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the genome sequence of the crenarchaeon acidilobus saccharovorans supports a new order, acidilobales, and suggests an important ecological role in terrestrial acidic hot springs.acidilobus saccharovorans is an anaerobic, organotrophic, thermoacidophilic crenarchaeon isolated from a terrestrial hot spring. we report the complete genome sequence of a. saccharovorans, which has permitted the prediction of genes for embden-meyerhof and entner-doudoroff pathways and genes associated with the oxidative tricarboxylic acid cycle. the electron transfer chain is branched with two sites of proton translocation and is linked to the reduction of elemental sulfur and thiosulfate. the ...201020581186
recognition of the amber uag stop codon by release factor rf1.we report the crystal structure of a termination complex containing release factor rf1 bound to the 70s ribosome in response to an amber (uag) codon at 3.6-a resolution. the amber codon is recognized in the 30s subunit-decoding centre directly by conserved elements of domain 2 of rf1, including t186 of the pvt motif. together with earlier structures, the mechanisms of recognition of all three stop codons by release factors rf1 and rf2 can now be described. our structure confirms that the backbon ...201020588254
using molecular dynamics to probe the structural basis for enhanced stability in thermal stable cytochromes p450.high-temperature molecular dynamics (md) has been used to assess if md can be employed as a useful tool for probing the structural basis for enhanced stability in thermal stable cytochromes p450. cyp119, the most thermal stable p450 known, unfolds more slowly during 500 k md simulations than p450s that melt at lower temperatures, p450cam and p450cin. a comparison of the 500 k md trajectories shows that the cys ligand loop, a critically important structural feature just under the heme, in both p4 ...201020593793
p. aeruginosa pilt structures with and without nucleotide reveal a dynamic type iv pilus retraction motor.type iv pili are bacterial extracellular filaments that can be retracted to create force and motility. retraction is accomplished by the motor protein pilt. crystal structures of pseudomonas aeruginosa pilt with and without bound beta,gamma-methyleneadenosine-5'-triphosphate have been solved at 2.6 a and 3.1 a resolution, respectively, revealing an interlocking hexamer formed by the action of a crystallographic 2-fold symmetry operator on three subunits in the asymmetric unit and held together b ...201020595000
the ccmc:heme:ccme complex in heme trafficking and cytochrome c biosynthesis.a superfamily of integral membrane proteins is characterized by a conserved tryptophan-rich region (called the wwd domain) in an external loop at the inner membrane surface. the three major members of this family (ccmc, ccmf, and ccsba) are each involved in cytochrome c biosynthesis, yet the function of the wwd domain is unknown. it has been hypothesized that the wwd domain binds heme to present it to an acceptor protein (apoccme for ccmc or apocytochrome c for ccmf and ccsba) such that the heme ...201020599545
properties of the c-terminal tail of human mitochondrial inner membrane protein oxa1l and its interactions with mammalian mitochondrial ribosomes.in humans the mitochondrial inner membrane protein oxa1l is involved in the biogenesis of membrane proteins and facilitates the insertion of both mitochondrial- and nuclear-encoded proteins from the mitochondrial matrix into the inner membrane. the c-terminal approximately 100-amino acid tail of oxa1l (oxa1l-ctt) binds to mitochondrial ribosomes and plays a role in the co-translational insertion of mitochondria-synthesized proteins into the inner membrane. contrary to suggestions made for yeast ...201020601428
structure of an archaeal non-discriminating glutamyl-trna synthetase: a missing link in the evolution of gln-trnagln formation.the molecular basis of the genetic code relies on the specific ligation of amino acids to their cognate trna molecules. however, two pathways exist for the formation of gln-trna(gln). the evolutionarily older indirect route utilizes a non-discriminating glutamyl-trna synthetase (nd-glurs) that can form both glu-trna(glu) and glu-trna(gln). the glu-trna(gln) is then converted to gln-trna(gln) by an amidotransferase. since the well-characterized bacterial nd-glurs enzymes recognize trna(glu) and t ...201020601684
bacillus anthracis surface-layer proteins assemble by binding to the secondary cell wall polysaccharide in a manner that requires csab and tago.bacillus anthracis, the causative agent of anthrax, requires surface (s)-layer proteins for the pathogenesis of infection. previous work characterized s-layer protein binding via the surface layer homology domain to a pyruvylated carbohydrate in the envelope of vegetative forms. the molecular identity of this carbohydrate and the mechanism of its display in the bacterial envelope are still unknown. analyzing acid-solubilized, purified carbohydrates by mass spectrometry and nmr spectroscopy, we i ...201020603129
the subunit composition of mitochondrial nadh:ubiquinone oxidoreductase (complex i) from pichia pastoris.respiratory complex i (nadh:quinone oxidoreductase) is an entry point to the electron transport chain in the mitochondria of many eukaryotes. it is a large, multisubunit enzyme with a hydrophilic domain in the matrix and a hydrophobic domain in the mitochondrial inner membrane. here we present a comprehensive analysis of the protein composition and post-translational modifications of complex i from pichia pastoris, using a combination of proteomic and bioinformatic approaches. forty-one subunits ...201020610779
evolution and thermodynamics of the slow unfolding of hyperstable monomeric proteins.the unfolding speed of some hyperthermophilic proteins is dramatically lower than that of their mesostable homologs. ribonuclease hii from the hyperthermophilic archaeon thermococcus kodakaraensis (tk-rnase hii) is stabilized by its remarkably slow unfolding rate, whereas rnase hi from the thermophilic bacterium thermus thermophilus (tt-rnase hi) unfolds rapidly, comparable with to that of rnase hi from escherichia coli (ec-rnase hi).201020615256
promoter melting triggered by bacterial rna polymerase occurs in three steps.rna synthesis, carried out by dna-dependent rna polymerase (rnap) in a process called transcription, involves several stages. in bacteria, transcription initiation starts with promoter recognition and binding of rnap holoenzyme, resulting in the formation of the closed (r.p(c)) rnap-promoter dna complex. subsequently, a transition to the open r.p(o) complex occurs, characterized by separation of the promoter dna strands in an approximately 12 base-pair region to form the transcription bubble. us ...201020615963
synthesis of glu-trna(gln) by engineered and natural aminoacyl-trna synthetases.a protein engineering approach to delineating which distinct elements of homologous trna synthetase architectures are responsible for divergent rna-amino acid pairing specificities is described. previously, we constructed a hybrid enzyme in which 23 amino acids from the catalytic domain of escherichia coli glutaminyl-trna synthetase (glnrs) were replaced with the corresponding residues of human glutamyl-trna synthetase (glurs). the engineered hybrid (glnrs s1/l1/l2) synthesizes glu-trna(gln) mor ...201020617848
a novel heme a insertion factor gene cotranscribes with the thermus thermophilus cytochrome ba3 oxidase locus.studying the biogenesis of the thermus thermophilus cytochrome ba(3) oxidase, we analyze heme a cofactor insertion into this membrane protein complex. only three proteins linked to oxidase maturation have been described for this extreme thermophile, and in particular, no evidence for a canonical surf1 homologue, required for heme a insertion, is available from genome sequence data. here, we characterize the product of an open reading frame, cbax, in the operon encoding subunits of the ba(3)-type ...201020622059
diversity of glycosyl hydrolases from cellulose-depleting communities enriched from casts of two earthworm species.the guts and casts of earthworms contain microbial assemblages that process large amounts of organic polymeric substrates from plant litter and soil; however, the enzymatic potential of these microbial communities remains largely unexplored. in the present work, we retrieved carbohydrate-modifying enzymes through the activity screening of metagenomic fosmid libraries from cellulose-depleting microbial communities established with the fresh casts of two earthworm species, aporrectodea caliginosa ...201020622123
structural studies of tri-functional human gart.human purine de novo synthesis pathway contains several multi-functional enzymes, one of which, tri-functional gart, contains three enzymatic activities in a single polypeptide chain. we have solved structures of two domains bearing separate catalytic functions: glycinamide ribonucleotide synthetase and aminoimidazole ribonucleotide synthetase. structures are compared with those of homologous enzymes from prokaryotes and analyzed in terms of the catalytic mechanism. we also report small angle x- ...201020631005
biosynthesis of compatible solutes in rhizobial strains isolated from phaseolus vulgaris nodules in tunisian fields.associated with appropriate crop and soil management, inoculation of legumes with microbial biofertilizers can improve food legume yield and soil fertility and reduce pollution by inorganic fertilizers. rhizospheric bacteria are subjected to osmotic stress imposed by drought and/or nacl, two abiotic constraints frequently found in semi-arid lands. osmostress response in bacteria involves the accumulation of small organic compounds called compatible solutes. whereas most studies on rhizobial osmo ...201020633304
dynamics of secy translocons with translocation-defective mutations.the secy/sec61 translocon complex, located in the endoplasmic reticulum membrane of eukaryotes (sec61) or the plasma membrane of prokaryotes (secy), mediates the transmembrane secretion or insertion of nascent proteins. mutations that permit the secretion of nascent proteins with defective signal sequences (prl-phenotype), or interfere with the transmembrane orientation of newly synthesized protein segments, can affect protein topogenesis. the crystallographic structure of secyebeta from methano ...201020637421
functional analysis of thermus thermophilus transcription factor nusg.transcription elongation factors from the nusg family are ubiquitous from bacteria to humans and play diverse roles in the regulation of gene expression. these proteins consist of at least two domains. the n-terminal domains directly bind to the largest, β' in bacteria, subunit of rna polymerase (rnap), whereas the c-terminal domains interact with other cellular components and serve as platforms for the assembly of large nucleoprotein complexes. escherichia coli nusg and its paralog rfah modify ...201020639538
rosr (cg1324), a hydrogen peroxide-sensitive marr-type transcriptional regulator of corynebacterium glutamicum.the cg1324 gene (rosr) of corynebacterium glutamicum encodes a marr-type transcriptional regulator. by a comparative transcriptome analysis with dna microarrays of a δrosr mutant and the wild type and subsequent emsas with purified rosr protein, direct target genes of rosr were identified. the narkghji operon, which encodes a nitrate/nitrite transporter and the dissimilatory nitrate reductase complex, was activated by rosr. all other target genes were repressed by rosr. they encode four putative ...201020643656
mitochondrial enzyme rhodanese is essential for 5 s ribosomal rna import into human mitochondria.5 s rrna is an essential component of ribosomes. in eukaryotic cells, it is distinguished by particularly complex intracellular traffic, including nuclear export and re-import. the finding that in mammalian cells 5 s rrna can eventually escape its usual circuit toward nascent ribosomes to get imported into mitochondria has made the scheme more complex, and it has raised questions about both the mechanism of 5 s rrna mitochondrial targeting and its function inside the organelle. previously, we sh ...201020663881
linkage isomerization in heme-nox compounds: understanding no, nitrite, and hyponitrite interactions with iron porphyrins.nitric oxide (no) and its derivatives such as nitrite and hyponitrite are biologically important species of relevance to human health. much of their physiological relevance stems from their interactions with the iron centers in heme proteins. the chemical reactivities displayed by the heme-nox species (nox = no, nitrite, hyponitrite) are a function of the binding modes of the nox ligands. hence, an understanding of the types of binding modes extant in heme-nox compounds is important if we are to ...201020666385
interference with histidyl-trna synthetase by a crispr spacer sequence as a factor in the evolution of pelobacter carbinolicus.pelobacter carbinolicus, a bacterium of the family geobacteraceae, cannot reduce fe(iii) directly or produce electricity like its relatives. how p. carbinolicus evolved is an intriguing problem. the genome of p. carbinolicus contains clustered regularly interspaced short palindromic repeats (crispr) separated by unique spacer sequences, which recent studies have shown to produce rna molecules that interfere with genes containing identical sequences.201020667132
the crystal structure of human transketolase and new insights into its mode of action.the crystal structure of human transketolase (tkt), a thiamine diphosphate (thdp) and ca(2+)-dependent enzyme that catalyzes the interketol transfer between ketoses and aldoses as part of the pentose phosphate pathway, has been determined to 1.75 å resolution. the recombinantly produced protein crystallized in space group c2 containing one monomer in the asymmetric unit. two monomers form the homodimeric biological assembly with two identical active sites at the dimer interface. although the pro ...201020667822
structural and functional characterization of salmonella enterica serovar typhimurium ycbl: an unusual type ii glyoxalase.ycbl has been annotated as either a metallo-β-lactamase or glyoxalase ii (glx2), both members of the zinc metallohydrolase superfamily, that contains many enzymes with a diverse range of activities. here, we report crystallographic and biochemical data for salmonella enterica serovar typhimurium ycbl that establishes it as glx2, which differs in certain structural and functional properties compared with previously known examples. these features include the insertion of an α-helix after residue 8 ...201020669241
insights into the nitric oxide reductase mechanism of flavodiiron proteins from a flavin-free enzyme.flavodiiron proteins (fdps) catalyze reductive scavenging of dioxygen and nitric oxide in air-sensitive microorganisms. fdps contain a distinctive non-heme diiron/flavin mononucleotide (fmn) active site. alternative mechanisms for the nitric oxide reductase (nor) activity consisting of either protonation of a diiron-bridging hyponitrite or "super-reduction" of a diferrous-dinitrosyl by the proximal fmnh(2) in the rate-determining step have been proposed. to test these alternative mechanisms, we ...201020669924
poxa, yjek, and elongation factor p coordinately modulate virulence and drug resistance in salmonella enterica.we report an interaction between poxa, encoding a paralog of lysyl trna-synthetase, and the closely linked yjek gene, encoding a putative 2,3-beta-lysine aminomutase, that is critical for virulence and stress resistance in salmonella enterica. salmonella poxa and yjek mutants share extensive phenotypic pleiotropy, including attenuated virulence in mice, an increased ability to respire under nutrient-limiting conditions, hypersusceptibility to a variety of diverse growth inhibitors, and altered e ...201020670890
dual biosynthesis pathway for longer-chain polyamines in the hyperthermophilic archaeon thermococcus kodakarensis.long-chain and/or branched-chain polyamines are unique polycations found in thermophiles. cytoplasmic polyamines were analyzed for cells cultivated at various growth temperatures in the hyperthermophilic archaeon thermococcus kodakarensis. spermidine [34] and n4-aminopropylspermine [3(3)43] were identified as major polyamines at 60°c, and the amounts of n4-aminopropylspermine [3(3)43] increased as the growth temperature rose. to identify genes involved in polyamine biosynthesis, a gene disruptio ...201020675472
functional role of thr-312 and thr-315 in the proton-transfer pathway in ba3 cytochrome c oxidase from thermus thermophilus.cytochrome ba(3) from thermus thermophilus is a member of the family of b-type heme-copper oxidases, which have a low degree of sequence homology to the well-studied mitochondrial-like a-type enzymes. recently, it was suggested that the ba(3) oxidase has only one pathway for the delivery of protons to the active site and that this pathway is spatially analogous to the k-pathway in the a-type oxidases [chang, h.-y., et al. (2009) proc. natl. acad. sci. u.s.a. 106, 16169-16173]. this suggested pat ...201020677778
origin of light-induced spin-correlated radical pairs in cryptochrome.blue-light excitation of cryptochromes and homologues uniformly triggers electron transfer (et) from the protein surface to the flavin adenine dinucleotide (fad) cofactor. a cascade of three conserved tryptophan residues has been considered to be critically involved in this photoreaction. if the fad is initially in its fully oxidized (diamagnetic) redox state, light-induced et via the tryptophan triad generates a series of short-lived spin-correlated radical pairs comprising an fad radical and a ...201020684534
mechanism of microrna-target interaction: molecular dynamics simulations and thermodynamics analysis.micrornas (mirnas) are endogenously produced approximately 21-nt riboregulators that associate with argonaute (ago) proteins to direct mrna cleavage or repress the translation of complementary rnas. capturing the molecular mechanisms of mirna interacting with its target will not only reinforce the understanding of underlying rna interference but also fuel the design of more effective small-interfering rna strands. to address this, in the present work the rna-bound (ago-mirna, ago-mirna-target) a ...201020686687
three distinct peptides from the n domain of translation termination factor erf1 surround stop codon in the ribosome.to study positioning of the polypeptide release factor erf1 toward a stop signal in the ribosomal decoding site, we applied photoactivatable mrna analogs, derivatives of oligoribonucleotides. the human erf1 peptides cross-linked to these short mrnas were identified. cross-linkers on the guanines at the second, third, and fourth stop signal positions modified fragment 31-33, and to lesser extent amino acids within region 121-131 (the "yxcxxxf loop") in the n domain. hence, both regions are involv ...201020688868
structural and functional studies of wlba: a dehydrogenase involved in the biosynthesis of 2,3-diacetamido-2,3-dideoxy-d-mannuronic acid .2,3-diacetamido-2,3-dideoxy-d-mannuronic acid (mannac3naca) is an unusual dideoxy sugar first identified nearly 30 years ago in the lipopolysaccharide of pseudomonas aeruginosa o:3a,d. it has since been observed in other organisms, including bordetella pertussis, the causative agent of whooping cough. five enzymes are required for the biosynthesis of udp-mannac3naca starting from udp-n-acetyl-d-glucosamine. here we describe a structural study of wlba, the nad-dependent dehydrogenase that catalyz ...201020690587
structures of hypoxanthine-guanine phosphoribosyltransferase (ttha0220) from thermus thermophilus hb8.hypoxanthine-guanine phosphoribosyltransferase (hgprtase), which is a key enzyme in the purine-salvage pathway, catalyzes the synthesis of imp or gmp from alpha-d-phosphoribosyl-1-pyrophosphate and hypoxanthine or guanine, respectively. structures of hgprtase from thermus thermophilus hb8 in the unliganded form, in complex with imp and in complex with gmp have been determined at 2.1, 1.9 and 2.2 a resolution, respectively. the overall fold of the imp complex was similar to that of the unliganded ...201020693661
mutations in 23s rrna at the peptidyl transferase center and their relationship to linezolid binding and cross-resistance.the oxazolidinone antibiotic linezolid targets the peptidyl transferase center (ptc) on the bacterial ribosome. thirteen single and four double 23s rrna mutations were introduced into a mycobacterium smegmatis strain with a single rrna operon. converting bacterial base identity by single mutations at positions 2032, 2453, and 2499 to human cytosolic base identity did not confer significantly reduced susceptibility to linezolid. the largest decrease in linezolid susceptibility for any of the intr ...201020696869
yeast 18 s rrna is directly involved in the ribosomal response to stringent aug selection during translation initiation.in eukaryotes, the 40 s ribosomal subunit serves as the platform of initiation factor assembly, to place itself precisely on the aug start codon. structural arrangement of the 18 s rrna determines the overall shape of the 40 s subunit. here, we present genetic evaluation of yeast 18 s rrna function using 10 point mutations altering the polysome profile. all the mutants reduce the abundance of the mutant 40 s, making it limiting for translation initiation. two of the isolated mutations, g875a, al ...201020699223
structural basis for the bacterial transcription-repair coupling factor/rna polymerase interaction.the transcription-repair coupling factor (trcf, the product of the mfd gene) is a widely conserved bacterial protein that mediates transcription-coupled dna repair. trcf uses its atp-dependent dna translocase activity to remove transcription complexes stalled at sites of dna damage, and stimulates repair by recruiting components of the nucleotide excision repair pathway to the site. a protein/protein interaction between trcf and the β-subunit of rna polymerase (rnap) is essential for trcf functi ...201020702425
a flexible loop in yeast ribosomal protein l11 coordinates p-site trna binding.high-resolution structures reveal that yeast ribosomal protein l11 and its bacterial/archael homologs called l5 contain a highly conserved, basically charged internal loop that interacts with the peptidyl-transfer rna (trna) t-loop. we call this the l11 'p-site loop'. chemical protection of wild-type ribosome shows that that the p-site loop is inherently flexible, i.e. it is extended into the ribosomal p-site when this is unoccupied by trna, while it is retracted into the terminal loop of 25s rr ...201020705654
the diheme cytochrome c(4) from vibrio cholerae is a natural electron donor to the respiratory cbb(3) oxygen reductase.the respiratory chain of vibrio cholerae contains three bd-type quinol oxygen reductases as well as one cbb(3) oxygen reductase. the cbb(3) oxygen reductase has been previously isolated and characterized; however, the natural mobile electron donor(s) that shuttles electrons between the bc(1) complex and the cbb(3) oxygen reductase is not known. the most likely candidates are the diheme cytochrome c(4) and monoheme cytochrome c(5), which have been previously shown to be present in the periplasm o ...201020715760
crystal structure of a transfer-ribonucleoprotein particle that promotes asparagine formation.four out of the 22 aminoacyl-trnas (aa-trnas) are systematically or alternatively synthesized by an indirect, two-step route requiring an initial mischarging of the trna followed by trna-dependent conversion of the non-cognate amino acid. during trna-dependent asparagine formation, trna(asn) promotes assembly of a ribonucleoprotein particle called transamidosome that allows channelling of the aa-trna from non-discriminating aspartyl-trna synthetase active site to the gatcab amidotransferase site ...201020717102
dna mismatch repair in eukaryotes and bacteria.dna mismatch repair (mmr) corrects mismatched base pairs mainly caused by dna replication errors. the fundamental mechanisms and proteins involved in the early reactions of mmr are highly conserved in almost all organisms ranging from bacteria to human. the significance of this repair system is also indicated by the fact that defects in mmr cause human hereditary nonpolyposis colon cancers as well as sporadic tumors. to date, 2 types of mmrs are known: the human type and escherichia coli type. t ...201020725617
multifactorial determinants of protein expression in prokaryotic open reading frames.a quantitative description of the relationship between protein expression levels and open reading frame (orf) nucleotide sequences is important for understanding natural systems, designing synthetic systems, and optimizing heterologous expression. codon identity, mrna secondary structure, and nucleotide composition within orfs markedly influence expression levels. bioinformatic analysis of orf sequences in 816 bacterial genomes revealed that these features show distinct regional trends. to inves ...201020727358
a mutation within the β subunit of escherichia coli rna polymerase impairs transcription from bacteriophage t4 middle promoters.during infection of escherichia coli, bacteriophage t4 usurps the host transcriptional machinery, redirecting it to the expression of early, middle, and late phage genes. middle genes, whose expression begins about 1 min postinfection, are transcribed both from the extension of early rna into middle genes and by the activation of t4 middle promoters. middle-promoter activation requires the t4 transcriptional activator mota and coactivator asia, which are known to interact with σ(70), the specifi ...201020729353
an extreme thermophile, thermus thermophilus, is a polyploid bacterium.an extremely thermophilic bacterium, thermus thermophilus hb8, is one of the model organisms for systems biology. its genome consists of a chromosome (1.85 mb), a megaplasmid (0.26 mb) designated ptt27, and a plasmid (9.3 kb) designated ptt8, and the complete sequence is available. we show here that t. thermophilus is a polyploid organism, harboring multiple genomic copies in a cell. in the case of the hb8 strain, the copy number of the chromosome was estimated to be four or five, and the copy n ...201020729360
molecular basis of vancomycin dependence in vana-type staphylococcus aureus vrsa-9.the vancomycin-resistant staphylococcus aureus vrsa-9 clinical isolate was partially dependent on glycopeptide for growth. the responsible vana operon had the same organization as that of tn1546 and was located on a plasmid. the chromosomal d-ala:d-ala ligase (ddl) gene had two point mutations that led to q260k and a283e substitutions, resulting in a 200-fold decrease in enzymatic activity compared to that of the wild-type strain vrsa-6. to gain insight into the mechanism of enzyme impairment, w ...201020729361
n-terminal fusion of a hyperthermophilic chitin-binding domain to xylose isomerase from thermotoga neapolitana enhances kinetics and thermostability of both free and immobilized enzymes.immobilization of a thermostable d-xylose isomerase (ec 5.3.1.5) from thermotoga neapolitana 5068 (tnxi) on chitin beads was accomplished via a n-terminal fusion with a chitin-binding domain (cbd) from a hyperthermophilic chitinase produced by pyrococcus furiosus (pf1233) to create a fusion protein (cbd-tnxi). the turnover numbers for glucose to fructose conversion for both unbound and immobilized cbd-tnxi were greater than the wild-type enzyme: k(cat) (min(-1)) was approximately 1,000, 3,800, a ...201020730758
binding of the dimeric deinococcus radiodurans single-stranded dna binding protein to single-stranded dna.deinococcus radiodurans single-stranded (ss) dna binding protein (drssb) originates from a radiation-resistant bacterium and participates in dna recombination, replication, and repair. although it functions as a homodimer, it contains four dna binding domains (ob-folds) and thus is structurally similar to the escherichia coli ssb (ecossb) homotetramer. we examined the equilibrium binding of drssb to ssdna for comparison with that of ecossb. we find that the occluded site size of drssb on poly(dt ...201020795631
the role of upf0157 in the folding of m. tuberculosis dephosphocoenzyme a kinase and the regulation of the latter by ctp.targeting the biosynthetic pathway of coenzyme a (coa) for drug development will compromise multiple cellular functions of the tubercular pathogen simultaneously. structural divergence in the organization of the penultimate and final enzymes of coa biosynthesis in the host and pathogen and the differences in their regulation mark out the final enzyme, dephosphocoenzyme a kinase (coae) as a potential drug target.200919876400
isolation and characterization of tirandamycins from a marine-derived streptomyces sp.the novel dienoyl tetramic acids tirandamycin c (1) and tirandamycin d (2) with activity against vancomycin-resistant enterococcus faecalis were isolated from the marine environmental isolate streptomyces sp. 307-9, which also produces the previously identified compounds tirandamycins a (3) and b (4). spectroscopic analysis of 1 and 2 indicated structural similarity to 3 and 4, with differences only in the pattern of pendant oxygenation on the bicyclic ketal system. the isolation of these putati ...200919883065
atp-induced conformational changes in hsp70: molecular dynamics and experimental validation of an in silico predicted conformation.the 70 kda heat shock proteins (hsp70s) play important roles in preventing the misfolding of proteins and repairing damage under stress by coupling atp binding and hydrolysis to protein substrate release and binding, respectively. atp binding is believed to induce closing of the hsp70 nucleotide binding domain (nbd) around the nucleotide. we report here a combined computational-experimental study of this open-closed transition. all-atom molecular dynamics simulations were performed for isolated ...200919883127
the structure of the ribosome with elongation factor g trapped in the posttranslocational state.elongation factor g (ef-g) is a guanosine triphosphatase (gtpase) that plays a crucial role in the translocation of transfer rnas (trnas) and messenger rna (mrna) during translation by the ribosome. we report a crystal structure refined to 3.6 angstrom resolution of the ribosome trapped with ef-g in the posttranslocational state using the antibiotic fusidic acid. fusidic acid traps ef-g in a conformation intermediate between the guanosine triphosphate and guanosine diphosphate forms. the interac ...200919833919
the crystal structure of the ribosome bound to ef-tu and aminoacyl-trna.the ribosome selects a correct transfer rna (trna) for each amino acid added to the polypeptide chain, as directed by messenger rna. aminoacyl-trna is delivered to the ribosome by elongation factor tu (ef-tu), which hydrolyzes guanosine triphosphate (gtp) and releases trna in response to codon recognition. the signaling pathway that leads to gtp hydrolysis upon codon recognition is critical to accurate decoding. here we present the crystal structure of the ribosome complexed with ef-tu and amino ...200919833920
v for victory--a v1-atpase structure revealed. 200919834508
redox characterization of the fes protein mitoneet and impact of thiazolidinedione drug binding.mitoneet is a small mitochondrial protein that has been identified recently as a target for the thiazolidinedione (tzd) class of diabetes drugs. mitoneet also binds a unique three-cys- and one-his-ligated [corrected] [2fe-2s] cluster. here we use protein film voltammetry (pfv) as a means to probe the redox properties of mitoneet and demonstrate the direct impact of tzd drug binding upon the redox chemistry of the fes cluster. when tzds bind, the midpoint potential at ph 7 is lowered by more than ...200919791753
mg(2+)-dependent gating of bacterial mgte channel underlies mg(2+) homeostasis.the mgte family of mg(2+) transporters is ubiquitously distributed in all phylogenetic domains. recent crystal structures of the full-length mgte and of its cytosolic domain in the presence and absence of mg(2+) suggested a mg(2+)-homeostasis mechanism, in which the mgte cytosolic domain acts as a 'mg(2+) sensor' to regulate the gating of the ion-conducting pore in response to the intracellular mg(2+) concentration. however, complementary functional analyses to confirm the proposed model have be ...200919798051
crystal structure of ynje from escherichia coli, a sulfurtransferase with three rhodanese domains.rhodaneses/sulfurtransferases are ubiquitous enzymes that catalyze the transfer of sulfane sulfur from a donor molecule to a thiophilic acceptor via an active site cysteine that is modified to a persulfide during the reaction. here, we present the first crystal structure of a triple-domain rhodanese-like protein, namely ynje from escherichia coli, in two states where its active site cysteine is either unmodified or present as a persulfide. compared to well-characterized tandem domain rhodaneses, ...200919798741
multiple biochemical and morphological factors underlie the production of methylketones in tomato trichomes.genetic analysis of interspecific populations derived from crosses between the wild tomato species solanum habrochaites f. sp. glabratum, which synthesizes and accumulates insecticidal methylketones (mk), mostly 2-undecanone and 2-tridecanone, in glandular trichomes, and cultivated tomato (solanum lycopersicum), which does not, demonstrated that several genetic loci contribute to mk metabolism in the wild species. a strong correlation was found between the shape of the glandular trichomes and th ...200919801397
two-dimensional pulsed electron spin resonance characterization of 15n-labeled archaeal rieske-type ferredoxin.two-dimensional electron spin-echo envelope modulation (eseem) analysis of the uniformly (15)n-labeled archaeal rieske-type [2fe-2s] ferredoxin (arf) from sulfolobus solfataricus p1 has been conducted in comparison with the previously characterized high-potential protein homologs. major differences among these proteins were found in the hyperfine sublevel correlation (hyscore) lineshapes and intensities of the signals in the (++) quadrant, which are contributed from weakly coupled (non-coordinat ...200919804777
the cytochrome ba3 oxygen reductase from thermus thermophilus uses a single input channel for proton delivery to the active site and for proton pumping.the heme-copper oxygen reductases are redox-driven proton pumps that generate a proton motive force in both prokaryotes and mitochondria. these enzymes have been divided into 3 evolutionarily related groups: the a-, b- and c-families. most experimental work on proton-pumping mechanisms has been performed with members of the a-family. these enzymes require 2 proton input pathways (d- and k-channels) to transfer protons used for oxygen reduction chemistry and for proton pumping, with the d-channel ...200919805275
structure of a trna-dependent kinase essential for selenocysteine decoding.compared to bacteria, archaea and eukaryotes employ an additional enzyme for the biosynthesis of selenocysteine (sec), the 21(st) natural amino acid (aa). an essential rna-dependent kinase, o-phosphoseryl-trna(sec) kinase (pstk), converts seryl-trna(sec) to o-phosphoseryl-trna(sec), the immediate precursor of selenocysteinyl-trna(sec). the sequence of methanocaldococcus jannaschii pstk (mjpstk) suggests an n-terminal kinase domain (177 aa) followed by a presumed trna binding region (75 aa). the ...200919805283
features of subunit nuom (nd4) in escherichia coli ndh-1: topology and implication of conserved glu144 for coupling site 1.the bacterial h(+)-pumping nadh-quinone oxidoreductase (ndh-1) is an l-shaped membrane-bound enzymatic complex. escherichia coli ndh-1 is composed of 13 subunits (nuoa-n). nuom (nd4) subunit is one of the hydrophobic subunits that constitute the membrane arm of ndh-1 and was predicted to bear 14 helices. we attempted to clarify the membrane topology of nuom by the introduction of histidine tags into different positions by chromosomal site-directed mutagenesis. from the data, we propose a topolog ...200919815558
structural insights into rna processing by the human risc-loading complex.targeted gene silencing by rna interference (rnai) requires loading of a short guide rna (small interfering rna (sirna) or microrna (mirna)) onto an argonaute protein to form the functional center of an rna-induced silencing complex (risc). in humans, argonaute2 (ago2) assembles with the guide rna-generating enzyme dicer and the rna-binding protein trbp to form a risc-loading complex (rlc), which is necessary for efficient transfer of nascent sirnas and mirnas from dicer to ago2. here, using sin ...200919820710
multistart simulated annealing refinement of the crystal structure of the 70s ribosome.a macromolecular x-ray crystal structure is usually represented as a single static model with a single set of temperature factors representing a simple approximation of motion and disorder of the structure. multiconformer representations of small proteins have been shown to better describe anisotropic motion and disorder and improve the quality of their electron density maps. here, we apply multistart simulated annealing crystallographic refinement to a 70s ribosome-rf1 translation termination c ...200919822758
a novel dimerization motif in the c-terminal domain of the thermus thermophilus dead box helicase hera confers substantial flexibility.dead box helicases are involved in nearly all aspects of rna metabolism. they share a common helicase core, and may comprise additional domains that contribute to rna binding. the thermus thermophilus helicase hera is the first dimeric dead box helicase. crystal structures of hera fragments reveal a bipartite c-terminal domain with a novel dimerization motif and an rna-binding module. we provide a first glimpse on the additional rna-binding module outside the hera helicase core. the dimerization ...200919050012
identification of a new endogenous metabolite and the characterization of its protein interactions through an immobilization approach.the emerging field of global mass-based metabolomics provides a platform for discovering unknown metabolites and their specific biochemical pathways. we report the identification of a new endogenous metabolite, n(4)-(n-acetylaminopropyl)spermidine and the use of a novel proteomics based method for the investigation of its protein interaction using metabolite immobilization on agarose beads. the metabolite was isolated from the organism pyrococcus furiosus, and structurally characterized through ...200919055353
a signal relay between ribosomal protein s12 and elongation factor ef-tu during decoding of mrna.codon recognition by aminoacyl-trna on the ribosome triggers a process leading to gtp hydrolysis by elongation factor tu (ef-tu) and release of aminoacyl-trna into the a site of the ribosome. the nature of this signal is largely unknown. here, we present genetic evidence that a specific set of direct interactions between ribosomal protein s12 and aminoacyl-trna, together with contacts between s12 and 16s rrna, provide a pathway for the signaling of codon recognition to ef-tu. three novel amino a ...200919095621
mitochondrial dna damage in iron overload.chronic iron overload has slow and insidious effects on heart, liver, and other organs. because iron-driven oxidation of most biologic materials (such as lipids and proteins) is readily repaired, this slow progression of organ damage implies some kind of biological "memory." we hypothesized that cumulative iron-catalyzed oxidant damage to mtdna might occur in iron overload, perhaps explaining the often lethal cardiac dysfunction. real time pcr was used to examine the "intactness" of mttdna in cu ...200919095657
identification of amino acids in the n-terminal domain of atypical methanogenic-type seryl-trna synthetase critical for trna recognition.seryl-trna synthetase (serrs) from methanogenic archaeon methanosarcina barkeri, contains an idiosyncratic n-terminal domain, composed of an antiparallel beta-sheet capped by a helical bundle, connected to the catalytic core by a short linker peptide. it is very different from the coiled-coil trna binding domain in bacterial-type serrs. because the crystal structure of the methanogenic-type serrsxtrna complex has not been obtained, a docking model was produced, which indicated that highly conser ...200919734148
biochemical properties and base excision repair complex formation of apurinic/apyrimidinic endonuclease from pyrococcus furiosus.apurinic/apyrimidinic (ap) sites are the most frequently found mutagenic lesions in dna, and they arise mainly from spontaneous base loss or modified base removal by damage-specific dna glycosylases. ap sites are cleaved by ap endonucleases, and the resultant gaps in the dna are repaired by dna polymerase/dna ligase reactions. we identified the gene product that is responsible for the ap endonuclease activity in the hyperthermophilic euryarchaeon, pyrococcus furiosus. furthermore, we detected th ...200919734344
srmb, a dead-box helicase involved in escherichia coli ribosome assembly, is specifically targeted to 23s rrna in vivo.dead-box proteins play specific roles in remodeling rna or ribonucleoprotein complexes. yet, in vitro, they generally behave as nonspecific rna-dependent atpases, raising the question of what determines their specificity in vivo. srmb, one of the five escherichia coli dead-box proteins, participates in the assembly of the large ribosomal subunit. moreover, when overexpressed, it compensates for a mutation in l24, the ribosomal protein (r-protein) thought to initiate assembly. here, using the tan ...200919734346
the interaction of bacillus subtilis sigmaa with rna polymerase.rna polymerase (rnap) is an essential and highly conserved enzyme in all organisms. the process of transcription initiation is fundamentally different between prokaryotes and eukaryotes. in prokaryotes, initiation is regulated by sigma factors, making the essential interaction between sigma factors and rnap an attractive target for antimicrobial agents. our objective was to achieve the first step in the process of developing novel antimicrobial agents, namely to prove experimentally that the int ...200919735077
continuous-wave and pulsed epr characterization of the [2fe-2s](cys)3(his)1 cluster in rat mitoneet.cw epr spectra of reduced [2fe-2s](cys)(3)(his)(1) clusters of mammalian mitoneet soluble domain appear to produce features resulting from the interaction of the electron spins of the two adjacent clusters, which can be explained by employing the local spin model. this model favors the reduction of the outermost iron with his87 and cys83 ligands, which is supported by orientation-selected hyperfine sublevel correlation (hyscore) characterization of the uniformly (15)n-labeled mitoneet showing on ...200919736979
characterization of rimo, a new member of the methylthiotransferase subclass of the radical sam superfamily.rimo, encoded by the ylig gene in escherichia coli, has been recently identified in vivo as the enzyme responsible for the attachment of a methylthio group on the beta-carbon of asp88 of the small ribosomal protein s12 [anton, b. p., saleh, l., benner, j. s., raleigh, e. a., kasif, s., and roberts, r. j. (2008) proc. natl. acad. sci. u.s.a. 105, 1826-1831]. to date, it is the only enzyme known to catalyze methylthiolation of a protein substrate; the four other naturally occurring methylthio modi ...200919736993
promiscuous substrate recognition in folding and assembly activities of the trigger factor chaperone.trigger factor (tf) is a molecular chaperone that binds to bacterial ribosomes where it contacts emerging nascent chains, but tf is also abundant free in the cytosol where its activity is less well characterized. in vitro studies show that tf promotes protein refolding. we find here that ribosome-free tf stably associates with and rescues from misfolding a large repertoire of full-length proteins. we identify over 170 members of this cytosolic escherichia coli tf substrate proteome, including ri ...200919737520
p38sj, a novel dingg protein protects neuronal cells from alcohol induced injury and death.ethanol induces neuronal cell injury and death by dysregulating several signaling events that are controlled, in part, by activation of mapk/erk1/2 and/or inactivation of its corresponding phosphatase, pp1. recently, we have purified a novel protein of 38 kda in size, p38sj, from a callus culture of hypericum perforatum, which belongs to an emerging dingg family of proteins with phosphate binding activity. here, we show that treatment of neuronal cells with p38sj protects cells against injury in ...200919739100
structural insights into the mechanism of the allosteric transitions of mycobacterium tuberculosis camp receptor protein.the camp receptor protein (crp) from mycobacterium tuberculosis is a camp-responsive global transcriptional regulator, responsible for the regulation of a multitude of diverse proteins. we have determined the crystal structures of the crp.camp and crp.n(6)-camp derivative-bound forms of the enzyme to 2.2- and 2.3 a-resolution, respectively, to investigate camp-mediated conformational and structural changes. the allosteric switch from the open, inactive conformation to the closed, active conforma ...200919740754
dual role of dna in regulating atp hydrolysis by the sopa partition protein.in bacteria, mitotic stability of plasmids and many chromosomes depends on replicon-specific systems, which comprise a centromere, a centromere-binding protein and an atpase. dynamic self-assembly of the atpase appears to enable active partition of replicon copies into cell-halves, but for walker-box partition atpases the molecular mechanism is unknown. atpase activity appears to be essential for this process. dna and centromere-binding proteins are known to stimulate the atpase activity but mol ...200919740757
in vitro metal uptake by recombinant human manganese superoxide dismutase.metal uptake by the antioxidant defense metalloenzyme manganese superoxide dismutase (mnsod) is an essential step in the functional maturation of the protein that is just beginning to be investigated in detail. we have extended earlier in vitro studies on metal binding by the dimeric escherichia coli apo-mnsod to investigate the mechanism of metal uptake by tetrameric human and thermus thermophilus apo-mnsods. like the e. coli apo-mnsod, these proteins also bind metal ions in vitro in a thermall ...200919755112
recognition of trnagln by helicobacter pylori glurs2--a trnagln-specific glutamyl-trna synthetase.accurate aminoacylation of trnas by the aminoacyl-trna synthetases (aarss) plays a critical role in protein translation. however, some of the aarss are missing in many microorganisms. helicobacter pylori does not have a glutaminyl-trna synthetase (glnrs) but has two divergent glutamyl-trna synthetases: glurs1 and glurs2. like a canonical glurs, glurs1 aminoacylates trna(glu1) and trna(glu2). in contrast, glurs2 only misacylates trna(gln) to form glu-trna(gln). it is not clear how glurs2 achieves ...200919755501
demonstration and characterization of the heterodimerization of znt5 and znt6 in the early secretory pathway.the majority of cdf/znt zinc transporters form homo-oligomers. however, znt5, znt6, and their orthologues form hetero-oligomers in the early secretory pathway where they load zinc onto zinc-requiring enzymes and maintain secretory pathway functions. the details of this hetero-oligomerization remain to be elucidated, and much more is known about homo-oligomerization that occurs in other cdf/znt family proteins. here, we addressed this issue using co-immunoprecipitation experiments, mutagenesis, a ...200919759014
the schizosaccharomyces pombe hsp104 disaggregase is unable to propagate the [psi] prion.the molecular chaperone hsp104 is a crucial factor in the acquisition of thermotolerance in yeast. under stress conditions, the disaggregase activity of hsp104 facilitates the reactivation of misfolded proteins. hsp104 is also involved in the propagation of fungal prions. for instance, the well-characterized [psi(+)] prion of saccharomyces cerevisiae does not propagate in deltahsp104 cells or in cells overexpressing hsp104. in this study, we characterized the functional homolog of hsp104 from sc ...200919759825
zinc-independent folate biosynthesis: genetic, biochemical, and structural investigations reveal new metal dependence for gtp cyclohydrolase ib.gtp cyclohydrolase i (gcyh-i) is an essential zn(2+)-dependent enzyme that catalyzes the first step of the de novo folate biosynthetic pathway in bacteria and plants, the 7-deazapurine biosynthetic pathway in bacteria and archaea, and the biopterin pathway in mammals. we recently reported the discovery of a new prokaryotic-specific gcyh-i (gcyh-ib) that displays no sequence identity to the canonical enzyme and is present in approximately 25% of bacteria, the majority of which lack the canonical ...200919767425
structure of d-alanine-d-alanine ligase from thermus thermophilus hb8: cumulative conformational change and enzyme-ligand interactions.d-alanine-d-alanine ligase (ddl) is one of the key enzymes in peptidoglycan biosynthesis and is an important target for drug discovery. the enzyme catalyzes the condensation of two d-ala molecules using atp to produce d-ala-d-ala, which is the terminal peptide of a peptidoglycan monomer. the structures of five forms of the enzyme from thermus thermophilus hb8 (ttddl) were determined: unliganded ttddl (2.3 a resolution), ttddl-adenylyl imidodiphosphate (2.6 a), ttddl-adp (2.2 a), ttddl-adp-d-ala ...200919770507
adenosine triphosphate stimulates aquifex aeolicus mutl endonuclease activity.background: human pms2 (hpms2) homologues act to nick 5' and 3' to misincorporated nucleotides during mismatch repair in organisms that lack muth. mn(++) was previously found to stimulate the endonuclease activity of these homologues. atp was required for the nicking activity of hpms2 and ypms1, but was reported to inhibit bacterial mutl proteins from thermus thermophilus and aquifex aeolicus that displayed homology to hpms2. mutational analysis has identified the dqha(x)(2)e(x)(4)e motif presen ...200919777055
increased expression of the oxidative pentose phosphate pathway and gluconeogenesis in anaerobically growing xylose-utilizing saccharomyces cerevisiae.fermentation of xylose to ethanol has been achieved in s. cerevisiae by genetic engineering. xylose utilization is however slow compared to glucose, and during anaerobic conditions addition of glucose has been necessary for cellular growth. in the current study, the xylose-utilizing strain tmb 3415 was employed to investigate differences between anaerobic utilization of glucose and xylose. this strain carried a xylose reductase (xyl1 k270r) engineered for increased nadh utilization and was capab ...200919778438
inter-subunit interaction and quaternary rearrangement defined by the central stalk of prokaryotic v1-atpase.v-type atpases (v-atpases) are categorized as rotary atp synthase/atpase complexes. the v-atpases are distinct from f-atpases in terms of their rotation scheme, architecture and subunit composition. however, there is no detailed structural information on v-atpases despite the abundant biochemical and biophysical research. here, we report a crystallographic study of v1-atpase, from thermus thermophilus, which is a soluble component consisting of a, b, d and f subunits. the structure at 4.5 a reso ...200919779483
ion-induced folding of a kink turn that departs from the conventional sequence.kink turns (k-turns) are important structural motifs that create a sharp axial bend in rna. most conform to a consensus in which a three-nucleotide bulge is followed by consecutive g*a and a*g base pairs, and when these g*a pairs are modified in vitro this generally leads to a failure to adopt the k-turn conformation. kt-23 in the 30s ribosomal subunit of thermus thermophilus is a rare exception in which the bulge-distal a*g pair is replaced by a non-watson-crick a*u pair. in the context of the ...200919783814
functional specialization of transcription elongation factors.elongation factors nusg and rfah evolved from a common ancestor and utilize the same binding site on rna polymerase (rnap) to modulate transcription. however, although nusg associates with rnap transcribing most escherichia coli genes, rfah regulates just a few operons containing ops, a dna sequence that mediates rfah recruitment. here, we describe the mechanism by which this specificity is maintained. we observe that rfah action is indeed restricted to those several operons that are devoid of n ...200919096362
functional specialization of transcription elongation factors.elongation factors nusg and rfah evolved from a common ancestor and utilize the same binding site on rna polymerase (rnap) to modulate transcription. however, although nusg associates with rnap transcribing most escherichia coli genes, rfah regulates just a few operons containing ops, a dna sequence that mediates rfah recruitment. here, we describe the mechanism by which this specificity is maintained. we observe that rfah action is indeed restricted to those several operons that are devoid of n ...200919096362
a novel insertion mutation in streptomyces coelicolor ribosomal s12 protein results in paromomycin resistance and antibiotic overproduction.we identified a novel paromomycin resistance-associated mutation in rpsl, caused by the insertion of a glycine residue at position 92, in streptomyces coelicolor ribosomal protein s12. this insertion mutation (gi92) resulted in a 20-fold increase in the paromomycin resistance level. in combination with another s12 mutation, k88e, the gi92 mutation markedly enhanced the production of the blue-colored polyketide antibiotic actinorhodin and the red-colored antibiotic undecylprodigiosin. the gene re ...200919104019
a novel insertion mutation in streptomyces coelicolor ribosomal s12 protein results in paromomycin resistance and antibiotic overproduction.we identified a novel paromomycin resistance-associated mutation in rpsl, caused by the insertion of a glycine residue at position 92, in streptomyces coelicolor ribosomal protein s12. this insertion mutation (gi92) resulted in a 20-fold increase in the paromomycin resistance level. in combination with another s12 mutation, k88e, the gi92 mutation markedly enhanced the production of the blue-colored polyketide antibiotic actinorhodin and the red-colored antibiotic undecylprodigiosin. the gene re ...200919104019
epr evidence of cyanide binding to the mn(mg) center of cytochrome c oxidase: support for cu(a)-mg involvement in proton pumping.we examined the anion binding behavior of the mg(mn) site in cytochrome c oxidase to test a possible role of this center in proton pumping. rhodobacter sphaeroides grown in a mn(ii)-rich medium replaces the intrinsic mg(ii) ion with an epr-detectable mn(ii) ion without change in activity. due to its close proximity and a shared ligand, oxidized cu(a) is spin-coupled to the mn(ii) ion, affecting the epr spectrum. an examination of both bovine and r.s. oxidase crystal structures reveals a hydrogen ...200919108635
characterization of two novel alpha-glucosidases from bifidobacterium breve ucc2003.two alpha-glucosidase-encoding genes (agl1 and agl2) from bifidobacterium breve ucc2003 were identified and characterized. based on their similarity to characterized carbohydrate hydrolases, the agl1 and agl2 enzymes are both assigned to a subgroup of the glycosyl hydrolase family 13, the alpha-1,6-glucosidases (ec 3.2.1.10). recombinant agl1 and agl2 into which a his(12) sequence was incorporated (agl1(his) and agl2(his), respectively) exhibited hydrolytic activity towards panose, isomaltose, i ...200919114534
characterization of two novel alpha-glucosidases from bifidobacterium breve ucc2003.two alpha-glucosidase-encoding genes (agl1 and agl2) from bifidobacterium breve ucc2003 were identified and characterized. based on their similarity to characterized carbohydrate hydrolases, the agl1 and agl2 enzymes are both assigned to a subgroup of the glycosyl hydrolase family 13, the alpha-1,6-glucosidases (ec 3.2.1.10). recombinant agl1 and agl2 into which a his(12) sequence was incorporated (agl1(his) and agl2(his), respectively) exhibited hydrolytic activity towards panose, isomaltose, i ...200919114534
the crystal structure of galacto-n-biose/lacto-n-biose i phosphorylase: a large deformation of a tim barrel scaffold.galacto-n-biose/lacto-n-biose i phosphorylase (glnbp) from bifidobacterium longum, a key enzyme for intestinal growth, phosphorolyses galacto-n-biose and lacto-n-biose i with anomeric inversion. glnbp homologues are often found in human pathogenic and commensal bacteria, and their substrate specificities potentially define the nutritional acquisition ability of these microbes in their habitat. we report the crystal structures of glnbp in five different ligand-binding forms. this is the first thr ...200919124470
architectural underpinnings of the genetic code for glutamine.structure-based mutational analysis was used to probe the architecture of the glutamine binding pocket in escherichia coli glutaminyl-trna synthetase (glnrs). crystallographic studies of several different glnrs complexes in a lattice that supports catalytic activity have shown that the glutamine amide group makes only ambiguous hydrogen-bonding interactions with a tyrosine hydroxyl and bound water molecule, rather than the highly specific hydrogen-bonding and electrostatic interactions made by t ...200919128026
dihydroorotase from the hyperthermophile aquifex aeolicus is activated by stoichiometric association with aspartate transcarbamoylase and forms a one-pot reactor for pyrimidine biosynthesis.in prokaryotes, the first three enzymes in pyrimidine biosynthesis, carbamoyl phosphate synthetase (cps), aspartate transcarbamoylase (atc), and dihydroorotase (dho), are commonly expressed separately and either function independently (escherichia coli) or associate into multifunctional complexes (aquifex aeolicus). in mammals the enzymes are expressed as a single polypeptide chain (cad) in the order cps-dho-atc and associate into a hexamer. this study presents the three-dimensional structure of ...200919128030
ribosome hijacking: a role for small protein b during trans-translation.tight recognition of codon-anticodon pairings by the ribosome ensures the accuracy and fidelity of protein synthesis. in eubacteria, translational surveillance and ribosome rescue are performed by the 'tmrna-smpb' system (transfer messenger rna-small protein b). remarkably, entry and accommodation of aminoacylated-tmrna into stalled ribosomes occur without a codon-anticodon interaction but in the presence of smpb. here, we show that within a stalled ribosome, smpb interacts with the three univer ...200919132006
combined microspectrophotometric and crystallographic examination of chemically reduced and x-ray radiation-reduced forms of cytochrome ba3 oxidase from thermus thermophilus: structure of the reduced form of the enzyme.three paths for obtaining crystals of reduced (ii-e4q/i-k258r) cytochrome ba(3) are described, and the structures of these are reported at approximately 2.8-3.0 a resolution. microspectrophotometry of single crystals of thermus ba(3) oxidase at 100 k was used to show that crystals of the oxidized enzyme are reduced in an intense x-ray (beam line 7-1 at the stanford synchrotron radiation laboratory), being nearly complete in 1 min. the previously reported structures of ba(3) (protein data bank en ...200919140675
a conserved active site tyrosine residue of proline dehydrogenase helps enforce the preference for proline over hydroxyproline as the substrate.proline dehydrogenase (prodh) catalyzes the oxidation of l-proline to delta-1-pyrroline-5-carboxylate. prodhs exhibit a pronounced preference for proline over hydroxyproline (trans-4-hydroxy-l-proline) as the substrate, but the basis for specificity is unknown. the goal of this study, therefore, is to gain insight into the structural determinants of substrate specificity of this class of enzyme, with a focus on understanding how prodhs discriminate between the two closely related molecules, prol ...200919140736
coarse-grained modeling of large rna molecules with knowledge-based potentials and structural filters.understanding the function of complex rna molecules depends critically on understanding their structure. however, creating three-dimensional (3d) structural models of rna remains a significant challenge. we present a protocol (the nucleic acid simulation tool [nast]) for rna modeling that uses an rna-specific knowledge-based potential in a coarse-grained molecular dynamics engine to generate plausible 3d structures. we demonstrate nast's capabilities by using only secondary structure and tertiar ...200919144906
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