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Post-translational modification by ß-lysylation is required for the activity of E. coli Elongation Factor P (EF-P).Bacterial elongation factor P (EF-P) is the ortholog of archaeal and eukaryotic initiation factor 5A (aIF5A and eIF5A). EF-P shares sequence homology and crystal structure with eIF5A, but unlike eIF5A, EF-P does not undergo hypusine modification. Recently, two bacterial genes, yjeA and yjeK, encoding truncated homologs of class II lysyl-tRNA synthetase and of lysine-2,3-aminomutase, respectively, have been implicated in the modification of EF-P to convert a specific lysine to a hypothetical ß- ...201122128152
Post-translational modification by ß-lysylation is required for the activity of E. coli Elongation Factor P (EF-P).Bacterial elongation factor P (EF-P) is the ortholog of archaeal and eukaryotic initiation factor 5A (aIF5A and eIF5A). EF-P shares sequence homology and crystal structure with eIF5A, but unlike eIF5A, EF-P does not undergo hypusine modification. Recently, two bacterial genes, yjeA and yjeK, encoding truncated homologs of class II lysyl-tRNA synthetase and of lysine-2,3-aminomutase, respectively, have been implicated in the modification of EF-P to convert a specific lysine to a hypothetical ß- ...201122128152
genomics of bacterial and archaeal viruses: dynamics within the prokaryotic virosphere.prokaryotes, bacteria and archaea, are the most abundant cellular organisms among those sharing the planet earth with human beings (among others). however, numerous ecological studies have revealed that it is actually prokaryotic viruses that predominate on our planet and outnumber their hosts by at least an order of magnitude. an understanding of how this viral domain is organized and what are the mechanisms governing its evolution is therefore of great interest and importance. the vast majorit ...201122126996
estimation of absolute protein quantities of unlabeled samples by selected reaction monitoring mass spectrometry.for many research questions in modern molecular and systems biology information about absolute protein quantities is imperative. these include, for example, kinetic modeling of processes, protein turnover determinations, stoichiometric investigations of protein complexes or quantitative comparisons of different proteins within one or across samples. to date, the vast majority of proteomic studies are limited to providing relative quantitative comparisons of protein levels between limited numbers ...201122101334
estimation of absolute protein quantities of unlabeled samples by selected reaction monitoring mass spectrometry.for many research questions in modern molecular and systems biology information about absolute protein quantities is imperative. these include, for example, kinetic modeling of processes, protein turnover determinations, stoichiometric investigations of protein complexes or quantitative comparisons of different proteins within one or across samples. to date, the vast majority of proteomic studies are limited to providing relative quantitative comparisons of protein levels between limited numbers ...201122101334
Biochemical and structural studies of the uncharacterized protein PA0743 from Pseudomonas aeruginosa revealed a NAD+-dependent L-serine dehydrogenase.The ß-hydroxyacid dehydrogenases form a large family of ubiquitous enzymes that catalyze oxidation of various ß-hydroxyacid substrates to corresponding semialdehydes. Several known enzymes include ß-hydroxyisobutyrate dehydrogenase, 6-phosphogluconate dehydrogenase, 2-(hydroxymethyl)glutarate dehydrogenase, and phenylserine dehydrogenase, but the vast majority of ß-hydroxyacid dehydrogenases remain uncharacterized. Here, we demonstrate that the predicted ß-hydroxyisobutyrate dehydrogenase PA0743 ...201122128181
Biochemical and structural studies of the uncharacterized protein PA0743 from Pseudomonas aeruginosa revealed a NAD+-dependent L-serine dehydrogenase.The ß-hydroxyacid dehydrogenases form a large family of ubiquitous enzymes that catalyze oxidation of various ß-hydroxyacid substrates to corresponding semialdehydes. Several known enzymes include ß-hydroxyisobutyrate dehydrogenase, 6-phosphogluconate dehydrogenase, 2-(hydroxymethyl)glutarate dehydrogenase, and phenylserine dehydrogenase, but the vast majority of ß-hydroxyacid dehydrogenases remain uncharacterized. Here, we demonstrate that the predicted ß-hydroxyisobutyrate dehydrogenase PA0743 ...201122128181
mass spectrometry of intact v-type atpases reveals bound lipids and the effects of nucleotide binding.the ability of electrospray to propel large viruses into a mass spectrometer is established and is rationalized by analogy to the atmospheric transmission of the common cold. much less clear is the fate of membrane-embedded molecular machines in the gas phase. here we show that rotary adenosine triphosphatases (atpases)/synthases from thermus thermophilus and enterococcus hirae can be maintained intact with membrane and soluble subunit interactions preserved in vacuum. mass spectra reveal subuni ...201122021858
translational diffusion of macromolecular assemblies measured using transverse-relaxation-optimized pulsed field gradient nmr.in structural biology, pulsed field gradient (pfg) nmr spectroscopy for the characterization of size and hydrodynamic parameters of macromolecular solutes has the advantage over other techniques that the measurements can be recorded with identical solution conditions as used for nmr structure determination or for crystallization trials. this paper describes two transverse-relaxation-optimized (tro) (15)n-filtered pfg stimulated-echo (ste) experiments for studies of macromolecular translational d ...201121919531
Comparative analyses of transport proteins encoded within the genomes of Mycobacterium tuberculosis and Mycobacterium leprae.The co-emergence of multidrug resistant pathogenic bacterial strains and the HIV pandemic has made tuberculosis a leading public health threat. The causative agent is Mycobacterium tuberculosis (Mtu), a facultative intracellular parasite. Mycobacterium leprae (Mle), a related organism that causes leprosy, is an obligate intracellular parasite. Given that different transporters are required for bacterial growth and persistence under a variety of growth conditions, we conducted comparative analyse ...201122179038
Comparative analyses of transport proteins encoded within the genomes of Mycobacterium tuberculosis and Mycobacterium leprae.The co-emergence of multidrug resistant pathogenic bacterial strains and the HIV pandemic has made tuberculosis a leading public health threat. The causative agent is Mycobacterium tuberculosis (Mtu), a facultative intracellular parasite. Mycobacterium leprae (Mle), a related organism that causes leprosy, is an obligate intracellular parasite. Given that different transporters are required for bacterial growth and persistence under a variety of growth conditions, we conducted comparative analyse ...201122179038
Structure and Function of the Small MutS-Related Domain.MutS family proteins are widely distributed in almost all organisms from bacteria to human and play central roles in various DNA transactions such as DNA mismatch repair and recombinational events. The small MutS-related (Smr) domain was originally found in the C-terminal domain of an antirecombination protein, MutS2, a member of the MutS family. MutS2 is thought to suppress homologous recombination by endonucleolytic resolution of early intermediates in the process. The endonuclease activity of ...201122091410
Probing conformational states of glutaryl-CoA dehydrogenase by fragment screening.Glutaric acidemia type 1 is an inherited metabolic disorder which can cause macrocephaly, muscular rigidity, spastic paralysis and other progressive movement disorders in humans. The defects in glutaryl-CoA dehydrogenase (GCDH) associated with this disease are thought to increase holoenzyme instability and reduce cofactor binding. Here, the first structural analysis of a GCDH enzyme in the absence of the cofactor flavin adenine dinucleotide (FAD) is reported. The apo structure of GCDH from Burkh ...201121904051
structural basis of free reduced flavin generation by flavin reductase from thermus thermophilus hb8.free reduced flavins are involved in a variety of biological functions. they are generated from nad(p)h by flavin reductase via co-factor flavin bound to the enzyme. although recent findings on the structure and function of flavin reductase provide new information about co-factor fad and substrate nad, there have been no reports on the substrate flavin binding site. here we report the structure of ttha0420 from thermus thermophilus hb8, which belongs to flavin reductase, and describe the dual bi ...201122052907
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
novel base triples in rna structures revealed by graph theoretical searching methods.highly hydrogen bonded base interactions play a major part in stabilizing the tertiary structures of complex rna molecules, such as transfer-rnas, ribozymes and ribosomal rnas.201122373013
a computational study of elongation factor g (efg) duplicated genes: diverged nature underlying the innovation on the same structural template.elongation factor g (efg) is a core translational protein that catalyzes the elongation and recycling phases of translation. a more complex picture of efg's evolution and function than previously accepted is emerging from analyzes of heterogeneous efg family members. whereas the gene duplication is postulated to be a prominent factor creating functional novelty, the striking divergence between efg paralogs can be interpreted in terms of innovation in gene function.201121829651
molecular evolution of urea amidolyase and urea carboxylase in fungi.urea amidolyase breaks down urea into ammonia and carbon dioxide in a two-step process, while another enzyme, urease, does this in a one step-process. urea amidolyase has been found only in some fungal species among eukaryotes. it contains two major domains: the amidase and urea carboxylase domains. a shorter form of urea amidolyase is known as urea carboxylase and has no amidase domain. eukaryotic urea carboxylase has been found only in several fungal species and green algae. in order to elucid ...201121447149
effect of lecithin on d-galactosamine induced hepatotoxicity through mitochondrial pathway involving bcl-2 and bax.twenty four wistar strain albino rats were used for the investigations. lecithin 50 and 100 mg/kg b wt was administered for 1 week by oral route. liver damage was induced by intra peritoneal administration of 400 mg/kg b wt d-galactosamine on the last day. at the end of the study animals were sacrificed and liver enzyme levels, histopathology, mitochondrial integrity, expression of p53, bax and bcl-2 mrna levels were studied. increases in the liver enzyme levels by d-galn were significantly inhi ...201123024474
a universal method for the identification of bacteria based on general pcr primers.the universal method (um) described here will allow the detection of any bacterial rdna leading to the identification of that bacterium. the method should allow prompt and accurate identification of bacteria. the principle of the method is simple; when a pure pcr product of the 16s gene is obtained, sequenced, and aligned against bacterial dna data base, then the bacterium can be identified. confirmation of identity may follow. in this work, several general 16s primers were designed, mixed and a ...201123024404
mycobacterium tuberculosis rv2419c, the missing glucosyl-3-phosphoglycerate phosphatase for the second step in methylglucose lipopolysaccharide biosynthesis.mycobacteria synthesize intracellular methylglucose lipopolysaccharides (mglp) proposed to regulate fatty acid synthesis. although their structures have been elucidated, the identity of most biosynthetic genes remains unknown. the first step in mglp biosynthesis is catalyzed by a glucosyl-3-phosphoglycerate synthase (gpgs, rv1208 in mycobacterium tuberculosis h37rv). however, a typical glucosyl-3-phosphoglycerate phosphatase (gpgp, ec3.1.3.70) for dephosphorylation of glucosyl-3-phosphoglycerate ...201122355692
crystallization and preliminary neutron diffraction studies of adp-ribose pyrophosphatase-i from thermus thermophilus hb8.adp-ribose pyrophosphatase-i from thermus thermophilus hb8 (ttadprase-i) prevents the intracellular accumulation of adp-ribose by hydrolyzing it to amp and ribose 5'-phosphate. to understand the catalytic mechanism of ttadprase-i, it is necessary to investigate the role of glutamates and metal ions as well as the coordination of water molecules located at the active site. a macroseeding method was developed in order to obtain a large ttadprase-i crystal which was suitable for a neutron diffracti ...201122232170
annotation of protein domains reveals remarkable conservation in the functional make up of proteomes across superkingdoms.the functional repertoire of a cell is largely embodied in its proteome, the collection of proteins encoded in the genome of an organism. the molecular functions of proteins are the direct consequence of their structure and structure can be inferred from sequence using hidden markov models of structural recognition. here we analyze the functional annotation of protein domain structures in almost a thousand sequenced genomes, exploring the functional and structural diversity of proteomes. we find ...201124710297
in vivo, in vitro, and x-ray crystallographic analyses suggest the involvement of an uncharacterized triose-phosphate isomerase (tim) barrel protein in protection against oxidative stress.accumulating genome sequences have revealed the existence of a large number of conserved hypothetical proteins. characterization of these proteins is considered essential in the elucidation of intracellular biological pathways. our previous transcriptomic analysis suggested that, in thermus thermophilus hb8, loss of an oxidized dna-repairing activity leads to the up-regulation of a function-unknown gene, tthb071, which is conserved in a wide range of bacteria. interestingly, the tthb071 gene pro ...201121984829
structural and mutational studies of a hyperthermophilic intein from dna polymerase ii of pyrococcus abyssi.protein splicing is a precise self-catalyzed process in which an intein excises itself from a precursor with the concomitant ligation of the flanking polypeptides (exteins). protein splicing proceeds through a four-step reaction but the catalytic mechanism is not fully understood at the atomic level. we report the solution nmr structures of the hyperthermophilic pyrococcus abyssi polii intein, which has a noncanonical c-terminal glutamine instead of an asparagine. the nmr structures were determi ...201121914805
Tilt-pair analysis of images from a range of different specimens in single-particle electron cryomicroscopy.The comparison of a pair of electron microscope images recorded at different specimen tilt angles provides a powerful approach for evaluating the quality of images, image-processing procedures, or three-dimensional structures. Here, we analyze tilt-pair images recorded from a range of specimens with different symmetries and molecular masses and show how the analysis can produce valuable information not easily obtained otherwise. We show that the accuracy of orientation determination of individua ...201121939668
isolation and characterization of the prochlorococcus carboxysome reveal the presence of the novel shell protein csos1d.cyanobacteria, including members of the genus prochlorococcus, contain icosahedral protein microcompartments known as carboxysomes that encapsulate multiple copies of the co(2)-fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco) in a thin protein shell that enhances the catalytic performance of the enzyme in part through the action of a shell-associated carbonic anhydrase. however, the exact mechanism by which compartmentation provides a catalytic advantage to the enzyme is n ...201122155772
bacteriophage t4 mota activator and the β-flap tip of rna polymerase target the same set of σ70 carboxyl-terminal residues.sigma factors, the specificity subunits of rna polymerase, are involved in interactions with promoter dna, the core subunits of rna polymerase, and transcription factors. the bacteriophage t4-encoded activator, mota, is one such factor, which engages the c terminus of the escherichia coli housekeeping sigma factor, σ(70). mota functions in concert with a phage-encoded co-activator, asia, as a molecular switch. this process, termed sigma appropriation, inhibits host transcription while activating ...201121911499
crystal structure of the bacteriophage t4 late-transcription coactivator gp33 with the β-subunit flap domain of escherichia coli rna polymerase.activated transcription of the bacteriophage t4 late genes, which is coupled to concurrent dna replication, is accomplished by an initiation complex containing the host rna polymerase associated with two phage-encoded proteins, gp55 (the basal promoter specificity factor) and gp33 (the coactivator), as well as the dna-mounted sliding-clamp processivity factor of the phage t4 replisome (gp45, the activator). we have determined the 3.0 å-resolution x-ray crystal structure of gp33 complexed with it ...201122135460
An ensemble of structures of Burkholderia pseudomallei 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase.Burkholderia pseudomallei is a soil-dwelling bacterium endemic to Southeast Asia and Northern Australia. Burkholderia is responsible for melioidosis, a serious infection of the skin. The enzyme 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase (PGAM) catalyzes the interconversion of 3-phosphoglycerate and 2-phosphoglycerate, a key step in the glycolytic pathway. As such it is an extensively studied enzyme and X-ray crystal structures of PGAM enzymes from multiple species have been elucid ...201121904048
identification of human fumarylacetoacetate hydrolase domain-containing protein 1 (fahd1) as a novel mitochondrial acylpyruvase.the human fumarylacetoacetate hydrolase (fah) domain-containing protein 1 (fahd1) is part of the fah protein superfamily, but its enzymatic function is unknown. in the quest for a putative enzymatic function of fahd1, we found that fahd1 exhibits acylpyruvase activity, demonstrated by the hydrolysis of acetylpyruvate and fumarylpyruvate in vitro, whereas several structurally related compounds were not hydrolyzed as efficiently. conserved amino acids asp-102 and arg-106 of fahd1 were found import ...201121878618
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
structural changes that occur upon photolysis of the fe(ii)(a3)-co complex in the cytochrome ba(3)-oxidase of thermus thermophilus: a combined x-ray crystallographic and infrared spectral study demonstrates co binding to cu(b).the purpose of the work was to provide a crystallographic demonstration of the venerable idea that co photolyzed from ferrous heme-a(3) moves to the nearby cuprous ion in the cytochrome c oxidases. crystal structures of co-bound cytochrome ba(3)-oxidase from thermus thermophilus, determined at ~2.8-3.2å resolution, reveal a fe-c distance of ~2.0å, a cu-o distance of 2.4å and a fe-c-o angle of ~126°. upon photodissociation at 100k, x-ray structures indicate loss of fe(a3)-co and appearance of cu( ...201122226917
structure of the pilus assembly protein tadz from eubacterium rectale: implications for polar localization.the tad (tight adherence) locus encodes a protein translocation system that produces a novel variant of type iv pili. the pilus assembly protein tadz (called cpae in caulobacter crescentus) is ubiquitous in tad loci, but is absent in other type iv pilus biogenesis systems. the crystal structure of tadz from eubacterium rectale (ertadz), in complex with atp and mg(2+) , was determined to 2.1 å resolution. ertadz contains an atypical atpase domain with a variant of a deviant walker-a motif that re ...201122211578
proline dehydrogenase contributes to pathogen defense in arabidopsis.l-proline (pro) catabolism is activated in plants recovering from abiotic stresses associated with water deprivation. in this catabolic pathway, pro is converted to glutamate by two reactions catalyzed by proline dehydrogenase (prodh) and ?(1)-pyrroline-5-carboxylate dehydrogenase (p5cdh), with ?(1)-pyrroline-5-carboxylate (p5c) as the intermediate. alternatively, under certain conditions, the p5c derived from pro is converted back to pro by p5c reductase, thus stimulating the pro-p5c cycle, whi ...201121311034
energetics of seca dimerization.transport of many proteins to extracytoplasmic locations occurs via the general secretion (sec) pathway. in escherichia coli, this pathway is composed of the secyeg protein-conducting channel and the seca atpase. seca plays a central role in binding the signal peptide region of preproteins, directing preproteins to membrane-bound secyeg and promoting translocation coupled with atp hydrolysis. although it is well established that seca is crucial for preprotein transport and thus cell viability, i ...201121315086
natural competence in the hyperthermophilic archaeon pyrococcus furiosus facilitates genetic manipulation: construction of markerless deletions of genes encoding the two cytoplasmic hydrogenases.in attempts to develop a method of introducing dna into pyrococcus furiosus, we discovered a variant within the wild-type population that is naturally and efficiently competent for dna uptake. a pyrf gene deletion mutant was constructed in the genome, and the combined transformation and recombination frequencies of this strain allowed marker replacement by direct selection using linear dna. we have demonstrated the use of this strain, designated com1, for genetic manipulation. using genetic sele ...201121317259
il-10 expression by primary tumor cells correlates with melanoma progression from radial to vertical growth phase and development of metastatic competence.downregulation of the immune system facilitates tumor progression at different stages of cutaneous melanoma. sentinel nodes, the first lymph nodes on lymphatics draining directly from a primary melanoma, are immune downregulated by tumor-generated immunosuppressive cytokines, including interleukin-10 (il-10). to better understand the kinetics of sentinel node suppression, we investigated il-10 expression by melanoma cells and tumor-associated macrophages and lymphocytes at different stages of pr ...201121317876
unusual outer membrane lipid composition of the gram-negative, lipopolysaccharide-lacking myxobacterium sorangium cellulosum so ce56.the gram-negative myxobacterium sorangium cellulosum so ce56 bears the largest bacterial genome published so far, coding for nearly 10,000 genes. careful analysis of this genome data revealed that part of the genes coding for the very well conserved biosynthesis of lipopolysaccharides (lps) are missing in this microbe. biochemical analysis gave no evidence for the presence of lps in the membranes of so ce56. by analyzing the lipid composition of its outer membrane sphingolipids were identified a ...201121321121
activity of and development of resistance to corallopyronin a, an inhibitor of rna polymerase.we explored the properties of corallopyronin a (cora), a poorly characterized inhibitor of bacterial rna polymerase (rnap). it displayed a 50% inhibitory concentration of 0.73 µm against rnap, compared with 11.5 nm for rifampin. the antibacterial activity of cora was also inferior to rifampin, and resistant mutants of staphylococcus aureus were easily selected. the mutations conferring resistance resided in the rpob and rpoc subunits of rnap. we conclude that cora is not a promising antibacteria ...201121321139
bacterial toxin rele mediates frequent codon-independent mrna cleavage from the 5' end of coding regions in vivo.the enzymatic activity of the rele bacterial toxin component of the escherichia coli relbe toxin-antitoxin system has been extensively studied in vitro and to a lesser extent in vivo. these earlier reports revealed that 1) rele alone does not exhibit mrna cleavage activity, 2) rele mediates mrna cleavage through its association with the ribosome, 3) rele-mediated mrna cleavage occurs at the ribosomal a site and, 4) cleavage of mrna by rele exhibits high codon specificity. more specifically, rele ...201121324908
a highly conserved protein of unknown function in sinorhizobium meliloti affects srna regulation similar to hfq.the smc01113/ybey protein, belonging to the upf0054 family, is highly conserved in nearly every bacterium. however, the function of these proteins still remains elusive. our results show that smc01113/ybey proteins share structural similarities with the mid domain of the argonaute (ago) proteins, and might similarly bind to a small-rna (srna) seed, making a special interaction with the phosphate on the 5'-side of the seed, suggesting they may form a component of the bacterial srna pathway. indee ...201121325267
avoiding premature oxidation during the binding of cu(ii) to a dithiolate site in bssco. a rapid freeze-quench epr study.the bacillus subtilis version of sco1 (bssco) is required for assembly of cu(a) in cytochrome c oxidase and may function in thiol-disulfide exchange and/or copper delivery. bssco binds cu(ii) with ligation by two cysteines, one histidine and one water. however, copper is a catalyst of cysteine oxidation and bssco must avoid this reaction to remain functional. time resolved, rapid freeze-quench (rfq) electron paramagnetic resonance of apo-bssco reacting with cu(ii) reveals an initial cu(ii) speci ...201121333651
organic hydroperoxide resistance protein and ergothioneine compensate for loss of mycothiol in mycobacterium smegmatis mutants.the msha::tn5 mutant of mycobacterium smegmatis does not produce mycothiol (msh) and was found to markedly overproduce both ergothioneine and an ~15-kda protein determined to be organic hydroperoxide resistance protein (ohr). an msha(g32d) mutant lacking msh overproduced ergothioneine but not ohr. comparison of the mutant phenotypes with those of the wild-type strain indicated the following: ohr protects against organic hydroperoxide toxicity, whereas ergothioneine does not; an additional msh-de ...201121335456
chlorite dismutases, dyps, and efeb: 3 microbial heme enzyme families comprise the cde structural superfamily.heme proteins are extremely diverse, widespread, and versatile biocatalysts, sensors, and molecular transporters. the chlorite dismutase family of hemoproteins received its name due to the ability of the first-isolated members to detoxify anthropogenic clo(2)(-), a function believed to have evolved only in the last few decades. family members have since been found in 15 bacterial and archaeal genera, suggesting ancient roots. a structure- and sequence-based examination of the family is presented ...201121354424
activity map of the escherichia coli rna polymerase bridge helix.transcription, the synthesis of rna from a dna template, is performed by multisubunit rna polymerases (rnaps) in all cellular organisms. the bridge helix (bh) is a distinct feature of all multisubunit rnaps and makes direct interactions with several active site-associated mobile features implicated in the nucleotide addition cycle and rna and dna binding. because the bh has been captured in both kinked and straight conformations in different crystals structures of rnap, recently supported by mol ...201121357417
correct assembly of iron-sulfur cluster fs0 into escherichia coli dimethyl sulfoxide reductase (dmsabc) is a prerequisite for molybdenum cofactor insertion.the fs0 [4fe-4s] cluster of the catalytic subunit (dmsa) of escherichia coli dimethyl sulfoxide reductase (dmsabc) plays a key role in the electron transfer relay. we have now established an additional role for the cluster in directing molybdenum cofactor assembly during enzyme maturation. epr spectroscopy indicates that fs0 has a high spin ground state (s = 3/2) in its reduced form, resulting in an epr spectrum with a peak at g ~ 5.0. the cluster is predicted to be in close proximity to the mol ...201121357619
an expanded collection and refined consensus model of glms ribozymes.self-cleaving glms ribozymes selectively bind glucosamine-6-phosphate (glcn6p) and use this metabolite as a cofactor to promote self-cleavage by internal phosphoester transfer. representatives of the glms ribozyme class are found in gram-positive bacteria where they reside in the 5' untranslated regions (utrs) of glms messenger rnas that code for the essential enzyme l-glutamine:d-fructose-6-phosphate aminotransferase. by using comparative sequence analyses, we have expanded the number of glms r ...201121367971
insertion domain within mammalian mitochondrial translation initiation factor 2 serves the role of eubacterial initiation factor 1.mitochondria have their own translational machineries for the synthesis of thirteen polypeptide chains that are components of the complexes that participate in the process of oxidative phosphorylation (or atp generation). translation initiation in mammalian mitochondria requires two initiation factors, if2(mt) and if3(mt), instead of the three that are present in eubacteria. the mammalian if2(mt) possesses a unique 37 amino acid insertion domain, which is known to be important for the formation ...201121368145
two rotary motors in f-atp synthase are elastically coupled by a flexible rotor and a stiff stator stalk.atp is synthesized by atp synthase (f(o)f(1)-atpase). its rotary electromotor (f(o)) translocates protons (in some organisms sodium cations) and generates torque to drive the rotary chemical generator (f(1)). elastic power transmission between f(o) and f(1) is essential for smoothing the cooperation of these stepping motors, thereby increasing their kinetic efficiency. a particularly compliant elastic domain is located on the central rotor (c(10-15)/e/?), right between the two sites of torque ge ...201121368147
e1- and ubiquitin-like proteins provide a direct link between protein conjugation and sulfur transfer in archaea.based on our recent work with haloferax volcanii, ubiquitin-like (ubl) proteins (samp1 and samp2) are known to be covalently attached to proteins in archaea. here, we investigated the enzymes required for the formation of these ubl-protein conjugates (sampylation) and whether this system is linked to sulfur transfer. markerless in-frame deletions were generated in h. volcanii target genes. the mutants were examined for: (i) the formation of ubl protein conjugates, (ii) growth under various condi ...201121368171
oxidative stress resistance in deinococcus radiodurans.deinococcus radiodurans is a robust bacterium best known for its capacity to repair massive dna damage efficiently and accurately. it is extremely resistant to many dna-damaging agents, including ionizing radiation and uv radiation (100 to 295 nm), desiccation, and mitomycin c, which induce oxidative damage not only to dna but also to all cellular macromolecules via the production of reactive oxygen species. the extreme resilience of d. radiodurans to oxidative stress is imparted synergistically ...201121372322
partial functional replacement of cyma by sircd in shewanella oneidensis mr-1.the gammaproteobacterium shewanella oneidensis mr-1 utilizes a complex electron transfer network composed primarily of c-type cytochromes to respire under anoxic conditions a variety of compounds, including fumarate, nitrate, and dimethyl sulfoxide (dmso), in addition to the minerals fe(iii) and mn(iv). central to several respiratory pathways is cyma, a cytoplasmic membrane-bound tetraheme c-type cytochrome that functions as the major hydroquinone dehydrogenase. to investigate functional redunda ...201121378180
structural insights into cognate versus near-cognate discrimination during decoding.the structural basis of the trna selection process is investigated by cryo-electron microscopy of ribosomes programmed with uga codons and incubated with ternary complex (tc) containing the near-cognate trp-trna(trp) in the presence of kirromycin. going through more than 350 000 images and employing image classification procedures, we find ~8% in which the tc is bound to the ribosome. the reconstructed 3d map provides a means to characterize the arrangement of the near-cognate aa-trna with respe ...201121378755
architecture of the rna polymerase-spt4/5 complex and basis of universal transcription processivity.related rna polymerases (rnaps) carry out cellular gene transcription in all three kingdoms of life. the universal conservation of the transcription machinery extends to a single rnap-associated factor, spt5 (or nusg in bacteria), which renders rnap processive and may have arisen early to permit evolution of long genes. spt5 associates with spt4 to form the spt4/5 heterodimer. here, we present the crystal structure of archaeal spt4/5 bound to the rnap clamp domain, which forms one side of the rn ...201121386817
mutagenesis of the l, m, and n subunits of complex i from escherichia coli indicates a common role in function.the membrane arm of complex i (nadh:ubiquinone oxidoreductase) contains three large, and closely related subunits, which are called l, m, and n in e. coli. these subunits are homologous to components of multi-subunit na(+)/h(+) antiporters, and so are implicated in proton translocation.201121387012
susceptibility and mode of binding of the mycobacterium tuberculosis cysteinyl transferase mycothiol ligase to trna synthetase inhibitors.the cysteinyl transferase mycothiol ligase, or mshc, catalyzes the fourth step in the biosynthesis of the small molecular weight thiol mycothiol. mshc is essential for growth of mycobacterium tuberculosis. two groups of known aminoacyl trna synthetase inhibitors were evaluated for inhibition of m. tuberculosis mshc including aminoacyl adenosine analogs and natural products. using enzyme assays, isothermal titration calorimetry and nmr, we show that mshc is selectively inhibited by cysteinyl sulf ...201121392992
cloning, expression, purification, crystallization and preliminary x-ray diffraction analysis of the regulatory domain of aspartokinase (rv3709c) from mycobacterium tuberculosis.the regulatory domain of mycobacterium tuberculosis aspartokinase (mtb-ak, mtb-ask, rv3709c) has been cloned, heterologously expressed in escherichia coli and purified using standard chromatographic techniques. screening for initial crystallization conditions using the regulatory domain (ak-+¦) in the presence of the potential feedback inhibitor threonine identified four conditions which yielded crystals suitable for x-ray diffraction analysis. from these four conditions five different crystal f ...201121393848
crystallization and preliminary x-ray analysis of mannosyl-3-phosphoglycerate phosphatase from thermus thermophilus hb27.mannosylglycerate (mg) is primarily known as an osmolyte and is widely distributed among (hyper)thermophilic marine microorganisms. the synthesis of mg via mannosyl-3-phosphoglycerate synthase (mpgs) and mannosyl-3-phosphoglycerate phosphatase (mpgp), the so-called two-step pathway, is the most prevalent route among these organisms. the phosphorylated intermediate mannosyl-3-phosphoglycerate is synthesized by the first enzyme and is subsequently dephosphorylated by the second. the structure of m ...201121393850
purification and biochemical properties of a cytochrome bc complex from the aerobic hyperthermophilic archaeon aeropyrum pernix.the bioenergetics of archaea with respect to the evolution of electron transfer systems is very interesting. in contrast to terminal oxidases, a canonical bc1 complex has not yet been isolated from archaea. in particular, c-type cytochromes have been reported only for a limited number of species.201121396131
the antibacterial threaded-lasso peptide capistruin inhibits bacterial rna polymerase.capistruin, a ribosomally synthesized, post-translationally modified peptide produced by burkholderia thailandensis e264, efficiently inhibits growth of burkholderia and closely related pseudomonas strains. the functional target of capistruin is not known. capistruin is a threaded-lasso peptide (lariat peptide) consisting of an n-terminal ring of nine amino acids and a c-terminal tail of 10 amino acids threaded through the ring. the structure of capistruin is similar to that of microcin j25 (mcc ...201121396375
biosynthesis of the rna polymerase inhibitor streptolydigin in streptomyces lydicus: tailoring modification of 3-methyl-aspartate.the asparaginyl-trna synthetase-like slgz and methyltransferase slgm enzymes are involved in the biosynthesis of the tetramic acid streptolydigin in streptomyces lydicus. inactivation of slgz led to a novel streptolydigin derivative. overexpression of slgz, slgm, or both in s. lydicus led to a considerable increase in streptolydigin production.201121398531
resolving stepping rotation in thermus thermophilus h(+)-atpase/synthase with an essentially drag-free probe.vacuole-type atpases (v(o)v1) and f(o)f1 atp synthases couple atp hydrolysis/synthesis in the soluble v(1) or f1 portion with proton (or na(+)) flow in the membrane-embedded v(o) or f(o) portion through rotation of one common shaft. here we show at submillisecond resolutions the atp-driven rotation of isolated v1 and the whole v(o)v1 from thermus thermophilus, by attaching a 40-nm gold bead for which viscous drag is almost negligible. v1 made 120° steps, commensurate with the presence of three c ...201121407199
genetic components of stringent response in vibrio cholerae.nutritional stress elicits stringent response in bacteria involving modulation of expression of several genes. this is mainly triggered by the intracellular accumulation of two small molecules, namely, guanosine 3'-diphosphate 5'-triphosphate and guanosine 3',5'-bis(diphosphate), collectively called (p)ppgpp. like in other gram-negative bacteria, the cellular level of (p)ppgpp is maintained in vibrio cholerae, the causative bacterial pathogen of the disease cholera, by the products of two genes ...201121415497
involvement of cara/litr and crp/fnr family transcriptional regulators in light-induced carotenoid production in thermus thermophilus.members of the cara/litr family are merr-type transcriptional regulators that contain a c-terminal cobalamin-binding domain. they are thought to be involved in light-induced transcriptional regulation in a wide variety of nonphototrophic bacteria. based on the distribution of this kind of regulator, the current study examined carotenoid production in thermus thermophilus, and it was found to occur in a light-induced manner. litr and carotenoid and cobalamin biosynthesis genes were all located on ...201121421762
substrate specificity of the bacillus licheniformis lyxose isomerase ydae and its application in in vitro catalysis for bioproduction of lyxose and glucose by two-step isomerization.enzymatic processes are useful for industrially important sugar production, and in vitro two-step isomerization has proven to be an efficient process in utilizing readily available sugar sources. a hypothetical uncharacterized protein encoded by ydae of bacillus licheniformis was found to have broad substrate specificities and has shown high catalytic efficiency on d-lyxose, suggesting that the enzyme is d-lyxose isomerase. escherichia coli bl21 expressing the recombinant protein, of 19.5 kda, s ...201121421786
insights into physiological and genetic mupirocin susceptibility in bifidobacteria.mupirocin is an antibiotic commonly used in selective media for the isolation of bifidobacteria. however, little is known about the genetic traits responsible for bifidobacterial resistance to mupirocin. our investigation demonstrates that all of the bifidobacteria tested exhibit a phenotype of generally high resistance to this antibiotic. the genotypic reason for bifidobacterial mupirocin resistance was further characterized by sequencing of the isoleucyl-trna synthetase gene (iles) coupled wit ...201121421794
a rigidifying salt-bridge favors the activity of thermophilic enzyme at high temperatures at the expense of low-temperature activity.thermophilic enzymes are often less active than their mesophilic homologues at low temperatures. one hypothesis to explain this observation is that the extra stabilizing interactions increase the rigidity of thermophilic enzymes and hence reduce their activity. here we employed a thermophilic acylphosphatase from pyrococcus horikoshii and its homologous mesophilic acylphosphatase from human as a model to study how local rigidity of an active-site residue affects the enzymatic activity.201121423654
phylogenetic distribution and evolutionary history of bacterial dead-box proteins.dead-box proteins are found in all domains of life and participate in almost all cellular processes that involve rna. the presence of dead and helicase_c conserved domains distinguish these proteins. dead-box proteins exhibit rna-dependent atpase activity in vitro, and several also show rna helicase activity. in this study, we analyzed the distribution and architecture of dead-box proteins among bacterial genomes to gain insight into the evolutionary pathways that have shaped their history. we i ...201121437710
nmr structure of the c-terminal domain of a tyrosyl-trna synthetase that functions in group i intron splicing.the mitochondrial tyrosyl-trna synthetases (mt tyrrss) of pezizomycotina fungi are bifunctional proteins that aminoacylate mitochondrial trna(tyr) and are structure-stabilizing splicing cofactors for group i introns. studies with the neurospora crassa synthetase (cyt-18 protein) showed that splicing activity is dependent upon pezizomycotina-specific structural adaptations that form a distinct group i intron-binding site in the n-terminal catalytic domain. although cyt-18's c-terminal domain also ...201121438536
unexpected diversity of chlorite dismutases: a catalytically efficient dimeric enzyme from nitrobacter winogradskyi.chlorite dismutase (cld) is a unique heme enzyme catalyzing the conversion of clo(2)(-) to cl(-) and o(2). cld is usually found in perchlorate- or chlorate-reducing bacteria but was also recently identified in a nitrite-oxidizing bacterium of the genus nitrospira. here we characterized a novel cld-like protein from the chemolithoautotrophic nitrite oxidizer nitrobacter winogradskyi which is significantly smaller than all previously known chlorite dismutases. its three-dimensional (3d) crystal st ...201121441524
solution structure of an alternate conformation of helix27 from escherichia coli16s rrna.helix (h)27 of 16s ribosomal (r)rna from escherichia coli was dubbed the "switch helix" when mutagenesis suggested that two alternative base pair registers may have distinct functional roles in the bacterial ribosome. although more recent genetic analyses suggest that h27 conformational switching is not required for translation, previous solution studies demonstrated that the isolated e. coli h27 can dynamically convert between the 885 and 888 conformations. here, we have solved the nuclear magn ...201121442607
a model of the proton translocation mechanism of complex i.despite decades of speculation, the proton pumping mechanism of complex i (nadh-ubiquinone oxidoreductase) is unknown and continues to be controversial. recent descriptions of the architecture of the hydrophobic region of complex i have resolved one vital issue: this region appears to have multiple proton transporters that are mechanically interlinked. thus, transduction of conformational changes to drive the transmembrane transporters linked by a "connecting rod" during the reduction of ubiquin ...201121454533
regulatory circuits of the aaa+ disaggregase hsp104.yeast hsp104 is an aaa+ chaperone that rescues proteins from the aggregated state. six protomers associate to form the functional hexamer. each protomer contains two aaa+ modules, nbd1 and nbd2. hsp104 converts energy provided by atp into mechanical force used to thread polypeptides through its axial channel, thereby disrupting protein aggregates. but how the action of its 12 aaa+ domains is co-ordinated to catalyze disaggregation remained unexplained. here, we identify a sophisticated allosteri ...201121454552
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