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Molecular basis of dihydrouridine formation on tRNA.Dihydrouridine (D) is a highly conserved modified base found in tRNAs from all domains of life. Dihydrouridine synthase (Dus) catalyzes the D formation of tRNA through reduction of uracil base with flavin mononucleotide (FMN) as a cofactor. Here, we report the crystal structures of Thermus thermophilus Dus (TthDus), which is responsible for D formation at positions 20 and 20a, in complex with tRNA and with a short fragment of tRNA (D-loop). Dus interacts extensively with the D-arm and recognizes ...201122123979
Crystallization and preliminary X-ray crystallographic analysis of putative tRNA-modification enzymes from Pyrococcus furiosus and Thermus thermophilus.Methyltransferases form a major class of tRNA-modifying enzymes that are needed for the proper functioning of tRNA. Here, the expression, purification and crystallization of two related putative tRNA methyltransferases from two kingdoms of life are reported. The protein encoded by the gene pf1002 from the archaeon Pyrococcus furiosus was crystallized in the monoclinic space group P2(1). A complete data set was collected to 2.2 Å resolution. The protein encoded by the gene ttc1157 from the eubact ...201122102250
Ratiometric pulse-chase amidination mass spectrometry as a probe of biomolecular complex formation.Selective chemical modification of protein side chains coupled with mass spectrometry is often most informative when used to compare residue-specific reactivities in a number of functional states or macromolecular complexes. Herein, we develop ratiometric pulse-chase amidination mass spectrometry (rPAm-MS) as a site-specific probe of lysine reactivities at equilibrium using the Cu(I)-sensing repressor CsoR from Bacillus subtilis as a model system. CsoR in various allosteric states was reacted ...201122007758
type-2 isopentenyl diphosphate isomerase: evidence for a stepwise mechanism.isopentenyl diphosphate isomerase (idi) catalyzes the interconversion of isopentenyl diphosphate (ipp) and dimethylallyl diphosphate (dmapp). these two molecules are the building blocks for construction of isoprenoid carbon skeletons in nature. two structurally unrelated forms of idi are known. a variety of studies support a proton addition/proton elimination mechanism for both enzymes. during studies with thermus thermophilus idi-2, we discovered that the olefinic hydrogens of a vinyl thiomethy ...201122047048
adaptation to trna acceptor stem structure by flexible adjustment in the catalytic domain of class i trna synthetases.class i aminoacyl-trna synthetases (aarss) use a rossmann-fold domain to catalyze the synthesis of aminoacyl-trnas required for decoding genetic information. while the rossmann-fold domain is conserved in evolution, the acceptor stem near the aminoacylation site varies among trna substrates, raising the question of how the conserved protein fold adapts to rna sequence variations. of interest is the existence of an unpaired c-a mismatch at the 1-72 position unique to bacterial initiator trna(fmet ...201122184460
Structural analysis of the Ras-like G protein MglA and its cognate GAP MglB and implications for bacterial polarity.The bacterium Myxococcus xanthus uses a G protein cycle to dynamically regulate the leading/lagging pole polarity axis. The G protein MglA is regulated by its GTPase-activating protein (GAP) MglB, thus resembling Ras family proteins. Here, we show structurally and biochemically that MglA undergoes a dramatic, GDP-GTP-dependent conformational change involving a screw-type forward movement of the central ß2-strand, never observed in any other G protein. This movement and complex formation with Mgl ...201121847100
termination of protein synthesis in mammalian mitochondria.all mechanisms of protein synthesis can be considered in four stages: initiation, elongation, termination, and ribosome recycling. remarkable progress has been made in understanding how these processes are mediated in the cytosol of many species; however, details of organellar protein synthesis remain sketchy. this is an important omission, as defects in human mitochondrial translation are known to cause disease and may contribute to the aging process itself. in this minireview, we focus on the ...201121873426
structural basis for leucine-induced allosteric activation of glutamate dehydrogenase.glutamate dehydrogenase (gdh) catalyzes reversible conversion between glutamate and 2-oxoglutarate using nad(p)(h) as a coenzyme. although mammalian gdh is regulated by gtp through the antenna domain, little is known about the mechanism of allosteric activation by leucine. an extremely thermophilic bacterium, thermus thermophilus, possesses gdh with a unique subunit configuration composed of two different subunits, gdha (regulatory subunit) and gdhb (catalytic subunit). t. thermophilus gdh is un ...201121900230
crystal structure of the central axis df complex of the prokaryotic v-atpase.v-atpases function as atp-dependent ion pumps in various membrane systems of living organisms. atp hydrolysis causes rotation of the central rotor complex, which is composed of the central axis d subunit and a membrane c ring that are connected by f and d subunits. here we determined the crystal structure of the df complex of the prokaryotic v-atpase of enterococcus hirae at 2.0-å resolution. the structure of the d subunit comprised a long left-handed coiled coil with a unique short β-hairpin re ...201122114184
Crystal structure of the NurA-dAMP-Mn2+ complex.Generation of the 3' overhang is a critical event during homologous recombination (HR) repair of DNA double strand breaks. A 5'-3' nuclease, NurA, plays an important role in generating 3' single-stranded DNA during archaeal HR, together with Mre11-Rad50 and HerA. We have determined the crystal structures of apo- and dAMP-Mn(2)(+)-bound NurA from Pyrococcus furiousus (Pf?NurA) to provide the basis for its cleavage mechanism. Pf?NurA forms a pyramid-shaped dimer containing a large central channel ...201122064858
Crystal structure of the NurA-dAMP-Mn2+ complex.Generation of the 3' overhang is a critical event during homologous recombination (HR) repair of DNA double strand breaks. A 5'-3' nuclease, NurA, plays an important role in generating 3' single-stranded DNA during archaeal HR, together with Mre11-Rad50 and HerA. We have determined the crystal structures of apo- and dAMP-Mn(2)(+)-bound NurA from Pyrococcus furiousus (Pf?NurA) to provide the basis for its cleavage mechanism. Pf?NurA forms a pyramid-shaped dimer containing a large central channel ...201122064858
structure and activity of the cas3 hd nuclease mj0384, an effector enzyme of the crispr interference.clustered regularly interspaced short palindromic repeats (crisprs) and cas proteins represent an adaptive microbial immunity system against viruses and plasmids. cas3 proteins have been proposed to play a key role in the crispr mechanism through the direct cleavage of invasive dna. here, we show that the cas3 hd domain protein mj0384 from methanocaldococcus jannaschii cleaves endonucleolytically and exonucleolytically (3'-5') single-stranded dnas and rnas, as well as 3'-flaps, splayed arms, and ...201122009198
Biogenesis of cbb(3)-type cytochrome c oxidase in Rhodobacter capsulatus.The cbb(3)-type cytochrome c oxidases (cbb(3)-Cox) constitute the second most abundant cytochrome c oxidase (Cox) group after the mitochondrial-like aa(3)-type Cox. They are present in bacteria only, and are considered to represent a primordial innovation in the domain of Eubacteria due to their phylogenetic distribution and their similarity to nitric oxide (NO) reductases. They are crucial for the onset of many anaerobic biological processes, such as anoxygenic photosynthesis or nitrogen fixati ...201122079199
Biogenesis of cbb(3)-type cytochrome c oxidase in Rhodobacter capsulatus.The cbb(3)-type cytochrome c oxidases (cbb(3)-Cox) constitute the second most abundant cytochrome c oxidase (Cox) group after the mitochondrial-like aa(3)-type Cox. They are present in bacteria only, and are considered to represent a primordial innovation in the domain of Eubacteria due to their phylogenetic distribution and their similarity to nitric oxide (NO) reductases. They are crucial for the onset of many anaerobic biological processes, such as anoxygenic photosynthesis or nitrogen fixati ...201122079199
Profile of Venkatraman Ramakrishnan. Interview by Prashant Nair. 201121914843
kinetic studies of the reactions of o(2) and no with reduced thermus thermophilus ba(3) and bovine aa(3) using photolabile carriers.the reactions of molecular oxygen (o(2)) and nitric oxide (no) with reduced thermus thermophilus (tt) ba(3) and bovine heart aa(3) were investigated by time-resolved optical absorption spectroscopy to establish possible relationships between the structural diversity of these enzymes and their reaction dynamics. to determine whether the photodissociated carbon monoxide (co) in the co flow-flash experiment affects the ligand binding dynamics, we monitored the reactions in the absence and presence ...201122201543
the structure of aquifex aeolicus ribosomal protein s8 reveals a unique subdomain that contributes to an extremely tight association with 16s rrna.the assembly of ribonucleoprotein complexes occurs under a broad range of conditions, but the principles that promote assembly and allow function at high temperature are poorly understood. the ribosomal protein s8 from aquifex aeolicus (as8) is unique in that there is a 41-residue insertion in the consensus s8 sequence. in addition, as8 exhibits an unusually high affinity for the 16s ribosomal rna, characterized by a picomolar dissociation constant that is approximately 26,000-fold tighter than ...201122079365
the structure of aquifex aeolicus ribosomal protein s8 reveals a unique subdomain that contributes to an extremely tight association with 16s rrna.the assembly of ribonucleoprotein complexes occurs under a broad range of conditions, but the principles that promote assembly and allow function at high temperature are poorly understood. the ribosomal protein s8 from aquifex aeolicus (as8) is unique in that there is a 41-residue insertion in the consensus s8 sequence. in addition, as8 exhibits an unusually high affinity for the 16s ribosomal rna, characterized by a picomolar dissociation constant that is approximately 26,000-fold tighter than ...201122079365
simultaneous polyhydroxyalkanoates and rhamnolipids production by thermus thermophilus hb8.abstract: the ability of thermus thermophilus hb8 to produce simultaneously two environmentally-friendly biodegradable products, polyhydroxyalkanoates (phas) and rhamnolipids (rls), using either sodium gluconate or glucose as sole carbon source, was demonstrated. the utilization of sodium gluconate resulted in higher levels of phas and rls production than when glucose was used as sole carbon source. the initial phosphate concentration (as po43-) influences both phas and rls productions that were ...201121906373
Properties and crystal structure of methylenetetrahydrofolate reductase from Thermus thermophilus HB8.Methylenetetrahydrofolate reductase (MTHFR) is one of the enzymes involved in homocysteine metabolism. Despite considerable genetic and clinical attention, the reaction mechanism and regulation of this enzyme are not fully understood because of difficult production and poor stability. While recombinant enzymes from thermophilic organisms are often stable and easy to prepare, properties of thermostable MTHFRs have not yet been reported.201121858212
bioinformatic analysis of leishmania donovani long-chain fatty acid-coa ligase as a novel drug target.fatty acyl-coa synthetase (fatty acid: coa ligase, amp-forming; (ec 6.2.1.3)) catalyzes the formation of fatty acyl-coa by a two-step process that proceeds through the hydrolysis of pyrophosphate. fatty acyl-coa represents bioactive compounds that are involved in protein transport, enzyme activation, protein acylation, cell signaling, and transcriptional control in addition to serving as substrates for beta oxidation and phospholipid biosynthesis. fatty acyl-coa synthetase occupies a pivotal rol ...201122091399
Mössbauer Spectroscopy on Respiratory Complex I: The Iron-Sulfur Cluster Ensemble in the NADH-Reduced Enzyme Is Partially Oxidized.In mitochondria, complex I (NADH:quinone oxidoreductase) couples electron transfer to proton translocation across an energy-transducing membrane. It contains a flavin mononucleotide to oxidize NADH, and an unusually long series of iron-sulfur (FeS) clusters that transfer the electrons to quinone. Understanding electron transfer in complex I requires spectroscopic and structural data to be combined to reveal the properties of individual clusters and of the ensemble. EPR studies on complex I from ...201122122402
Mössbauer Spectroscopy on Respiratory Complex I: The Iron-Sulfur Cluster Ensemble in the NADH-Reduced Enzyme Is Partially Oxidized.In mitochondria, complex I (NADH:quinone oxidoreductase) couples electron transfer to proton translocation across an energy-transducing membrane. It contains a flavin mononucleotide to oxidize NADH, and an unusually long series of iron-sulfur (FeS) clusters that transfer the electrons to quinone. Understanding electron transfer in complex I requires spectroscopic and structural data to be combined to reveal the properties of individual clusters and of the ensemble. EPR studies on complex I from ...201122122402
Histidine 66 in E. coli elongation factor Tu selectively stabilizes aminoacyl-tRNAs.The universally conserved H66 of Elongation Factor Tu stacks on the side chain of the esterified Phe of Phe-tRNA(Phe). The affinities of eight aminoacyl-tRNAs were differentially destabilized by the introduction of the H66A mutation into E. coli EF-Tu while Ala-tRNA(Ala) and Gly-tRNA(Gly) were unaffected. The H66F and H66W proteins each show a different pattern of binding of ten different aa-tRNAs, clearly showing that this position is critical in establishing the specificity of EF-Tu for differ ...201122105070
Histidine 66 in E. coli elongation factor Tu selectively stabilizes aminoacyl-tRNAs.The universally conserved H66 of Elongation Factor Tu stacks on the side chain of the esterified Phe of Phe-tRNA(Phe). The affinities of eight aminoacyl-tRNAs were differentially destabilized by the introduction of the H66A mutation into E. coli EF-Tu while Ala-tRNA(Ala) and Gly-tRNA(Gly) were unaffected. The H66F and H66W proteins each show a different pattern of binding of ten different aa-tRNAs, clearly showing that this position is critical in establishing the specificity of EF-Tu for differ ...201122105070
substrate specificity of bacterial prolyl-trna synthetase editing domain is controlled by a tunable hydrophobic pocket.aminoacyl-trna synthetases catalyze the covalent attachment of amino acids onto their cognate trnas. high fidelity in this reaction is crucial to the accurate decoding of genetic information and is ensured, in part, by proofreading of the newly synthesized aminoacyl-trnas. prolyl-trna synthetases (prors) mischarge trna(pro) with alanine or cysteine due to their smaller or similar sizes relative to cognate proline. mischarged ala-trna(pro) is hydrolyzed by an editing domain (ins) present in most ...201122128149
substrate specificity of bacterial prolyl-trna synthetase editing domain is controlled by a tunable hydrophobic pocket.aminoacyl-trna synthetases catalyze the covalent attachment of amino acids onto their cognate trnas. high fidelity in this reaction is crucial to the accurate decoding of genetic information and is ensured, in part, by proofreading of the newly synthesized aminoacyl-trnas. prolyl-trna synthetases (prors) mischarge trna(pro) with alanine or cysteine due to their smaller or similar sizes relative to cognate proline. mischarged ala-trna(pro) is hydrolyzed by an editing domain (ins) present in most ...201122128149
thermostable multicopper oxidase from thermus thermophilus hb27: crystallization and preliminary x-ray diffraction analysis of apo and holo forms.a thermostable multicopper oxidase from thermus thermophilus hb27 (tth-mco) was successfully crystallized using the sitting-drop and hanging-drop vapour-diffusion methods. crystallization conditions and preliminary x-ray diffraction data to 1.5 å resolution obtained using synchrotron radiation at 100 k are reported. the crystals belonged to space group c222(1), with unit-cell parameters a = 93.6, b = 110.3, c = 96.3 å. a monomer in the asymmetric unit yielded a matthews coefficient (v(m)) of 2.6 ...201122139175
modular pathways for editing non-cognate amino acids by human cytoplasmic leucyl-trna synthetase.to prevent potential errors in protein synthesis, some aminoacyl-transfer rna (trna) synthetases have evolved editing mechanisms to hydrolyze misactivated amino acids (pre-transfer editing) or misacylated trnas (post-transfer editing). class ia leucyl-trna synthetase (leurs) may misactivate various natural and non-protein amino acids and then mischarge trna(leu). it is known that the fidelity of prokaryotic leurs depends on multiple editing pathways to clear the incorrect intermediates and produ ...201120805241
modular pathways for editing non-cognate amino acids by human cytoplasmic leucyl-trna synthetase.to prevent potential errors in protein synthesis, some aminoacyl-transfer rna (trna) synthetases have evolved editing mechanisms to hydrolyze misactivated amino acids (pre-transfer editing) or misacylated trnas (post-transfer editing). class ia leucyl-trna synthetase (leurs) may misactivate various natural and non-protein amino acids and then mischarge trna(leu). it is known that the fidelity of prokaryotic leurs depends on multiple editing pathways to clear the incorrect intermediates and produ ...201120805241
quantitative mapping of reversible mitochondrial complex i cysteine oxidation in a parkinson disease mouse model.differential cysteine oxidation within mitochondrial complex i has been quantified in an in vivo oxidative stress model of parkinson disease. we developed a strategy that incorporates rapid and efficient immunoaffinity purification of complex i followed by differential alkylation and quantitative detection using sensitive mass spectrometry techniques. this method allowed us to quantify the reversible cysteine oxidation status of 34 distinct cysteine residues out of a total 130 present in murine ...201121196577
stress-induced evolution of escherichia coli points to original concepts in respiratory cofactor selectivity.bacterial metabolism is characterized by a remarkable capacity to rapidly adapt to environmental changes. we restructured the central metabolic network in escherichia coli to force a higher production of nadph, and then grew this strain in conditions favoring adaptive evolution. a six-fold increase in growth capacity was attained that could be attributed in multiple clones, after whole genome mutation mapping, to a specific single mutation. each clone had an evolved nuof*(e183a) enzyme in the re ...201121205901
archaeal 3'-phosphate rna splicing ligase characterization identifies the missing component in trna maturation.intron removal from trna precursors involves cleavage by a trna splicing endonuclease to yield trna 3'-halves beginning with a 5'-hydroxyl, and 5'-halves ending in a 2',3'-cyclic phosphate. a trna ligase then incorporates this phosphate into the internucleotide bond that joins the two halves. although this 3'-p rna splicing ligase activity was detected almost three decades ago in extracts from animal and later archaeal cells, the protein responsible was not yet identified. here we report the pur ...201121209330
fidelity escape by the unnatural amino acid β-hydroxynorvaline: an efficient substrate for escherichia coli threonyl-trna synthetase with toxic effects on growth.in all living systems, the fidelity of translation is maintained in part by the editing mechanisms of aminoacyl-trna synthetases (arss). some nonproteogenic amino acids, including β-hydroxynorvaline (hnv) are nevertheless efficiently aminoacylated and become incorporated into proteins. to investigate the basis of hnv's ability to function in protein synthesis, the utilization of hnv by escherichia coli threonyl-trna synthetase (thrrs) was investigated through both in vitro functional experiments ...201121222438
rtcb is the rna ligase component of an escherichia coli rna repair operon.rna 2',3'-cyclic phosphate ends play important roles in rna metabolism as substrates for rna ligases during trna restriction-repair and trna splicing. diverse bacteria from multiple phyla encode a two-component rna repair cassette, comprising pnkp (polynucleotide kinase-phosphatase-ligase) and hen1 (rna 3'-terminal ribose 2'-o-methyltransferase), that heals and then seals broken trnas with 2',3'-cyclic phosphate and 5'-oh ends. the pnkp-hen1 repair operon is absent in the majority of bacterial s ...201121224389
exploration of the cytochrome c oxidase pathway puzzle and examination of the origin of elusive mutational effects.gaining detailed understanding of the energetics of the proton-pumping process in cytochrome c oxidase (cco) is a problem of great current interest. despite promising mechanistic proposals, so far, a physically consistent model that would reproduce all the relevant barriers needed to create a working pump has not been presented. in addition, there are major problems in elucidating the origin of key mutational effects and in understanding the nature of the apparent pk(a) values associated with th ...201121232525
the biomolecular interaction network database in psi-mi 2.5.the biomolecular interaction network database (bind) is a major source of curated biomolecular interactions, which has been unmaintained for the last few years, a trend which will eventually result in the loss of a significant amount of unique biomolecular interaction information, mostly as database identifiers become out of date. to help reverse this trend, we converted bind to a standard format, proteomics standard initiative-molecular interaction 2.5, starting from the last curated data relea ...201121233089
biochemical and structural characterization of wlba from bordetella pertussis and chromobacterium violaceum: enzymes required for the biosynthesis of 2,3-diacetamido-2,3-dideoxy-d-mannuronic acid.the unusual sugar 2,3-diacetamido-2,3-dideoxy-d-mannuronic acid, or mannac3naca, has been observed in the lipopolysaccharides of both pathogenic and nonpathogenic gram-negative bacteria. it is added to the lipopolysaccharides of these organisms by glycosyltransferases that use as substrates udp-mannac3naca. five enzymes are ultimately required for the biosynthesis of udp-mannac3naca starting from udp-n-acetylglucosamine. the second enzyme in the pathway, encoded by the wlba gene and referred to ...201121241053
crystal structure of a bacterial phosphoglucomutase, an enzyme involved in the virulence of multiple human pathogens.the crystal structure of the enzyme phosphoglucomutase from salmonella typhimurium (stpgm) is reported at 1.7 a resolution. this is the first high-resolution structural characterization of a bacterial protein from this large enzyme family, which has a central role in metabolism and is also important to bacterial virulence and infectivity. a comparison of the active site of stpgm with that of other phosphoglucomutases reveals conserved residues that are likely involved in catalysis and ligand bin ...201121246636
escherichia coli class ib ribonucleotide reductase contains a dimanganese(iii)-tyrosyl radical cofactor in vivo.escherichia coli class ib ribonucleotide reductase (rnr) converts nucleoside 5'-diphosphates to deoxynucleoside 5'-diphosphates in iron-limited and oxidative stress conditions. we have recently demonstrated in vitro that this rnr is active with both diferric-tyrosyl radical (fe(iii)(2)-y(•)) and dimanganese(iii)-y(•) (mn(iii)(2)-y(•)) cofactors in the β2 subunit, nrdf [cotruvo, j. a., jr., and stubbe, j. (2010) biochemistry 49, 1297-1309]. here we demonstrate, by purification of this protein fro ...201121250660
superfolder gfp is fluorescent in oxidizing environments when targeted via the sec translocon.the ability to study proteins in live cells using genetically encoded fluorescent proteins (fps) has revolutionized cell biology (1-3). researchers have created numerous fp biosensors and optimized fps for specific organisms and subcellular environments in a rainbow of colors (4,5). however, expressing fps in oxidizing environments such as the eukaryotic endoplasmic reticulum (er) or the bacterial periplasm can impair folding, thereby preventing fluorescence (6,7). a substantial fraction of enha ...201121255213
identification of macrodomain proteins as novel o-acetyl-adp-ribose deacetylases.sirtuins are a family of protein lysine deacetylases, which regulate gene silencing, metabolism, life span, and chromatin structure. sirtuins utilize nad(+) to deacetylate proteins, yielding o-acetyl-adp-ribose (oaadpr) as a reaction product. the macrodomain is a ubiquitous protein module known to bind adp-ribose derivatives, which diverged through evolution to support many different protein functions and pathways. the observation that some sirtuins and macrodomains are physically linked as fusi ...201121257746
manipulations in the peripheral stalk of the saccharomyces cerevisiae f1f0-atp synthase.the saccharomyces cerevisiae f(1)f(0)-atp synthase peripheral stalk is composed of the oscp, h, d, and b subunits. the b subunit has two membrane-spanning domains and a large hydrophilic domain that extends along one side of the enzyme to the top of f(1). in contrast, the escherichia coli peripheral stalk has two identical b subunits, and subunits with substantially altered lengths can be incorporated into a functional f(1)f(0)-atp synthase. the differences in subunit structure between the eukar ...201121257750
identification of critical residues of the mycobacterial dephosphocoenzyme a kinase by site-directed mutagenesis.dephosphocoenzyme a kinase performs the transfer of the ?-phosphate of atp to dephosphocoenzyme a, catalyzing the last step of coenzyme a biosynthesis. this enzyme belongs to the p-loop-containing ntp hydrolase superfamily, all members of which posses a three domain topology consisting of a coa domain that binds the acceptor substrate, the nucleotide binding domain and the lid domain. differences in the enzymatic organization and regulation between the human and mycobacterial counterparts, have ...201121264299
bacillus pumilus laccase: a heat stable enzyme with a wide substrate spectrum.laccases are multi-copper oxidases that catalyze the one electron oxidation of a broad range of compounds. laccase substrates include substituted phenols, arylamines and aromatic thiols. such compounds are activated by the enzyme to the corresponding radicals. owing to their broad substrate range laccases are considered to be versatile biocatalysts which are capable of oxidizing natural and non-natural industrial compounds, with water as sole by-product.201121266052
poly-alpha-glutamic acid synthesis using a novel catalytic activity of rimk from escherichia coli k-12.poly-l-a-amino acids have various applications because of their biodegradable properties and biocompatibility. microorganisms contain several enzymes that catalyze the polymerization of l-amino acids in an atp-dependent manner, but the products from these reactions contain amide linkages at the side residues of amino acids: e.g., poly-?-glutamic acid, poly-e-lysine, and cyanophycin. in this study, we found a novel catalytic activity of rimk, a ribosomal protein s6-modifying enzyme derived from e ...201121278279
selective inhibitors of methionyl-trna synthetase have potent activity against trypanosoma brucei infection in mice.human african trypanosomiasis continues to be an important public health threat in extensive regions of sub-saharan africa. treatment options for infected patients are unsatisfactory due to toxicity, difficult administration regimes, and poor efficacy of available drugs. the aminoacyl-trna synthetases were selected as attractive drug targets due to their essential roles in protein synthesis and cell survival. comparative sequence analysis disclosed differences between the trypanosome and mammali ...201121282428
crystal structure of the synergistic antibiotic pair, lankamycin and lankacidin, in complex with the large ribosomal subunit.the structures of the large ribosomal subunit of deinococcus radiodurans (d50s) in complex with the antibiotic lankamycin (3.2 å) and a double antibiotic complex of lankamycin and lankacidin c (3.45 å) have been determined, in continuation of previous crystallographic studies on lankacidin-d50s complex. these two drugs have been previously reported to inhibit ribosomal function with mild synergistic effect. lankamycin, a member of the macrolide family, binds in a similar manner to erythromycin. ...201121282615
structure and function of pilq, a secretin of the dna transporter from the thermophilic bacterium thermus thermophilus hb27.secretins are a family of large bacterial outer membrane protein complexes mediating the transport of complex structures, such as type iv pili, dna and filamentous phage, or various proteins, such as extracellular enzymes and pathogenicity determinants. pilq of the thermophilic bacterium thermus thermophilus hb27 is a member of the secretin family required for natural transformation. here we report the isolation, structural, and functional analyses of a unique pilq from t. thermophilus. native p ...201121285351
two novel classes of enzymes are required for the biosynthesis of aurofusarin in fusarium graminearum.previous studies have reported the functional characterization of 9 out of 11 genes found in the gene cluster responsible for biosynthesis of the polyketide pigment aurofusarin in fusarium graminearum. here we reanalyze the function of a putative aurofusarin pump (aurt) and the two remaining orphan genes, aurz and aurs. targeted gene replacement of aurz resulted in the discovery that the compound ywa1, rather than nor-rubrofusarin, is the primary product of f. graminearum polyketide synthase 12 ...201121296881
molecular breeding of polymerases for resistance to environmental inhibitors.potent inhibitors limit the use of pcr assays in a wide spectrum of specimens. here, we describe the engineering of polymerases with a broad resistance to complex environmental inhibitors using molecular breeding of eight different polymerase orthologues from the genus thermus and directed evolution by csr in the presence of inhibitors. selecting for resistance to the inhibitory effects of neomylodon bone powder, we isolated 2d9, a chimeric polymerase comprising sequence elements derived from dn ...201121297114
structure of the dimeric form of ctp synthase from sulfolobus solfataricus.ctp synthase catalyzes the last committed step in de novo pyrimidine-nucleotide biosynthesis. active ctp synthase is a tetrameric enzyme composed of a dimer of dimers. the tetramer is favoured in the presence of the substrate nucleotides atp and utp; when saturated with nucleotide, the tetramer completely dominates the oligomeric state of the enzyme. furthermore, phosphorylation has been shown to regulate the oligomeric states of the enzymes from yeast and human. the crystal structure of a dimer ...201121301086
crystallization and preliminary x-ray crystallographic studies of ß-transaminase from mesorhizobium sp. strain luk.ß-transaminase (ß-ta) catalyzes the transamination reaction between ß-aminocarboxylic acids and keto acids. this enzyme is a particularly suitable candidate for use as a biocatalyst for the asymmetric synthesis of enantiochemically pure ß-amino acids for pharmaceutical purposes. the ß-ta from mesorhizobium sp. strain luk (ß-tams) belongs to a novel class in that it shows ß-transaminase activity with a broad and unique substrate specificity. in this study, ß-tams was overexpressed in escherichia ...201121301093
understanding ribosome assembly: the structure of in vivo assembled immature 30s subunits revealed by cryo-electron microscopy.four decades after early in vitro assembly studies demonstrated that ribosome assembly is a controlled process, our understanding of ribosome assembly is still incomplete. just as structure determination has been so important to understanding ribosome function, so too will it be critical to sorting out the assembly process. here, we used a viable deletion in the yjeq gene, a recognized ribosome assembly factor, to isolate and structurally characterize immature 30s subunits assembled in vivo. the ...201121303937
Genomic and proteomic characterization of the large Myoviridae bacteriophage ?TMA of the extreme thermophile Thermus thermophilus.A lytic phage, designated as ?TMA, was isolated from a Japanese hot spring using Thermus thermophilus HB27 as an indicator strain. Electron microscopic examination showed that ?TMA had an icosahedral head and a contractile tail. The circular double-stranded DNA sequence of ?TMA was 151,483 bp in length, and its organization was essentially same as that of ?YS40 except that the ?TMA genome contained genes for a pair of transposase and resolvase, and a gene for a serine to asparagine substituted o ...201122164349
evidence for atp-dependent structural rearrangement of nuclease catalytic site in dna mismatch repair endonuclease mutl.dna mismatch repair (mmr) greatly contributes to genome integrity via the correction of mismatched bases that are mainly generated by replication errors. postreplicative mmr excises a relatively long tract of error-containing single-stranded dna. mutl is a widely conserved nicking endonuclease that directs the excision reaction to the error-containing strand of the duplex by specifically nicking the daughter strand. because mutl apparently exhibits nonspecific nicking endonuclease activity in vi ...201121953455
comparison of two yeast mnsods: mitochondrial saccharomyces cerevisiae versus cytosolic candida albicans.human mnsod is significantly more product-inhibited than bacterial mnsods at high concentrations of superoxide (o(2)(-)). this behavior limits the amount of h(2)o(2) produced at high [o(2)(-)]; its desirability can be explained by the multiple roles of h(2)o(2) in mammalian cells, particularly its role in signaling. to investigate the mechanism of product inhibition in mnsod, two yeast mnsods, one from saccharomyces cerevisiae mitochondria (scmnsod) and the other from candida albicans cytoso ...201122077216
Crystal structure of phosphopantetheine adenylyltransferase from Enterococcus faecalis in the ligand-unbound state and in complex with ATP and pantetheine.Phosphopantetheine adenylyltransferase (PPAT) catalyzes the reversible transfer of an adenylyl group from ATP to 4'-phosphopantetheine (Ppant) to form dephospho-CoA (dPCoA) and pyrophosphate in the Coenzyme A (CoA) biosynthetic pathway. Importantly, PPATs are the potential target for developing antibiotics because bacterial and mammalian PPATs share little sequence homology. Previous structural studies revealed the mechanism of the recognizing substrates and products. The binding modes of ATP, A ...201121912874
The rate-limiting step in O(2) reduction by cytochrome ba(3) from Thermus thermophilus.Cytochrome ba(3) (ba(3)) of Thermus thermophilus (T. thermophilus) is a member of the heme-copper oxidase family, which has a binuclear catalytic center comprised of a heme (heme a(3)) and a copper (Cu(B)). The heme-copper oxidases generally catalyze the four electron reduction of molecular oxygen in a sequence involving several intermediates. We have investigated the reaction of the fully reduced ba(3) with O(2) using stopped-flow techniques. Transient visible absorption spectra indicated that ...201122138627
binding and inhibition of human spermidine synthase by decarboxylated s-adenosylhomocysteine.aminopropyltransferases are essential enzymes that form polyamines in eukaryotic and most prokaryotic cells. spermidine synthase (spds) is one of the most well-studied enzymes in this biosynthetic pathway. the enzyme uses decarboxylated s-adenosylmethionine and a short-chain polyamine (putrescine) to make a medium-chain polyamine (spermidine) and 5'-deoxy-5'-methylthioadenosine as a byproduct. here, we report a new spermidine synthase inhibitor, decarboxylated s-adenosylhomocysteine (dcsah). the ...201121898642
Multifaceted SlyD from Helicobacter pylori: implication in [NiFe] hydrogenase maturation.SlyD belongs to the FK506-binding protein (FKBP) family with both peptidylprolyl isomerase (PPIase) and chaperone activities, and is considered to be a ubiquitous cytosolic protein-folding facilitator in bacteria. It possesses a histidine- and cysteine-rich C-terminus binding to selected divalent metal ions (e.g., Ni(2+), Zn(2+)), which is important for its involvement in the maturation processes of metalloenzymes. We have determined the solution structure of C-terminus-truncated SlyD from Helic ...201122045417
Multifaceted SlyD from Helicobacter pylori: implication in [NiFe] hydrogenase maturation.SlyD belongs to the FK506-binding protein (FKBP) family with both peptidylprolyl isomerase (PPIase) and chaperone activities, and is considered to be a ubiquitous cytosolic protein-folding facilitator in bacteria. It possesses a histidine- and cysteine-rich C-terminus binding to selected divalent metal ions (e.g., Ni(2+), Zn(2+)), which is important for its involvement in the maturation processes of metalloenzymes. We have determined the solution structure of C-terminus-truncated SlyD from Helic ...201122045417
5' End-independent RNase J1 endonuclease cleavage of Bacillus subtilis model RNA.Bacillus subtilis trp leader RNA is a small (140-nucleotide) RNA that results from attenuation of trp operon transcription upon binding of the regulatory TRAP complex. Previously, endonucleolytic cleavage by ribonuclease RNase J1 in a 3'-proximal, single-stranded region was shown to be critical for initiation of trp leader RNA decay. RNase J1 is a dual-specificity enzyme, with both 5' exonucleolytic and endonucleolytic activities. Here, we provide in vivo and in vitro evidence that RNase J1 acce ...201121862575
new biotechnological perspectives of a nadh oxidase variant from thermus thermophilus hb27 as nad+-recycling enzyme.the number of biotransformations that use nicotinamide recycling systems is exponentially growing. for this reason one of the current challenges in biocatalysis is to develop and optimize more simple and efficient cofactor recycling systems. one promising approach to regenerate nad+ pools is the use of nadh-oxidases that reduce oxygen to hydrogen peroxide while oxidizing nadh to nad+. this class of enzymes may be applied to asymmetric reduction of prochiral substrates in order to obtain enantiop ...201122053761
inhibition of mycobacterium tuberculosis rna polymerase by binding of a gre factor homolog to secondary channel.because of the essential nature, each step of transcription viz, initiation, elongation and termination is subjected to elaborate regulation. a number of transcription factors modulate the rates of transcription at these different steps in addition to several inhibitors which shut down the process. many modulators including small molecules and proteinaceous inhibitors bind rna polymerase (rnap) secondary channel to control transcription. we describe here the first small protein inhibitor of tran ...201122194445
Biogeography and phylogenetic diversity of a cluster of exclusively marine myxobacteria.Myxobacteria are common in terrestrial habitats and well known for their formation of fruiting bodies and production of secondary metabolites. We studied a cluster of myxobacteria consisting only of sequences of marine origin (marine myxobacteria cluster, MMC) in sediments of the North Sea. Using a specific PCR, MMC sequences were detected in North Sea sediments down to 2.2?m depth, but not in the limnetic section of the Weser estuary and other freshwater habitats. In the water column, this clus ...201122189493
Biogeography and phylogenetic diversity of a cluster of exclusively marine myxobacteria.Myxobacteria are common in terrestrial habitats and well known for their formation of fruiting bodies and production of secondary metabolites. We studied a cluster of myxobacteria consisting only of sequences of marine origin (marine myxobacteria cluster, MMC) in sediments of the North Sea. Using a specific PCR, MMC sequences were detected in North Sea sediments down to 2.2?m depth, but not in the limnetic section of the Weser estuary and other freshwater habitats. In the water column, this clus ...201122189493
dehydratase mediated 1-propanol production in metabolically engineered escherichia coli.with the increasing consumption of fossil fuels, the question of meeting the global energy demand is of great importance in the near future. as an effective solution, production of higher alcohols from renewable sources by microorganisms has been proposed to address both energy crisis and environmental concerns. higher alcohols contain more than two carbon atoms and have better physiochemical properties than ethanol as fuel substitutes.201122074179
in vivo and in silico determination of essential genes of campylobacter jejuni.in the united kingdom, the thermophilic campylobacter species c. jejuni and c. coli are the most frequent causes of food-borne gastroenteritis in humans. while campylobacteriosis is usually a relatively mild infection, it has a significant public health and economic impact, and possible complications include reactive arthritis and the autoimmune diseases guillain-barré syndrome. the rapid developments in "omics" technologies have resulted in the availability of diverse datasets allowing predicti ...201122044676
Chloroplast RNase J compensates for inefficient transcription termination by removal of antisense RNA.Ribonuclease J is an essential enzyme, and the Bacillus subtilis ortholog possesses both endoribonuclease and 5' ? 3' exoribonuclease activities. Chloroplasts also contain RNase J, which has been postulated to participate, as both an exo- and endonuclease, in the maturation of polycistronic mRNAs. Here we have examined recombinant Arabidopsis RNase J and found both 5' ? 3' exoribonuclease and endonucleolytic activities. Virus-induced gene silencing was used to reduce RNase J expression in Arabid ...201122033332
proline utilization by bacillus subtilis: uptake and catabolism.l-proline can be used by bacillus subtilis as a sole source of carbon or nitrogen. we traced l-proline utilization genetically to the putbcp (ycgmno) locus. the putbcp gene cluster encodes a high-affinity proline transporter (putp) and two enzymes, the proline dehydrogenase putb and the δ(1)-pyrroline-5-carboxylate dehydrogenase putc, which jointly catabolize l-proline to l-glutamate. northern blotting, primer extension, and putb-trea reporter gene fusion analysis showed that the putbcp locus is ...201122139509
effects of pressure and temperature on the binding of reca protein to single-stranded dna.the binding and polymerization of reca protein to dna is required for recombination, which is an essential function of life. we study the pressure and temperature dependence of reca binding to single-stranded dna in the presence of adenosine 5'-[γ-thio]triphosphate (atp[γ-s]), in a temperature regulated high pressure cell using fluorescence anisotropy. measurements were possible at temperatures between 5-60 °c and pressures up to 300 mpa. experiments were performed on escherichia coli reca and r ...201122123983
arrangement of electron transport chain components in bovine mitochondrial supercomplex i1iii2iv1.the respiratory chain in the inner mitochondrial membrane contains three large multi-enzyme complexes that together establish the proton gradient for atp synthesis, and assemble into a supercomplex. a 19-å 3d map of the 1.7-mda amphipol-solubilized supercomplex i(1)iii(2)iv(1) from bovine heart obtained by single-particle electron cryo-microscopy reveals an amphipol belt replacing the membrane lipid bilayer. a precise fit of the x-ray structures of complex i, the complex iii dimer, and monomeric ...201121909073
Crystal structure of the hybrid state of ribosome in complex with the guanosine triphosphatase release factor 3.Protein release factor 3 (RF3), a guanosine triphosphatase, binds to ribosome after release of the nascent peptide and promotes dissociation of the class I release factors during the termination of protein synthesis. Here we present the crystal structure of the 70S ribosome with RF3 in the presence of a nonhydrolyzable GTP analogue, guanosine 5'-ß,?-methylenetriphosphate (GDPCP), refined to 3.8 Å resolution. The structure shows that the subunits of the ribosome are rotated relative to each other ...201121903932
Manganese superoxide dismutase is a mitochondrial fidelity protein that protects Pol? against UV-induced inactivation.Manganese superoxide dismutase is a nuclear encoded primary antioxidant enzyme localized exclusively in the mitochondrial matrix. Genotoxic agents, such as ultraviolet (UV) radiation, generates oxidative stress and cause mitochondrial DNA (mtDNA) damage. The mtDNA polymerase (Pol?), a major constituent of nucleoids, is responsible for the replication and repair of the mitochondrial genome. Recent studies suggest that the mitochondria contain fidelity proteins and MnSOD constitutes an integral pa ...201121909133
Manganese superoxide dismutase is a mitochondrial fidelity protein that protects Pol? against UV-induced inactivation.Manganese superoxide dismutase is a nuclear encoded primary antioxidant enzyme localized exclusively in the mitochondrial matrix. Genotoxic agents, such as ultraviolet (UV) radiation, generates oxidative stress and cause mitochondrial DNA (mtDNA) damage. The mtDNA polymerase (Pol?), a major constituent of nucleoids, is responsible for the replication and repair of the mitochondrial genome. Recent studies suggest that the mitochondria contain fidelity proteins and MnSOD constitutes an integral pa ...201121909133
a novel mechanism of sulfur transfer catalyzed by o-acetylhomoserine sulfhydrylase in the methionine-biosynthetic pathway of wolinella succinogenes.o-acetylhomoserine sulfhydrylase (oahs) is a pyridoxal 5'-phosphate (plp) dependent sulfide-utilizing enzyme in the l-cysteine and l-methionine biosynthetic pathways of various enteric bacteria and fungi. oahs catalyzes the conversion of o-acetylhomoserine to homocysteine using sulfide in a process known as direct sulfhydrylation. however, the source of the sulfur has not been identified and no structures of oahs have been reported in the literature. here, the crystal structure of wolinella succ ...201121931214
Characterization of benzoxaborole-based antifungal resistance mutations demonstrates that editing depends on electrostatic stabilization of the leucyl-tRNA synthetase editing cap.The broad-spectrum benzoxaborole antifungal AN2690 blocks protein synthesis by inhibiting leucyl-tRNA synthetase (LeuRS) via a novel oxaborole tRNA trapping mechanism in the editing site. Herein, one set of resistance mutations is at Asp487 outside the LeuRS hydrolytic editing pocket, in a region of unknown function. It is located within a eukaryote/archaea specific insert I4, which forms part of a cap over a benzoxaborole-AMP that is bound in the LeuRS CP1 domain editing active site. Mutational ...201121856301
Small-angle X-ray Scattering Studies of the Oligomeric State and Quaternary Structure of the Trifunctional Proline Utilization A (PutA) Flavoprotein from Escherichia coli.The trifunctional flavoprotein proline utilization A (PutA) links metabolism and gene regulation in Gram-negative bacteria by catalyzing the two-step oxidation of proline to glutamate and repressing transcription of the proline utilization regulon. Small-angle x-ray scattering (SAXS) and domain deletion analysis were used to obtain solution structural information for the 1320-residue PutA from Escherichia coli. Shape reconstructions show that PutA is a symmetric V-shaped dimer having dimensions ...201122013066
a temperature-dependent conformational change of nadh oxidase from thermus thermophilus hb8.using molecular dynamics simulations and steady-state fluorescence spectroscopy, we have identified a conformational change in the active site of a thermophilic flavoenzyme, nadh oxidase from thermus thermophilus hb8 (nox). the enzyme's far-uv circular dichroism spectrum, intrinsic tryptophan fluorescence, and apparent molecular weight measured by dynamic light scattering varied little between 25 and 75°c. however, the fluorescence of the tightly bound fad cofactor increased approximately fourfo ...201122081476
"stealth" melanoma cells in histology-negative sentinel lymph nodes.a proportion of patients who develop regional and distant recurrences of melanoma after a pathologically negative sentinel lymph node (sn) biopsy are reported to have enhanced signals for melanoma-associated messenger ribonucleic acid (mrna) when sensitive molecular approaches such as reverse transcriptase polymerase chain reaction (rt-pcr) are used to evaluate their sn tissue. the significance of these findings remains controversial, because the cellular source of the augmented signals cannot b ...201121997686
The structure and allosteric regulation of mammalian glutamate dehydrogenase.Glutamate dehydrogenase (GDH) is a homohexameric enzyme that catalyzes the reversible oxidative deamination of l-glutamate to 2-oxoglutarate. Only in the animal kingdom is this enzyme heavily allosterically regulated by a wide array of metabolites. The major activators are ADP and leucine, while the most important inhibitors include GTP, palmitoyl CoA, and ATP. Recently, spontaneous mutations in the GTP inhibitory site that lead to the hyperinsulinism/hyperammonemia (HHS) syndrome have shed ligh ...201122079166
The structure and allosteric regulation of mammalian glutamate dehydrogenase.Glutamate dehydrogenase (GDH) is a homohexameric enzyme that catalyzes the reversible oxidative deamination of l-glutamate to 2-oxoglutarate. Only in the animal kingdom is this enzyme heavily allosterically regulated by a wide array of metabolites. The major activators are ADP and leucine, while the most important inhibitors include GTP, palmitoyl CoA, and ATP. Recently, spontaneous mutations in the GTP inhibitory site that lead to the hyperinsulinism/hyperammonemia (HHS) syndrome have shed ligh ...201122079166
Trimming down a protein structure to its bare foldons: spatial organisation of the cooperative unit.Folding of the ribosomal protein S6 is a malleable process controlled by two competing, and partly overlapping, folding nuclei. Together, these nuclei extend over most of the S6 structure, save the edge strand ß2, which is consistently missing in the folding transition states: despite being part of the S6 four-stranded sheet, ß2 seems not to be part of the protein's cooperative unit. The question is then if ß2 can be removed from the S6 structure without compromising folding cooperativity or ...201122117065
Trimming down a protein structure to its bare foldons: spatial organisation of the cooperative unit.Folding of the ribosomal protein S6 is a malleable process controlled by two competing, and partly overlapping, folding nuclei. Together, these nuclei extend over most of the S6 structure, save the edge strand ß2, which is consistently missing in the folding transition states: despite being part of the S6 four-stranded sheet, ß2 seems not to be part of the protein's cooperative unit. The question is then if ß2 can be removed from the S6 structure without compromising folding cooperativity or ...201122117065
yeast cytochrome c oxidase: a model system to study mitochondrial forms of the haem-copper oxidase superfamily.the known subunits of yeast mitochondrial cytochrome c oxidase are reviewed. the structures of all eleven of its subunits are explored by building homology models based on the published structures of the homologous bovine subunits and similarities and differences are highlighted, particularly of the core functional subunit i. yeast genetic techniques to enable introduction of mutations into the three core mitochondrially-encoded subunits are reviewed. this article is part of a special issue enti ...201121925484
yeast cytochrome c oxidase: a model system to study mitochondrial forms of the haem-copper oxidase superfamily.the known subunits of yeast mitochondrial cytochrome c oxidase are reviewed. the structures of all eleven of its subunits are explored by building homology models based on the published structures of the homologous bovine subunits and similarities and differences are highlighted, particularly of the core functional subunit i. yeast genetic techniques to enable introduction of mutations into the three core mitochondrially-encoded subunits are reviewed. this article is part of a special issue enti ...201121925484
crystallization and preliminary crystallographic analysis of a putative glucokinase/hexokinase from thermus thermophilus.glucokinase/hexokinase catalyzes the phosphorylation of glucose to glucose 6-phosphate, which is the first step of glycolysis. the open reading frame ttha0299 of the extreme thermophile thermus thermophilus encodes a putative glucokinase/hexokinase which contains the consensus sequence for proteins from the repressors, open reading frames and sugar kinases family. in this study, the glucokinase/hexokinase from t. thermophilus was purified and crystallized using polyethylene glycol 8000 as a prec ...201122139166
High-resolution structure of shikimate dehydrogenase from Thermotoga maritima reveals a tightly closed conformation.Shikimate dehydrogenase (SDH), which catalyses the NADPH-dependent reduction of 3-dehydroshikimate to shikimate in the shikimate pathway, is an attractive target for the development of herbicides and antimicrobial agents. Structural analysis of a SDH from Thermotoga maritima encoded by the Tm0346 gene was performed to facilitate further structural comparisons between the various shikimate dehydrogenases. The crystal structure of SDH from T. maritima was determined at 1.45 SDH from T. maritima sh ...201122095087
High-resolution structure of shikimate dehydrogenase from Thermotoga maritima reveals a tightly closed conformation.Shikimate dehydrogenase (SDH), which catalyses the NADPH-dependent reduction of 3-dehydroshikimate to shikimate in the shikimate pathway, is an attractive target for the development of herbicides and antimicrobial agents. Structural analysis of a SDH from Thermotoga maritima encoded by the Tm0346 gene was performed to facilitate further structural comparisons between the various shikimate dehydrogenases. The crystal structure of SDH from T. maritima was determined at 1.45 SDH from T. maritima sh ...201122095087
functional genomic and advanced genetic studies reveal novel insights into the metabolism, regulation, and biology of haloferax volcanii.the genome sequence of haloferax volcanii is available and several comparative genomic in silico studies were performed that yielded novel insight for example into protein export, rna modifications, small non-coding rnas, and ubiquitin-like small archaeal modifier proteins. the full range of functional genomic methods has been established and results from transcriptomic, proteomic and metabolomic studies are discussed. notably, hfx. volcanii is together with halobacterium salinarum the only prok ...201122190865
structure and molecular evolution of cdgsh iron-sulfur domains.the recently discovered cdgsh iron-sulfur domains (cisds) are classified into seven major types with a wide distribution throughout the three domains of life. the type 1 protein mitoneet has been shown to fold into a dimer with the signature cdgsh motif binding to a [2fe-2s] cluster. however, the structures of all other types of cisds were unknown. here we report the crystal structures of type 3, 4, and 6 cisds determined at 1.5 å, 1.8 å and 1.15 å resolution, respectively. the type 3 and 4 cisd ...201121949752
orphan macrodomain protein (human c6orf130) is an o-acyl-adp-ribose deacylase: solution structure and catalytic properties.post-translational modification of proteins/histones by lysine acylation has profound effects on the physiological function of modified proteins. deacylation by nad(+)-dependent sirtuin reactions yields as a product o-acyl-adp-ribose, which has been implicated as a signaling molecule in modulating cellular processes. macrodomain-containing proteins are reported to bind nad(+)-derived metabolites. here, we describe the structure and function of an orphan macrodomain protein, human c6orf130. this ...201121849506
interaction of complexes i, iii, and iv within the bovine respirasome by single particle cryoelectron tomography.the respirasome is a multisubunit supercomplex of the respiratory chain in mitochondria. here we report the 3d reconstruction of the bovine heart respirasome, composed of dimeric complex iii and single copies of complex i and iv, at about 2.2-nm resolution, determined by cryoelectron tomography and subvolume averaging. fitting of x-ray structures of single complexes i, iii(2), and iv with high fidelity allows interpretation of the model at the level of secondary structures and shows how the indi ...201121876144
a mycobacterium leprae hsp65 mutant as a candidate for mitigating lupus aggravation in mice.hsp60 is an abundant and highly conserved family of intracellular molecules. increased levels of this family of proteins have been observed in the extracellular compartment in chronic inflammation. administration of m. leprae hsp65 [wt] in [nzbxnzw]f(1) mice accelerates the systemic lupus erythematosus [sle] progression whereas the point mutated k(409)a hsp65 protein delays the disease. here, the biological effects of m. leprae hsp65 leader pep and k(409)a pep synthetic peptides, which cover res ...201121961033
structural comparison of trna m1a58 methyltransferases revealed different molecular strategies to maintain their oligomeric architecture under extreme conditions.abstract: background: trna m1a58 methyltransferases (trmi) catalyze the transfer of a methyl group from s-adenosyl-l-methionine to nitrogen 1 of adenine 58 in the t-loop of trnas from all three domains of life. the m1a58 modification has been shown to be essential for cell growth in yeast and for adaptation to high temperatures in thermophilic organisms. these enzymes were shown to be active as tetramers. the crystal structures of five trmis from hyperthermophilic archaea and thermophilic or me ...201122168821
cloning, expression, and homology modeling of groel protein from leptospira interrogans serovar autumnalis strain n2.leptospirosis is an infectious bacterial disease caused by leptospira species. in this study, we cloned and sequenced the gene encoding the immunodominant protein groel from l. interrogans serovar autumnalis strain n2, which was isolated from the urine of a patient during an outbreak of leptospirosis in chennai, india. this groel gene encodes a protein of 60 kda with a high degree of homology (99% similarity) to those of other leptospiral serovars. recombinant groel was overexpressed in escheric ...201122196358
a novel metagenomic short-chain dehydrogenase/reductase attenuates pseudomonas aeruginosa biofilm formation and virulence on caenorhabditis elegans.in pseudomonas aeruginosa, the expression of a number of virulence factors, as well as biofilm formation, are controlled by quorum sensing (qs). n-acylhomoserine lactones (ahls) are an important class of signaling molecules involved in bacterial qs and in many pathogenic bacteria infection and host colonization are ahl-dependent. the ahl signaling molecules are subject to inactivation mainly by hydrolases (enzyme commission class number ec 3) (i.e. n-acyl-homoserine lactonases and n-acyl-homoser ...201122046268
rational design of an evolutionary precursor of glutaminyl-trna synthetase.the specificity of most aminoacyl-trna synthetases for an amino acid and cognate trna pair evolved before the divergence of the three domains of life. glutaminyl-trna synthetase (glnrs) evolved later and is derived from the archaeal-type nondiscriminating glutamyl-trna synthetase (glurs), an enzyme with relaxed trna specificity capable of forming both glu-trna(glu) and glu-trna(gln). the archaea lack glnrs and use a specialized amidotransferase to convert glu-trna(gln) to gln-trna(gln) needed fo ...201122158897
membrane proteins in four acts: function precedes structure determination.studies on four membrane protein systems, which combine information derived from crystal structures and biophysical studies have emphasized, as a precursor to crystallization, demonstration of functional activity. these assays have relied on sensitive spectrophotometric, electrophysiological, and microbiological assays of activity to select purification procedures that lead to functional complexes and with greater likelihood to successful crystallization: (i), hetero-oligomeric proteins involved ...201122079407
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