysis and computational docking. we maintain that, although an alternative explanation can be provided for any single observation reported by us, taken together our results support a model in which tgt acts by trapping the trigger loop (tl) in an inactive state (artsimovitch et al.). this model is consistent with all the data collected with a physiolo ...201222414748
the binding site and mechanism of the rna polymerase inhibitor tagetitoxin: an issue open to debate.in a recent paper in the journal of biological chemistry, artsimovitch et al. report a major revision of a crystallographic model and proposed mechanism of the rna polymerase inhibitor, tagetitoxin. this reassessment is based on theoretical modeling using molecular dynamics simulations. here, we argue that this theoretical model contradicts experimental results and a published crystal structure cannot exclude several mechanistically distinct alternative models and does not support some major con ...201222414754
rationalization and prediction of selective decoding of pseudouridine-modified nonsense and sense codons.a stop or nonsense codon is an in-frame triplet within a messenger rna that signals the termination of translation. one common feature shared among all three nonsense codons (uaa, uag, and uga) is a uridine present at the first codon position. it has been recently shown that the conversion of this uridine into pseudouridine (ψ) suppresses translation termination, both in vitro and in vivo. furthermore, decoding of the pseudouridylated nonsense codons is accompanied by the incorporation of two sp ...201222282339
mapping hidden potential identity elements by computing the average discriminating power of individual trna positions.the recently published discrete mathematical method, extended consensus partition (ecp), identifies nucleotide types at each position that are strictly absent from a given sequence set, while occur in other sets. these are defined as discriminating elements (des). in this study using the ecp approach, we mapped potential hidden identity elements that discriminate the 20 different trna identities. we filtered the tdna data set for the obligatory presence of well-established trna features, and the ...201222378766
cross-talk between phosphorylation and lysine acetylation in a genome-reduced bacterium.protein post-translational modifications (ptms) represent important regulatory states that when combined have been hypothesized to act as molecular codes and to generate a functional diversity beyond genome and transcriptome. we systematically investigate the interplay of protein phosphorylation with other post-transcriptional regulatory mechanisms in the genome-reduced bacterium mycoplasma pneumoniae. systematic perturbations by deletion of its only two protein kinases and its unique protein ph ...201222373819
4,6-α-glucanotransferase activity occurs more widespread in lactobacillus strains and constitutes a separate gh70 subfamily.family 70 glycoside hydrolase glucansucrase enzymes exclusively occur in lactic acid bacteria and synthesize a wide range of α-d-glucan (abbreviated as α-glucan) oligo- and polysaccharides. of the 47 characterized gh70 enzymes, 46 use sucrose as glucose donor. a single gh70 enzyme was recently found to be inactive with sucrose and to utilize maltooligosaccharides [(1→4)-α-d-glucooligosaccharides] as glucose donor substrates for α-glucan synthesis, acting as a 4,6-α-glucanotransferase (4,6-αgt) e ...201222361861
4,6-α-glucanotransferase activity occurs more widespread in lactobacillus strains and constitutes a separate gh70 subfamily.family 70 glycoside hydrolase glucansucrase enzymes exclusively occur in lactic acid bacteria and synthesize a wide range of α-d-glucan (abbreviated as α-glucan) oligo- and polysaccharides. of the 47 characterized gh70 enzymes, 46 use sucrose as glucose donor. a single gh70 enzyme was recently found to be inactive with sucrose and to utilize maltooligosaccharides [(1→4)-α-d-glucooligosaccharides] as glucose donor substrates for α-glucan synthesis, acting as a 4,6-α-glucanotransferase (4,6-αgt) e ...201222361861
subunit interactions at the v1-vo interface in yeast vacuolar atpase.eukaryotic vacuolar atpase (v-atpase) is regulated by a reversible dissociation mechanism that involves breaking and reforming of protein-protein interactions at the interface of the v(1)-atpase and v(o)-proton channel domains. we found previously that the head domain of the single copy c subunit (c(head)) binds one subunit eg heterodimer with high affinity (oot, r.a. and wilkens, s. (2010) j. biol. chem. 285, 24654-24664). here we generated a water-soluble construct of the n-terminal domain of ...201222367203
investigating the entire course of telithromycin binding to escherichia coli ribosomes.applying kinetics and footprinting analysis, we show that telithromycin, a ketolide antibiotic, binds to escherichia coli ribosomes in a two-step process. during the first, rapidly equilibrated step, telithromycin binds to a low-affinity site (k(t) = 500 nm), in which the lactone ring is positioned at the upper portion of the peptide exit tunnel, while the alkyl-aryl side chain of the drug inserts a groove formed by nucleotides a789 and u790 of 23s rrna. during the second step, telithromycin shi ...201222362747
the asparagine-transamidosome from helicobacter pylori: a dual-kinetic mode in non-discriminating aspartyl-trna synthetase safeguards the genetic code.helicobacter pylori catalyzes asn-trna(asn) formation by use of the indirect pathway that involves charging of asp onto trna(asn) by a non-discriminating aspartyl-trna synthetase (nd-asprs), followed by conversion of the mischarged asp into asn by the gatcab amidotransferase. we show that the partners of asparaginylation assemble into a dynamic asn-transamidosome, which uses a different strategy than the gln-transamidosome to prevent the release of the mischarged aminoacyl-trna intermediate. the ...201222362756
crystal structures of the trna:m2g6 methyltransferase trm14/trmn from two domains of life.methyltransferases (mtases) form a major class of trna-modifying enzymes needed for the proper functioning of trna. recently, rna mtases from the trmn/trm14 family that are present in archaea, bacteria and eukaryota have been shown to specifically modify trna(phe) at guanosine 6 in the trna acceptor stem. here, we report the first x-ray crystal structures of the trna m(2)g6 (n(2)-methylguanosine) mtase (ttc)trmn from thermus thermophilus and its ortholog (pf)trm14 from pyrococcus furiosus. struc ...201222362751
cytosolic ni(ii) sensor in cyanobacterium: nickel detection follows nickel affinity across four families of metal sensors.efflux of surplus ni(ii) across the outer and inner membranes of synechocystis pcc 6803 is mediated by the nrs system under the control of a sensor of periplasmic ni(ii), nrss. here, we show that the product of orf sll0176, which encodes a csor/rcnr-like protein now designated inrs (for internal nickel-responsive sensor), represses nrsd (nrsd is deduced to efflux ni(ii) across the inner membrane) from a cryptic promoter between the final two orfs in the nrs operon. transcripts initiated from the ...201222356910
cloning, purification, crystallization and preliminary x-ray analysis of the burkholderia pseudomallei l1 ribosomal protein.the gene encoding the l1 ribosomal protein from burkholderia pseudomallei strain d286 has been cloned into the petblue-1 vector system, overexpressed in escherichia coli and purified. crystals of the native protein were grown by the hanging-drop vapour-diffusion technique using peg 3350 as a precipitant and diffracted to beyond 1.65 å resolution. the crystals belonged to space group p2(1)2(1)2, with unit-cell parameters a = 53.6, b = 127.1, c = 31.8 å and with a single molecule in the asymmetric ...201222442241
crystallization and preliminary x-ray crystallographic analysis of the catalytic domain of human dihydrouridine synthase.dihydrouridine synthases catalyse the reduction of uridine to dihydrouridine in the d-loop and variable loop of trna. the human dihydrouridine synthase hsdus2l has been implicated in the development of pulmonary carcinogenesis. here, the purification, crystallization and preliminary x-ray characterization of the hsdus2l catalytic domain are reported. the crystals belonged to space group p2(1) and contained a single molecule of hsdus2l in the asymmetric unit. a complete data set was collected to ...201222442237
the dynamic stator stalk of rotary atpases.rotary atpases couple atp hydrolysis/synthesis with proton translocation across biological membranes and so are central components of the biological energy conversion machinery. their peripheral stalks are essential components that counteract torque generated by rotation of the central stalk during atp synthesis or hydrolysis. here we present a 2.25-å resolution crystal structure of the peripheral stalk from thermus thermophilus a-type atpase/synthase. we identify bending and twisting motions in ...201222353718
recycling of methylthioadenosine is essential for normal vascular development and reproduction in arabidopsis.5'-methylthioadenosine (mta) is the common by-product of polyamine (pa), nicotianamine (na), and ethylene biosynthesis in arabidopsis (arabidopsis thaliana). the methylthiol moiety of mta is salvaged by 5'-methylthioadenosine nucleosidase (mtn) in a reaction producing methylthioribose (mtr) and adenine. the mtn double mutant, mtn1-1mtn2-1, retains approximately 14% of the mtn enzyme activity present in the wild type and displays a pleiotropic phenotype that includes altered vasculature and impai ...201222345506
discovery and analysis of cofactor-dependent phosphoglycerate mutase homologs as novel phosphoserine phosphatases in hydrogenobacter thermophilus.phosphoserine phosphatase (psp) catalyzes the dephosphorylation of phosphoserine to serine and inorganic phosphate. psps, which have been found in all three domains of life, belong to the haloacid dehalogenase-like hydrolase superfamily. however, certain organisms, particularly bacteria, lack a classical psp gene, although they appear to possess a functional phosphoserine synthetic pathway. the apparent lack of a psp ortholog in hydrogenobacter thermophilus, an obligately chemolithoautotrophic a ...201222337887
the tyrosyl-trna synthetase like gene located in the tyramine biosynthesis cluster of enterococcus durans is transcriptionally regulated by tyrosine concentration and extracellular ph.the tyramine producer enterococcus durans ipla655 contains all the necessary genes for tyramine biosynthesis, grouped in the tdc cluster. this cluster includes tyrs, an aminoacyl-trna synthetase like gene.201222333391
heme-copper terminal oxidase using both cytochrome c and ubiquinol as electron donors.the cytochrome c oxidase cox2 has been purified from native membranes of the hyperthermophilic eubacterium aquifex aeolicus. it is a cytochrome ba(3) oxidase belonging to the family b of the heme-copper containing terminal oxidases. it consists of three subunits, subunit i (coxa2, 63.9 kda), subunit ii (coxb2, 16.8 kda), and an additional subunit iia of 5.2 kda. surprisingly it is able to oxidize both reduced cytochrome c and ubiquinol in a cyanide sensitive manner. cox2 is part of a respiratory ...201222334648
structural basis of the substrate specificity of bacillus cereus adenosine phosphorylase.purine nucleoside phosphorylases catalyze the phosphorolytic cleavage of the glycosidic bond of purine (2'-deoxy)nucleosides, generating the corresponding free base and (2'-deoxy)-ribose 1-phosphate. two classes of pnps have been identified: homotrimers specific for 6-oxopurines and homohexamers that accept both 6-oxopurines and 6-aminopurines. bacillus cereus adenosine phosphorylase (adop) is a hexameric pnp; however, it is highly specific for 6-aminopurines. to investigate the structural basis ...201222349225
the 2'-o-methylation status of a single guanosine controls transfer rna-mediated toll-like receptor 7 activation or inhibition.foreign rna serves as pathogen-associated molecular pattern (pamp) and is a potent immune stimulator for innate immune receptors. however, the role of single bacterial rna species in immune activation has not been characterized in detail. we analyzed the immunostimulatory potential of transfer rna (trna) from different bacteria. interestingly, bacterial trna induced type i interferon (ifn) and inflammatory cytokines in mouse dendritic cells (dcs) and human peripheral blood mononuclear cells (pbm ...201222312111
thermodynamic basis of selectivity in guide-target-mismatched rna interference.silencing in rnai is strongly affected by guide-strand/target-mrna mismatches. target nucleation is thought to occur at positions 2-8 of the guide ("seed region"); successful hybridization in this region is the primary determinant of target-binding affinity and hence target cleavage. to define a molecular basis for the target sequence selectivity in rnai, we studied all possible distinct single mismatches in seven positions of the seed region-a total of 21 substitutions. we report results from s ...201222275138
activity, stability, and structure of metagenome-derived lc11-rnase h1, a homolog of sulfolobus tokodaii rnase h1.metagenome-derived lc11-rnase h1 is a homolog of sulfolobus tokodaii rnase h1 (sto-rnase h1). it lacks a c-terminal tail, which is responsible for hyperstabilization of sto-rnase h1. sto-rnase h1 is characterized by its ability to cleave not only an rna/dna hybrid but also a double-stranded rna (dsrna). to examine whether lc11-rnase h1 also exhibits both rnase h and dsrnase activities, lc11-rnase h1 was overproduced in escherichia coli, purified, and characterized. lc11-rnase h1 exhibited rnase ...201222389131
selenocysteine insertion sequence (secis)-binding protein 2 alters conformational dynamics of residues involved in trna accommodation in 80 s ribosomes.sec-trna(sec) is site-specifically delivered at defined uga codons in selenoprotein mrnas. this recoding event is specified by the selenocysteine insertion sequence (secis) element and requires the selenocysteine (sec)-specific elongation factor, eefsec, and the secis binding protein, sbp2. sec-trna(sec) is delivered to the ribosome by eefsec-gtp, but this ternary complex is not sufficient for sec incorporation, indicating that its access to the ribosomal a-site is regulated. sbp2 stably associa ...201222308032
role of apoptosis-inducing factor, proline dehydrogenase, and nadph oxidase in apoptosis and oxidative stress.flavoproteins catalyze a variety of reactions utilizing flavin mononucleotide or flavin adenine dinucleotide as cofactors. the oxidoreductase properties of flavoenzymes implicate them in redox homeostasis, oxidative stress, and various cellular processes, including programmed cell death. here we explore three critical flavoproteins involved in apoptosis and redox signaling, ie, apoptosis-inducing factor (aif), proline dehydrogenase, and nadph oxidase. these proteins have diverse biochemical func ...201222593641
argonaute and the nuclear rnas: new pathways for rna-mediated control of gene expression.small rnas are a commonly used tool for gene silencing and a promising platform for nucleic acid drug development. they are almost exclusively used to silence gene expression post-transcriptionally through degradation of mrna. small rnas, however, can have a broader range of function by binding to argonaute proteins and associating with complementary rna targets in the nucleus, including long noncoding rnas (lncrnas) and pre-mrna. argonaute-rna complexes can regulate nuclear events like transcri ...201222283730
crystal structure of release factor rf3 trapped in the gtp state on a rotated conformation of the ribosome.the class ii release factor rf3 is a gtpase related to elongation factor ef-g, which catalyzes release of class i release factors rf1 and rf2 from the ribosome after termination of protein synthesis. the 3.3 å crystal structure of the rf3·gdpnp·ribosome complex provides a high-resolution description of interactions and structural rearrangements that occur when binding of this translational gtpase induces large-scale rotational movements in the ribosome. rf3 induces a 7° rotation of the body and ...201222187675
fidelity of trna 5'-maturation: a possible basis for the functional dependence of archaeal and eukaryal rnase p on multiple protein cofactors.rnase p, which catalyzes trna 5'-maturation, typically comprises a catalytic rnase p rna (rpr) and a varying number of rnase p proteins (rpps): 1 in bacteria, at least 4 in archaea and 9 in eukarya. the four archaeal rpps have eukaryotic homologs and function as heterodimers (pop5•rpp30 and rpp21•rpp29). by studying the archaeal methanocaldococcus jannaschii rpr's cis cleavage of precursor trna(gln) (pre-trna(gln)), which lacks certain consensus structures/sequences needed for substrate recognit ...201222298511
the structural basis for substrate versatility of chloramphenicol acetyltransferase cati.novel antibiotics are needed to overcome the challenge of continually evolving bacterial resistance. this has led to a renewed interest in mechanistic studies of once popular antibiotics like chloramphenicol (cam). chloramphenicol acetyltransferases (cats) are enzymes that covalently modify cam, rendering it inactive against its target, the ribosome, and thereby causing resistance to cam. of the three major types of cat (cat(i-iii)), the cam-specific cat(iii) has been studied extensively. much l ...201222294317
trna-controlled nuclear import of a human trna synthetase.aminoacyl-trna synthetases, essential components of the cytoplasmic translation apparatus, also have nuclear functions that continue to be elucidated. however, little is known about how the distribution between cytoplasmic and nuclear compartments is controlled. using a combination of methods, here we showed that human tyrosyl-trna synthetase (tyrrs) distributes to the nucleus and that the nuclear import of human tyrrs is regulated by its cognate trna(tyr). we identified a hexapeptide motif in t ...201222291016
a deeply branching thermophilic bacterium with an ancient acetyl-coa pathway dominates a subsurface ecosystem.a nearly complete genome sequence of candidatus 'acetothermum autotrophicum', a presently uncultivated bacterium in candidate division op1, was revealed by metagenomic analysis of a subsurface thermophilic microbial mat community. phylogenetic analysis based on the concatenated sequences of proteins common among 367 prokaryotes suggests that ca. 'a. autotrophicum' is one of the earliest diverging bacterial lineages. it possesses a folate-dependent wood-ljungdahl (acetyl-coa) pathway of co(2) fix ...201222303444
crystal structure of urea carboxylase provides insights into the carboxyltransfer reaction.urea carboxylase (uc) is conserved in many bacteria, algae, and fungi and catalyzes the conversion of urea to allophanate, an essential step in the utilization of urea as a nitrogen source in these organisms. uc belongs to the biotin-dependent carboxylase superfamily and shares the biotin carboxylase (bc) and biotin carboxyl carrier protein (bccp) domains with these other enzymes, but its carboxyltransferase (ct) domain is distinct. currently, there is no information on the molecular basis of ca ...201222277658
multistep assembly of chloroplast nadh dehydrogenase-like subcomplex a requires several nucleus-encoded proteins, including crr41 and crr42, in arabidopsis.chloroplast nadh dehydrogenase-like complex (ndh) mediates photosystem i cyclic electron transport and chlororespiration in thylakoids. recently, substantial progress has been made in understanding the structure of ndh, but our knowledge of its assembly has been limited. in this study, a series of interactive proteomic analyses identified several stroma-localized factors required for the assembly of a stroma-protruding arm of ndh (subcomplex a). in addition to further characterization of the pre ...201222274627
biocatalyst development by directed evolution.biocatalysis has emerged as a great addition to traditional chemical processes for production of bulk chemicals and pharmaceuticals. to overcome the limitations of naturally occurring enzymes, directed evolution has become the most important tool for improving critical traits of biocatalysts such as thermostability, activity, selectivity, and tolerance towards organic solvents for industrial applications. recent advances in mutant library creation and high-throughput screening have greatly facil ...201222310212
ribosome clearance by fusb-type proteins mediates resistance to the antibiotic fusidic acid.resistance to the antibiotic fusidic acid (fa) in the human pathogen staphylococcus aureus usually results from expression of fusb-type proteins (fusb or fusc). these proteins bind to elongation factor g (ef-g), the target of fa, and rescue translation from fa-mediated inhibition by an unknown mechanism. here we show that the fusb family are two-domain metalloproteins, the c-terminal domain of which contains a four-cysteine zinc finger with a unique structural fold. this domain mediates a high-a ...201222308410
visual tracking of plant virus infection and movement using a reporter myb transcription factor that activates anthocyanin biosynthesis.insertion of reporter genes into plant virus genomes is a common experimental strategy to research many aspects of the viral infection dynamics. their numerous advantages make fluorescent proteins the markers of choice in most studies. however, the use of fluorescent proteins still has some limitations, such as the need of specialized material and facilities to detect the fluorescence. here, we demonstrate a visual reporter marker system to track virus infection and movement through the plant. t ...201222238422
classification of protein functional surfaces using structural characteristics.protein structure and function are closely related, especially in functional surfaces, which are local spatial regions that perform the biological functions. also, protein structures tend to evolve more slowly than amino acid sequences. we have therefore developed a method to classify proteins using the structures of functional surfaces; we call it protein surface classification (psc). psc may reflect functional relationships among proteins and may detect evolutionary relationships among highly ...201222238424
epr-endor characterization of (17o, 1h, 2h) water in manganese catalase and its relevance to the oxygen-evolving complex of photosystem ii.the synthesis of efficient water-oxidation catalysts demands insight into the only known, naturally occurring water-oxidation catalyst, the oxygen-evolving complex (oec) of photosystem ii (psii). understanding the water oxidation mechanism requires knowledge of where and when substrate water binds to the oec. mn catalase in its mn(iii)-mn(iv) state is a protein model of the oec's s(2) state. from (17)o-labeled water exchanged into the di-μ-oxo di-mn(iii,iv) coordination sphere of mn catalase, cw ...201222142421
the roles of rhodobacter sphaeroides copper chaperones pcu(a)c and sco (prrc) in the assembly of the copper centers of the aa(3)-type and the cbb(3)-type cytochrome c oxidases.the α proteobacter rhodobacter sphaeroides accumulates two cytochrome c oxidases (cco) in its cytoplasmic membrane during aerobic growth: a mitochondrial-like aa(3)-type cco containing a di-copper cu(a) center and mono-copper cu(b), plus a cbb(3)-type cco that contains cu(b) but lacks cu(a). three copper chaperones are located in the periplasm of r. sphaeroides, pcu(a)c, prrc (sco) and cox11. cox11 is required to assemble cu(b) of the aa(3)-type but not the cbb(3)-type cco. prrc is homologous to ...201222248670
a-site residues move independently from p-site residues in all-atom molecular dynamics simulations of the 70s bacterial ribosome.the ribosome is a large macromolecular machine, and correlated motion between residues is necessary for coordinating function across multiple protein and rna chains. we ran two all-atom, explicit solvent molecular dynamics simulations of the bacterial ribosome and calculated correlated motion between residue pairs by using mutual information. because of the short timescales of our simulation (ns), we expect that dynamics are largely local fluctuations around the crystal structure. we hypothesize ...201222235290
the antibiotic thermorubin inhibits protein synthesis by binding to inter-subunit bridge b2a of the ribosome.thermorubin is a small-molecule inhibitor of bacterial protein synthesis, but relatively little is known about the molecular mechanism by which it blocks translation. the structure of the complex between thermorubin and the 70s ribosome from thermus thermophilus reported here shows that thermorubin interacts with the ribosome in a way that is distinct from any other known class of ribosome inhibitor. though it is structurally similar to tetracycline, it binds to the ribosome at an entirely diffe ...201222240456
the fpg/nei family of dna glycosylases: substrates, structures, and search for damage.during the initial stages of the base excision dna repair pathway, dna glycosylases are responsible for locating and removing the majority of endogenous oxidative base lesions. the bifunctional formamidopyrimidine dna glycosylase (fpg) and endonuclease viii (nei) are members of the fpg/nei family, one of the two families of glycosylases that recognize oxidized dna bases, the other being the hhh/gpd (or nth) superfamily. structural and biochemical developments over the past decades have led to no ...201222749143
an exit cavity was crucial to the polymerase activity of the early ribosome.the emergence of an rna entity capable of synthesizing peptides was a key prebiotic development. it is hypothesized that a precursor of the modern ribosomal exit tunnel was associated with this rna entity (e.g., "protoribosome" or "bonding entity") from the earliest time and played an essential role. various compounds that can bind and activate amino acids, including extremely short rna chains carrying amino acids, and possibly di- or tripeptides, would have associated with the internal cavity o ...201222191510
learning from bacteriophages - advantages and limitations of phage and phage-encoded protein applications.the emergence of bacteria resistance to most of the currently available antibiotics has become a critical therapeutic problem. the bacteria causing both hospital and community-acquired infections are most often multidrug resistant. in view of the alarming level of antibiotic resistance between bacterial species and difficulties with treatment, alternative or supportive antibacterial cure has to be developed. the presented review focuses on the major characteristics of bacteriophages and phage-en ...201223305359
characterization and structure of the aquifex aeolicus protein duf752: a bacterial trna-methyltransferase (mnmc2) functioning without the usually fused oxidase domain (mnmc1).post-transcriptional modifications of the wobble uridine (u34) of trnas play a critical role in reading nna/g codons belonging to split codon boxes. in a subset of escherichia coli trna, this wobble uridine is modified to 5-methylaminomethyluridine (mnm(5)u34) through sequential enzymatic reactions. uridine 34 is first converted to 5-carboxymethylaminomethyluridine (cmnm(5)u34) by the mnme-mnmg enzyme complex. the cmnm(5)u34 is further modified to mnm(5)u by the bifunctional mnmc protein. in the ...201223091054
redundancy and modularity in membrane-associated dissimilatory nitrate reduction in bacillus.the genomes of two phenotypically denitrifying type strains of the genus bacillus were sequenced and the pathways for dissimilatory nitrate reduction were reconstructed. results suggest that denitrification proceeds in the periplasmic space and in an analogous fashion as in gram-negative organisms, yet with the participation of proteins that tend to be membrane-bound or membrane-associated. a considerable degree of functional redundancy was observed with marked differences between b. azotoforman ...201223087684
the crystal structures of the α-subunit of the α(2)β (2) tetrameric glycyl-trna synthetase.aminoacyl-trna synthetases (aarss) are ligases (ec.6.1.1.-) that catalyze the acylation of amino acids to their cognate trnas in the process of translating genetic information from mrna to protein. their amino acid and trna specificity are crucial for correctly translating the genetic code. glycine is the smallest amino acid and the glycyl-trna synthetase (glyrs) belongs to class ii aarss. the enzyme is unusual because it can assume different quaternary structures. in eukaryotes, archaebacteria ...201223054484
specific metal recognition in nickel trafficking.nickel is an essential metal for a number of bacterial species that have developed systems for acquiring, delivering, and incorporating the metal into target enzymes and controlling the levels of nickel in cells to prevent toxic effects. as with other transition metals, these trafficking systems must be able to distinguish between the desired metal and other transition metal ions with similar physical and chemical properties. because there are few enzymes (targets) that require nickel for activi ...201222970729
the role of bacterial enhancer binding proteins as specialized activators of σ54-dependent transcription.bacterial enhancer binding proteins (bebps) are transcriptional activators that assemble as hexameric rings in their active forms and utilize atp hydrolysis to remodel the conformation of rna polymerase containing the alternative sigma factor σ(54). we present a comprehensive and detailed summary of recent advances in our understanding of how these specialized molecular machines function. the review is structured by introducing each of the three domains in turn: the central catalytic domain, the ...201222933558
double-stranded endonuclease activity in bacillus halodurans clustered regularly interspaced short palindromic repeats (crispr)-associated cas2 protein.the crispr (clustered regularly interspaced short palindromic repeats) system is a prokaryotic rna-based adaptive immune system against extrachromosomal genetic elements. cas2 is a universally conserved core crispr-associated protein required for the acquisition of new spacers for crispr adaptation. it was previously characterized as an endoribonuclease with preference for single-stranded (ss)rna. here, we show using crystallography, mutagenesis, and isothermal titration calorimetry that the bac ...201222942283
phylogenetic position of aquificales based on the whole genome sequences of six aquificales species.species belonging to the order aquificales are believed to be an early branching lineage within the bacteria. however, the branching order of this group in single-gene phylogenetic trees is highly variable; for example, it has also been proposed that the aquificales should be grouped with ε-proteobacteria. to investigate the phylogenetic position of aquificales at the whole-genome level, here we reconstructed the phylogenetic trees of 18 bacteria including six aquificales species based on the co ...201222844640
structural dynamics of the aminoacylation and proofreading functional cycle of bacterial leucyl-trna synthetase.leucyl-trna synthetase (leurs) produces error-free leucyl-trna(leu) by coordinating translocation of the 3' end of (mis-)charged trnas from its synthetic site to a separate proofreading site for editing. here we report cocrystal structures of the escherichia coli leurs-trna(leu) complex in the aminoacylation or editing conformations, showing that translocation involves correlated rotations of four flexibly linked leurs domains. this pivots the trna to guide its charged 3' end from the closed ami ...201222683997
rational design and directed evolution of a bacterial-type glutaminyl-trna synthetase precursor.protein biosynthesis requires aminoacyl-transfer rna (trna) synthetases to provide aminoacyl-trna substrates for the ribosome. most bacteria and all archaea lack a glutaminyl-trna synthetase (glnrs); instead, gln-trna(gln) is produced via an indirect pathway: a glutamyl-trna synthetase (glurs) first attaches glutamate (glu) to trna(gln), and an amidotransferase converts glu-trna(gln) to gln-trna(gln). the human pathogen helicobacter pylori encodes two glurs enzymes, with glurs2 specifically amin ...201222661575
structure of escherichia coli aspartate α-decarboxylase asn72ala: probing the role of asn72 in pyruvoyl cofactor formation.the crystal structure of the asn72ala site-directed mutant of escherichia coli aspartate α-decarboxylase (adc) has been determined at 1.7 å resolution. the refined structure is consistent with the presence of a hydrolysis product serine in the active site in place of the pyruvoyl group required for catalysis, which suggests that the role of asn72 is to protect the ester formed during adc activation from hydrolysis. in previously determined structures of activated adc, including the wild type and ...201222505409
chemoinformatic identification of novel inhibitors against mycobacterium tuberculosis l-aspartate α-decarboxylase.l-aspartate α-decarboxylase (adc) belongs to a class of pyruvoyl dependent enzymes and catalyzes the conversion of aspartate to β-alanine in the pantothenate pathway, which is critical for the growth of several micro-organisms, including mycobacterium tuberculosis (mtb). its presence only in micro-organisms, fungi and plants and its absence in animals, particularly human, make it a promising drug target. we have followed a chemoinformatics-based approach to identify potential drug-like inhibitor ...201222470451
genetic recombination in bacillus subtilis: a division of labor between two single-strand dna-binding proteins.we have investigated the structural, biochemical and cellular roles of the two single-stranded (ss) dna-binding proteins from bacillus subtilis, ssba and ssbb. during transformation, ssbb localizes at the dna entry pole where it binds and protects internalized ssdna. the 2.8-å resolution structure of ssbb bound to ssdna reveals a similar overall protein architecture and ssdna-binding surface to that of escherichia coli ssb. ssba, which binds ssdna with higher affinity than ssbb, co-assembles ont ...201222373918
new insight into the transcarbamylase family: the structure of putrescine transcarbamylase, a key catalyst for fermentative utilization of agmatine.transcarbamylases reversibly transfer a carbamyl group from carbamylphosphate (cp) to an amine. although aspartate transcarbamylase and ornithine transcarbamylase (otc) are well characterized, little was known about putrescine transcarbamylase (ptc), the enzyme that generates cp for atp production in the fermentative catabolism of agmatine. we demonstrate that ptc (from enterococcus faecalis), in addition to using putrescine, can utilize l-ornithine as a poor substrate. crystal structures at 2.5 ...201222363663
substrate channeling in proline metabolism.proline metabolism is an important pathway that has relevance in several cellular functions such as redox balance, apoptosis, and cell survival. results from different groups have indicated that substrate channeling of proline metabolic intermediates may be a critical mechanism. one intermediate is pyrroline-5-carboxylate (p5c), which upon hydrolysis opens to glutamic semialdehyde (gsa). recent structural and kinetic evidence indicate substrate channeling of p5c/gsa occurs in the proline catabol ...201222201749
crystal structure of rlmm, the 2'o-ribose methyltransferase for c2498 of escherichia coli 23s rrna.rlmm (ygde) catalyzes the s-adenosyl methionine (adomet)-dependent 2'o methylation of c2498 in 23s ribosomal rna (rrna) of escherichia coli. previous experiments have shown that rlmm is active on 23s rrna from an rlmm knockout strain but not on mature 50s subunits from the same strain. here, we demonstrate rlmm methyltransferase (mtase) activity on in vitro transcribed 23s rrna and its domain v. we have solved crystal structures of e. coli rlmm at 1.9 å resolution and of an rlmm-adomet complex a ...201222923526
frequency, spectrum, and nonzero fitness costs of resistance to myxopyronin in staphylococcus aureus.the antibiotic myxopyronin (myx) functions by inhibiting bacterial rna polymerase (rnap). the binding site on rnap for myx-the rnap "switch region sw1/sw2 subregion"-is different from the binding site on rnap for the rnap inhibitor currently used in broad-spectrum antibacterial therapy, rifampin (rif). here, we report the frequency, spectrum, and fitness costs of myx resistance in staphylococcus aureus. the resistance rate for myx is 4 × 10(-8) to 7 × 10(-8) per generation, which is equal within ...201223006749
the porphyrias: advances in diagnosis and treatment.the inborn errors of heme biosynthesis, the porphyrias, are 8 genetically distinct metabolic disorders that can be classified as "acute hepatic," "hepatic cutaneous," and "erythropoietic cutaneous" diseases. recent advances in understanding their pathogenesis and molecular genetic heterogeneity have led to improved diagnosis and treatment. these advances include dna-based diagnoses for all the porphyrias, new understanding of the pathogenesis of the acute hepatic porphyrias, identification of th ...201222791288
chemosensory signaling controls motility and subcellular polarity in myxococcus xanthus.myxococcus xanthus is a model system for the study of dynamic protein localization and cell polarity in bacteria. m. xanthus cells are motile on solid surfaces enabled by two forms of motility. motility is controlled by the che-like frz pathway, which is essential for fruiting body formation and differentiation. the frz signal is mediated by a gtpase/gap protein pair that establishes cell polarity and directs the motility systems. pilus driven motility at the leading pole of the cell requires dy ...201223142584
function and regulation of clustered regularly interspaced short palindromic repeats (crispr) / crispr associated (cas) systems.phages are the most abundant biological entities on earth and pose a constant challenge to their bacterial hosts. thus, bacteria have evolved numerous 'innate' mechanisms of defense against phage, such as abortive infection or restriction/modification systems. in contrast, the clustered regularly interspaced short palindromic repeats (crispr) systems provide acquired, yet heritable, sequence-specific 'adaptive' immunity against phage and other horizontally-acquired elements, such as plasmids. re ...201223202464
interaction of card with rna polymerase mediates mycobacterium tuberculosis viability, rifampin resistance, and pathogenesis.mycobacterium tuberculosis infection continues to cause substantial human suffering. new chemotherapeutic strategies, which require insight into the pathways essential for m. tuberculosis pathogenesis, are imperative. we previously reported that depletion of the card protein in mycobacteria compromises viability, resistance to oxidative stress and fluoroquinolones, and pathogenesis. card associates with the rna polymerase (rnap), but it has been unknown which of the diverse functions of card are ...201222904282
whole genome analysis of leptospira licerasiae provides insight into leptospiral evolution and pathogenicity.the whole genome analysis of two strains of the first intermediately pathogenic leptospiral species to be sequenced (leptospira licerasiae strains var010 and mmd0835) provides insight into their pathogenic potential and deepens our understanding of leptospiral evolution. comparative analysis of eight leptospiral genomes shows the existence of a core leptospiral genome comprising 1547 genes and 452 conserved genes restricted to infectious species (including l. licerasiae) that are likely to be pa ...201223145189
naxd is a deacetylase required for lipid a modification and francisella pathogenesis.modification of specific gram-negative bacterial cell envelope components, such as capsule, o-antigen and lipid a, are often essential for the successful establishment of infection. francisella species express lipid a molecules with unique characteristics involved in circumventing host defences, which significantly contribute to their virulence. in this study, we show that naxd, a member of the highly conserved ydjc superfamily, is a deacetylase required for an important modification of the oute ...201222966934
architecture and conservation of the bacterial dna replication machinery, an underexploited drug target.new antibiotics with novel modes of action are required to combat the growing threat posed by multi-drug resistant bacteria. over the last decade, genome sequencing and other high-throughput techniques have provided tremendous insight into the molecular processes underlying cellular functions in a wide range of bacterial species. we can now use these data to assess the degree of conservation of certain aspects of bacterial physiology, to help choose the best cellular targets for development of n ...201222206257
rna polymerase-promoter interactions determining different stability of the escherichia coli and thermus aquaticus transcription initiation complexes.transcription initiation complexes formed by bacterial rna polymerases (rnaps) exhibit dramatic species-specific differences in stability, leading to different strategies of transcription regulation. the molecular basis for this diversity is unclear. promoter complexes formed by rnap from thermus aquaticus (taq) are considerably less stable than escherichia coli rnap promoter complexes, particularly at temperatures below 37°c. here, we used a fluorometric rnap molecular beacon assay to discern p ...201223087380
interplay of dna repair with transcription: from structures to mechanisms.many dna transactions are crucial for maintaining genomic integrity and faithful transfer of genetic information but remain poorly understood. an example is the interplay between nucleotide excision repair (ner) and transcription, also known as transcription-coupled dna repair (tcr). discovered decades ago, the mechanisms for tcr have remained elusive, not in small part due to the scarcity of structural studies of key players. here we summarize recent structural information on ner/tcr factors, f ...201223084398
altered large-ring cyclodextrin product profile due to a mutation at tyr-172 in the amylomaltase of corynebacterium glutamicum.corynebacterium glutamicum amylomaltase (cgam) catalyzes the formation of large-ring cyclodextrins (lr-cds) with a degree of polymerization of 19 and higher. the cloned cgam gene was ligated into the pet-17b vector and used to transform escherichia coli bl21(de3). site-directed mutagenesis of tyr-172 in cgam to alanine (y172a) was performed to determine its role in the control of lr-cd production. both the recombinant wild-type (wt) and y172a enzymes were purified to apparent homogeneity and cha ...201222865069
basic mechanisms of rna polymerase ii activity and alteration of gene expression in saccharomyces cerevisiae.transcription by rna polymerase ii (pol ii), and all rna polymerases for that matter, may be understood as comprising two cycles. the first cycle relates to the basic mechanism of the transcription process wherein pol ii must select the appropriate nucleoside triphosphate (ntp) substrate complementary to the dna template, catalyze phosphodiester bond formation, and translocate to the next position on the dna template. performing this cycle in an iterative fashion allows the synthesis of rna chai ...201223022618
basic mechanisms of rna polymerase ii activity and alteration of gene expression in saccharomyces cerevisiae.transcription by rna polymerase ii (pol ii), and all rna polymerases for that matter, may be understood as comprising two cycles. the first cycle relates to the basic mechanism of the transcription process wherein pol ii must select the appropriate nucleoside triphosphate (ntp) substrate complementary to the dna template, catalyze phosphodiester bond formation, and translocate to the next position on the dna template. performing this cycle in an iterative fashion allows the synthesis of rna chai ...201223022618
dna stabilization at the bacillus subtilis polx core--a binding model to coordinate polymerase, ap-endonuclease and 3'-5' exonuclease activities.family x dna polymerases (polxs) are involved in dna repair. their binding to gapped dnas relies on two conserved helix-hairpin-helix motifs, one located at the 8-kda domain and the other at the fingers subdomain. bacterial/archaeal polxs have a specifically conserved third helix-hairpin-helix motif (gfgxk) at the fingers subdomain whose putative role in dna binding had not been established. here, mutagenesis at the corresponding residues of bacillus subtilis polx (polxbs), gly130, gly132 and ly ...201222844091
structural basis for translation termination by archaeal rf1 and gtp-bound ef1α complex.when a stop codon appears at the ribosomal a site, the class i and ii release factors (rfs) terminate translation. in eukaryotes and archaea, the class i and ii rfs form a heterodimeric complex, and complete the overall translation termination process in a gtp-dependent manner. however, the structural mechanism of the translation termination by the class i and ii rf complex remains unresolved. in archaea, archaeal elongation factor 1 alpha (aef1α), a carrier gtpase for trna, acts as a class ii r ...201222772989
draft genome sequence of thermus sp. strain rl, isolated from a hot water spring located atop the himalayan ranges at manikaran, india.thermus sp. strain rl was isolated from a hot water spring (90°c to 98°c) at manikaran, himachal pradesh, india. here we report the draft genome sequence (20,36,600 bp) of this strain. the draft genome sequence consists of 17 contigs and 1,986 protein-coding sequences and has an average g+c content of 68.77%.201222689228
distinct functions of regions 1.1 and 1.2 of rna polymerase σ subunits from escherichia coli and thermus aquaticus in transcription initiation.rna polymerase (rnap) from thermophilic thermus aquaticus is characterized by higher temperature of promoter opening, lower promoter complex stability, and higher promoter escape efficiency than rnap from mesophilic escherichia coli. we demonstrate that these differences are in part explained by differences in the structures of the n-terminal regions 1.1 and 1.2 of the e. coli σ(70) and t. aquaticus σ(a) subunits. in particular, region 1.1 and, to a lesser extent, region 1.2 of the e. coli σ(70) ...201222605342
closely related archaeal haloarcula hispanica icosahedral viruses hhiv-2 and sh1 have nonhomologous genes encoding host recognition functions.studies on viral capsid architectures and coat protein folds have revealed the evolutionary lineages of viruses branching to all three domains of life. a widespread group of icosahedral tailless viruses, the prd1-adenovirus lineage, was the first to be established. a double β-barrel fold for a single major capsid protein is characteristic of these viruses. similar viruses carrying genes coding for two major capsid proteins with a more complex structure, such as thermus phage p23-77 and haloarcha ...201222357274
characterization of multi-functional properties and conformational analysis of muts2 from thermotoga maritima msb8.the muts2 homologues have received attention because of their unusual activities that differ from those of muts. in this work, we report on the functional characteristics and conformational diversities of thermotoga maritima muts2 (tmmuts2). various biochemical features of the protein were demonstrated via diverse techniques such as scanning probe microscopy (spm), atpase assays, analytical ultracentrifugation, dna binding assays, size chromatography, and limited proteolytic analysis. dimeric tm ...201222545085
related bifunctional restriction endonuclease-methyltransferase triplets: tspdti, tth111ii/tthhb27i and tsoi with distinct specificities.we previously defined a family of restriction endonucleases (reases) from thermus sp., which share common biochemical and biophysical features, such as the fusion of both the nuclease and methyltransferase (mtase) activities in a single polypeptide, cleavage at a distance from the recognition site, large molecular size, modulation of activity by s-adenosylmethionine (sam), and incomplete cleavage of the substrate dna. members include related thermophilic reases with five distinct specificities: ...201222489904
a novel phage-encoded transcription antiterminator acts by suppressing bacterial rna polymerase pausing.gp39, a small protein encoded by thermus thermophilus phage p23-45, specifically binds the host rna polymerase (rnap) and inhibits transcription initiation. here, we demonstrate that gp39 also acts as an antiterminator during transcription through intrinsic terminators. the antitermination activity of gp39 relies on its ability to suppress transcription pausing at poly(u) tracks. gp39 also accelerates transcription elongation by decreasing rnap pausing and backtracking but does not significantly ...201222238378
structure of ribose 5-phosphate isomerase from the probiotic bacterium lactobacillus salivarius ucc118.the structure of ribose 5-phosphate isomerase from the probiotic bacterium lactobacillus salivarius ucc188 has been determined at 1.72 å resolution. the structure was solved by molecular replacement, which identified the functional homodimer in the asymmetric unit. despite only showing 57% sequence identity to its closest homologue, the structure adopted the typical α and β d-ribose 5-phosphate isomerase fold. comparison to other related structures revealed high homology in the active site, allo ...201223192019
high-resolution structures of thermus thermophilus enoyl-acyl carrier protein reductase in the apo form, in complex with nad+ and in complex with nad+ and triclosan.enoyl-acyl carrier protein reductase (enr; the product of the fabi gene) is an important enzyme that is involved in the type ii fatty-acid-synthesis pathway of bacteria, plants, apicomplexan protozoa and mitochondria. harmful pathogens such as mycobacterium tuberculosis and plasmodium falciparum use the type ii fatty-acid-synthesis system, but not mammals or fungi, which contain a type i fatty-acid-synthesis pathway consisting of one or two multifunctional enzymes. for this reason, specific inhi ...201223027736
molecular evolution of hydrogen peroxide degrading enzymes.for efficient removal of intra- and/or extracellular hydrogen peroxide by dismutation to harmless dioxygen and water (2h(2)o(2) → o(2) + 2h(2)o), nature designed three metalloenzyme families that differ in oligomeric organization, monomer architecture as well as active site geometry and catalytic residues. here we report on the updated reconstruction of the molecular phylogeny of these three gene families. ubiquitous typical (monofunctional) heme catalases are found in all domains of life showin ...201222330759
glutamine versus ammonia utilization in the nad synthetase family.nad is a ubiquitous and essential metabolic redox cofactor which also functions as a substrate in certain regulatory pathways. the last step of nad synthesis is the atp-dependent amidation of deamido-nad by nad synthetase (nads). members of the nads family are present in nearly all species across the three kingdoms of life. in eukaryotic nads, the core synthetase domain is fused with a nitrilase-like glutaminase domain supplying ammonia for the reaction. this two-domain nads arrangement enabling ...201222720044
staphylococcus aureus fabi: inhibition, substrate recognition, and potential implications for in vivo essentiality.methicillin-resistant staphylococcus aureus (mrsa) infections constitute a serious health threat worldwide, and novel antibiotics are therefore urgently needed. the enoyl-acp reductase (safabi) is essential for the s. aureus fatty acid biosynthesis and, hence, serves as an attractive drug target. we have obtained a series of snapshots of this enzyme that provide a mechanistic picture of ligand and inhibitor binding, including a dimer-tetramer transition combined with extensive conformational cha ...201222579249
aggregate reactivation mediated by the hsp100 chaperones.hsp100 family of molecular chaperones shows a unique capability to resolubilize and reactivate aggregated proteins. the hsp100-mediated protein disaggregation is linked to the activity of other chaperones from the hsp70 and hsp40 families. the best-studied members of the hsp100 family are the bacterial clpb and hsp104 from yeast. hsp100 chaperones are members of a large super-family of energy-driven conformational "machines" known as aaa+ atpases. this review describes the current mechanistic mo ...201222306514
spectral identification of intermediates generated during the reaction of dioxygen with the wild-type and eq(i-286) mutant of rhodobacter sphaeroides cytochrome c oxidase.cytochrome c oxidase from rhodobacter sphaeroides is frequently used to model the more complex mitochondrial enzyme. the o(2) reduction in both enzymes is generally described by a unidirectional mechanism involving the sequential formation of the ferrous-oxy complex (compound a), the p(r) state, the oxyferryl f form, and the oxidized state. in this study we investigated the reaction of dioxygen with the wild-type reduced r. sphaeroides cytochrome oxidase and the eq(i-286) mutant using the co flo ...201223057757
roles of subunit nuok (nd4l) in the energy-transducing mechanism of escherichia coli ndh-1 (nadh:quinone oxidoreductase).the bacterial h(+)-translocating nadh:quinone oxidoreductase (ndh-1) catalyzes electron transfer from nadh to quinone coupled with proton pumping across the cytoplasmic membrane. the nuok subunit (counterpart of the mitochondrial nd4l subunit) is one of the seven hydrophobic subunits in the membrane domain and bears three transmembrane segments (tm1-3). two glutamic residues located in the adjacent transmembrane helices of nuok are important for the energy coupled activity of ndh-1. in particula ...201223105119
cell-free protein synthesis: the state of the art.cell-free protein synthesis harnesses the synthetic power of biology, programming the ribosomal translational machinery of the cell to create macromolecular products. like pcr, which uses cellular replication machinery to create a dna amplifier, cell-free protein synthesis is emerging as a transformative technology with broad applications in protein engineering, biopharmaceutical development, and post-genomic research. by breaking free from the constraints of cell-based systems, it takes the nex ...201223086573
cell-free protein synthesis: the state of the art.cell-free protein synthesis harnesses the synthetic power of biology, programming the ribosomal translational machinery of the cell to create macromolecular products. like pcr, which uses cellular replication machinery to create a dna amplifier, cell-free protein synthesis is emerging as a transformative technology with broad applications in protein engineering, biopharmaceutical development, and post-genomic research. by breaking free from the constraints of cell-based systems, it takes the nex ...201223086573
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