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an unusual n-terminal ααβαββα fold of pilq from t. thermophilus mediates ring formation and is essential for piliation.dna translocators of natural transformation systems are complex systems critical for the uptake of free dna and provide a powerful mechanism for adaptation to changing environmental conditions. in natural transformation machineries, outer membrane secretins are suggested to form a multimeric pore for the uptake of external dna. recently, we reported on a novel structure of the dna translocator secretin complex, pilq, in thermus thermophilus hb27 comprising a stable cone and cup structure and six ...201222253437
expression and cellular localization of inducible nitric oxide synthase in lipopolysaccharide-treated rat kidneys. 201222260992
multistep assembly of chloroplast nadh dehydrogenase-like subcomplex a requires several nucleus-encoded proteins, including crr41 and crr42, in arabidopsis.chloroplast nadh dehydrogenase-like complex (ndh) mediates photosystem i cyclic electron transport and chlororespiration in thylakoids. recently, substantial progress has been made in understanding the structure of ndh, but our knowledge of its assembly has been limited. in this study, a series of interactive proteomic analyses identified several stroma-localized factors required for the assembly of a stroma-protruding arm of ndh (subcomplex a). in addition to further characterization of the pre ...201222274627
thermodynamic basis of selectivity in guide-target-mismatched rna interference.silencing in rnai is strongly affected by guide-strand/target-mrna mismatches. target nucleation is thought to occur at positions 2-8 of the guide ("seed region"); successful hybridization in this region is the primary determinant of target-binding affinity and hence target cleavage. to define a molecular basis for the target sequence selectivity in rnai, we studied all possible distinct single mismatches in seven positions of the seed region-a total of 21 substitutions. we report results from s ...201222275138
crystal structure of urea carboxylase provides insights into the carboxyltransfer reaction.urea carboxylase (uc) is conserved in many bacteria, algae, and fungi and catalyzes the conversion of urea to allophanate, an essential step in the utilization of urea as a nitrogen source in these organisms. uc belongs to the biotin-dependent carboxylase superfamily and shares the biotin carboxylase (bc) and biotin carboxyl carrier protein (bccp) domains with these other enzymes, but its carboxyltransferase (ct) domain is distinct. currently, there is no information on the molecular basis of ca ...201222277658
rationalization and prediction of selective decoding of pseudouridine-modified nonsense and sense codons.a stop or nonsense codon is an in-frame triplet within a messenger rna that signals the termination of translation. one common feature shared among all three nonsense codons (uaa, uag, and uga) is a uridine present at the first codon position. it has been recently shown that the conversion of this uridine into pseudouridine (ψ) suppresses translation termination, both in vitro and in vivo. furthermore, decoding of the pseudouridylated nonsense codons is accompanied by the incorporation of two sp ...201222282339
argonaute and the nuclear rnas: new pathways for rna-mediated control of gene expression.small rnas are a commonly used tool for gene silencing and a promising platform for nucleic acid drug development. they are almost exclusively used to silence gene expression post-transcriptionally through degradation of mrna. small rnas, however, can have a broader range of function by binding to argonaute proteins and associating with complementary rna targets in the nucleus, including long noncoding rnas (lncrnas) and pre-mrna. argonaute-rna complexes can regulate nuclear events like transcri ...201222283730
trna-controlled nuclear import of a human trna synthetase.aminoacyl-trna synthetases, essential components of the cytoplasmic translation apparatus, also have nuclear functions that continue to be elucidated. however, little is known about how the distribution between cytoplasmic and nuclear compartments is controlled. using a combination of methods, here we showed that human tyrosyl-trna synthetase (tyrrs) distributes to the nucleus and that the nuclear import of human tyrrs is regulated by its cognate trna(tyr). we identified a hexapeptide motif in t ...201222291016
the structural basis for substrate versatility of chloramphenicol acetyltransferase cati.novel antibiotics are needed to overcome the challenge of continually evolving bacterial resistance. this has led to a renewed interest in mechanistic studies of once popular antibiotics like chloramphenicol (cam). chloramphenicol acetyltransferases (cats) are enzymes that covalently modify cam, rendering it inactive against its target, the ribosome, and thereby causing resistance to cam. of the three major types of cat (cat(i-iii)), the cam-specific cat(iii) has been studied extensively. much l ...201222294317
mathematical modeling and comparison of protein size distribution in different plant, animal, fungal and microbial species reveals a negative correlation between protein size and protein number, thus providing insight into the evolution of proteomes.the sizes of proteins are relevant to their biochemical structure and for their biological function. the statistical distribution of protein lengths across a diverse set of taxa can provide hints about the evolution of proteomes.201222296664
fidelity of trna 5'-maturation: a possible basis for the functional dependence of archaeal and eukaryal rnase p on multiple protein cofactors.rnase p, which catalyzes trna 5'-maturation, typically comprises a catalytic rnase p rna (rpr) and a varying number of rnase p proteins (rpps): 1 in bacteria, at least 4 in archaea and 9 in eukarya. the four archaeal rpps have eukaryotic homologs and function as heterodimers (pop5•rpp30 and rpp21•rpp29). by studying the archaeal methanocaldococcus jannaschii rpr's cis cleavage of precursor trna(gln) (pre-trna(gln)), which lacks certain consensus structures/sequences needed for substrate recognit ...201222298511
a deeply branching thermophilic bacterium with an ancient acetyl-coa pathway dominates a subsurface ecosystem.a nearly complete genome sequence of candidatus 'acetothermum autotrophicum', a presently uncultivated bacterium in candidate division op1, was revealed by metagenomic analysis of a subsurface thermophilic microbial mat community. phylogenetic analysis based on the concatenated sequences of proteins common among 367 prokaryotes suggests that ca. 'a. autotrophicum' is one of the earliest diverging bacterial lineages. it possesses a folate-dependent wood-ljungdahl (acetyl-coa) pathway of co(2) fix ...201222303444
structural, bioinformatic, and in vivo analyses of two treponema pallidum lipoproteins reveal a unique trap transporter.treponema pallidum, the bacterial agent of syphilis, is predicted to encode one tripartite atp-independent periplasmic transporter (trap-t). trap-ts typically employ a periplasmic substrate-binding protein (sbp) to deliver the cognate ligand to the transmembrane symporter. herein, we demonstrate that the genes encoding the putative trap-t components from t. pallidum, tp0957 (the sbp), and tp0958 (the symporter), are in an operon with an uncharacterized third gene, tp0956. we determined the cryst ...201222306465
aggregate reactivation mediated by the hsp100 chaperones.hsp100 family of molecular chaperones shows a unique capability to resolubilize and reactivate aggregated proteins. the hsp100-mediated protein disaggregation is linked to the activity of other chaperones from the hsp70 and hsp40 families. the best-studied members of the hsp100 family are the bacterial clpb and hsp104 from yeast. hsp100 chaperones are members of a large super-family of energy-driven conformational "machines" known as aaa+ atpases. this review describes the current mechanistic mo ...201222306514
selenocysteine insertion sequence (secis)-binding protein 2 alters conformational dynamics of residues involved in trna accommodation in 80 s ribosomes.sec-trna(sec) is site-specifically delivered at defined uga codons in selenoprotein mrnas. this recoding event is specified by the selenocysteine insertion sequence (secis) element and requires the selenocysteine (sec)-specific elongation factor, eefsec, and the secis binding protein, sbp2. sec-trna(sec) is delivered to the ribosome by eefsec-gtp, but this ternary complex is not sufficient for sec incorporation, indicating that its access to the ribosomal a-site is regulated. sbp2 stably associa ...201222308032
ribosome clearance by fusb-type proteins mediates resistance to the antibiotic fusidic acid.resistance to the antibiotic fusidic acid (fa) in the human pathogen staphylococcus aureus usually results from expression of fusb-type proteins (fusb or fusc). these proteins bind to elongation factor g (ef-g), the target of fa, and rescue translation from fa-mediated inhibition by an unknown mechanism. here we show that the fusb family are two-domain metalloproteins, the c-terminal domain of which contains a four-cysteine zinc finger with a unique structural fold. this domain mediates a high-a ...201222308410
biocatalyst development by directed evolution.biocatalysis has emerged as a great addition to traditional chemical processes for production of bulk chemicals and pharmaceuticals. to overcome the limitations of naturally occurring enzymes, directed evolution has become the most important tool for improving critical traits of biocatalysts such as thermostability, activity, selectivity, and tolerance towards organic solvents for industrial applications. recent advances in mutant library creation and high-throughput screening have greatly facil ...201222310212
the 2'-o-methylation status of a single guanosine controls transfer rna-mediated toll-like receptor 7 activation or inhibition.foreign rna serves as pathogen-associated molecular pattern (pamp) and is a potent immune stimulator for innate immune receptors. however, the role of single bacterial rna species in immune activation has not been characterized in detail. we analyzed the immunostimulatory potential of transfer rna (trna) from different bacteria. interestingly, bacterial trna induced type i interferon (ifn) and inflammatory cytokines in mouse dendritic cells (dcs) and human peripheral blood mononuclear cells (pbm ...201222312111
the bridge helix of rna polymerase acts as a central nanomechanical switchboard for coordinating catalysis and substrate movement.the availability of in vitro assembly systems to produce recombinant archaeal rna polymerases (rnaps) offers one of the most powerful experimental tools for investigating the still relatively poorly understood molecular mechanisms underlying rnap function. over the last few years, we pioneered new robot-based high-throughput mutagenesis approaches to study structure/function relationships within various domains surrounding the catalytic center. the bridge helix domain, which appears in numerous ...201122312317
the bridge helix of rna polymerase acts as a central nanomechanical switchboard for coordinating catalysis and substrate movement.the availability of in vitro assembly systems to produce recombinant archaeal rna polymerases (rnaps) offers one of the most powerful experimental tools for investigating the still relatively poorly understood molecular mechanisms underlying rnap function. over the last few years, we pioneered new robot-based high-throughput mutagenesis approaches to study structure/function relationships within various domains surrounding the catalytic center. the bridge helix domain, which appears in numerous ...201122312317
in silico analysis of a therapeutic target in leishmania infantum: the guanosine-diphospho-d-mannose pyrophosphorylase.leishmaniases are tropical and sub-tropical diseases for which classical drugs (i.e. antimonials) exhibit toxicity and drug resistance. such a situation requires to find new chemical series with antileishmanial activity. this work consists in analyzing the structure of a validated target in leishmania: the gdp-mannose pyrophosphorylase (gdp-mp), an enzyme involved in glycosylation and essential for amastigote survival. by comparing both human and l. infantum gdp-mp 3d homology models, we identif ...201222314241
molecular evolution of hydrogen peroxide degrading enzymes.for efficient removal of intra- and/or extracellular hydrogen peroxide by dismutation to harmless dioxygen and water (2h(2)o(2) → o(2) + 2h(2)o), nature designed three metalloenzyme families that differ in oligomeric organization, monomer architecture as well as active site geometry and catalytic residues. here we report on the updated reconstruction of the molecular phylogeny of these three gene families. ubiquitous typical (monofunctional) heme catalases are found in all domains of life showin ...201222330759
structural aspects of phenylalanylation and quality control in three major forms of phenylalanyl-trna synthetase.aminoacyl-trna synthetases (aarss) are a canonical set of enzymes that specifically attach corresponding amino acids to their cognate transfer rnas in the cytoplasm, mitochondria, and nucleus. the aarss display great differences in primary sequence, subunit size, and quaternary structure. existence of three types of phenylalanyl-trna synthetase (phers)-bacterial (αβ)(2), eukaryotic/archaeal cytosolic (αβ)(2), and mitochondrial α-is a prominent example of structural diversity within the aarss fam ...201022331999
the tyrosyl-trna synthetase like gene located in the tyramine biosynthesis cluster of enterococcus durans is transcriptionally regulated by tyrosine concentration and extracellular ph.the tyramine producer enterococcus durans ipla655 contains all the necessary genes for tyramine biosynthesis, grouped in the tdc cluster. this cluster includes tyrs, an aminoacyl-trna synthetase like gene.201222333391
heme-copper terminal oxidase using both cytochrome c and ubiquinol as electron donors.the cytochrome c oxidase cox2 has been purified from native membranes of the hyperthermophilic eubacterium aquifex aeolicus. it is a cytochrome ba(3) oxidase belonging to the family b of the heme-copper containing terminal oxidases. it consists of three subunits, subunit i (coxa2, 63.9 kda), subunit ii (coxb2, 16.8 kda), and an additional subunit iia of 5.2 kda. surprisingly it is able to oxidize both reduced cytochrome c and ubiquinol in a cyanide sensitive manner. cox2 is part of a respiratory ...201222334648
domain iii of the t. thermophilus 23s rrna folds independently to a near-native state.the three-dimensional structure of the ribosomal large subunit (lsu) reveals a single morphological element, although the 23s rrna is contained in six secondary structure domains. based upon maps of inter- and intra-domain interactions and proposed evolutionary pathways of development, we hypothesize that domain iii is a truly independent structural domain of the lsu. domain iii is primarily stabilized by intra-domain interactions, negligibly perturbed by inter-domain interactions, and is not pe ...201222334759
discovery and analysis of cofactor-dependent phosphoglycerate mutase homologs as novel phosphoserine phosphatases in hydrogenobacter thermophilus.phosphoserine phosphatase (psp) catalyzes the dephosphorylation of phosphoserine to serine and inorganic phosphate. psps, which have been found in all three domains of life, belong to the haloacid dehalogenase-like hydrolase superfamily. however, certain organisms, particularly bacteria, lack a classical psp gene, although they appear to possess a functional phosphoserine synthetic pathway. the apparent lack of a psp ortholog in hydrogenobacter thermophilus, an obligately chemolithoautotrophic a ...201222337887
regulation of expression and catalytic activity of escherichia coli rsmg methyltransferase.rsmg is an adomet-dependent methyltransferase responsible for the synthesis of m(7)g527 in the 530 loop of bacterial 16s rrna. this loop is universally conserved, plays a key role in ribosomal accuracy, and is a target for streptomycin binding. loss of the m(7)g527 modification confers low-level streptomycin resistance and may affect ribosomal functioning. here, we explore the mechanisms controlling rsmg expression and activity, which may somehow respond to the demand set by the amount of rrna. ...201222337945
the open reading frame ttc1157 of thermus thermophilus hb27 encodes the methyltransferase forming n²-methylguanosine at position 6 in trna.n(2)-methylguanosine (m(2)g) is found at position 6 in the acceptor stem of thermus thermophilus trna(phe). in this article, we describe the cloning, expression, and characterization of the t. thermophilus hb27 methyltransferase (mtase) encoded by the ttc1157 open reading frame that catalyzes the formation of this modified nucleoside. s-adenosyl-l-methionine is used as donor of the methyl group. the enzyme behaves as a monomer in solution. it contains an n-terminal thump domain predicted to bind ...201222337946
thermus thermophilus nucleoside phosphorylases active in the synthesis of nucleoside analogues.cells extracts from thermus thermophilus hb27 express phosphorolytic activities on purines and pyrimidine nucleosides. five putative encoding genes were cloned and expressed in escherichia coli, and the corresponding recombinant proteins were purified and studied. two of these showed phosphorolytic activities against purine nucleosides, and third one showed phosphorolytic activity against pyrimidine nucleosides in vitro, and the three were named ttpnpi, ttpnpii, and ttpynp, respectively. the opt ...201222344645
molecular determinants of the cofactor specificity of ribitol dehydrogenase, a short-chain dehydrogenase/reductase.ribitol dehydrogenase from zymomonas mobilis (zmrdh) catalyzes the conversion of ribitol to d-ribulose and concomitantly reduces nad(p)(+) to nad(p)h. a systematic approach involving an initial sequence alignment-based residue screening, followed by a homology model-based screening and site-directed mutagenesis of the screened residues, was used to study the molecular determinants of the cofactor specificity of zmrdh. a homologous conserved amino acid, ser156, in the substrate-binding pocket of ...201222344653
recycling of methylthioadenosine is essential for normal vascular development and reproduction in arabidopsis.5'-methylthioadenosine (mta) is the common by-product of polyamine (pa), nicotianamine (na), and ethylene biosynthesis in arabidopsis (arabidopsis thaliana). the methylthiol moiety of mta is salvaged by 5'-methylthioadenosine nucleosidase (mtn) in a reaction producing methylthioribose (mtr) and adenine. the mtn double mutant, mtn1-1mtn2-1, retains approximately 14% of the mtn enzyme activity present in the wild type and displays a pleiotropic phenotype that includes altered vasculature and impai ...201222345506
distribution of hepatitis c virus genotypes in canada: results from the lcdc sentinel health unit surveillance system.in a sentinel hepatitis surveillance study conducted by sentinel health units, 1469 patients were enrolled, and 959 (65.3%) were positive for antibody to hepatitis c virus (hcv). samples from 387 patients (40.4%) were tested for hcv rna, and 289 (74.7%) were positive for rna. the major risk factor for hcv infection was injection drug use, reported in 71% of cases. the genotyping of hcv isolates showed that subtype 1a (48%) was predominant in canada. the other subtypes detected were 1b (19%), 2a ...199922346372
recent advances in laboratory diagnosis of hepatitis c virus infection. 199422346511
structural basis of the substrate specificity of bacillus cereus adenosine phosphorylase.purine nucleoside phosphorylases catalyze the phosphorolytic cleavage of the glycosidic bond of purine (2'-deoxy)nucleosides, generating the corresponding free base and (2'-deoxy)-ribose 1-phosphate. two classes of pnps have been identified: homotrimers specific for 6-oxopurines and homohexamers that accept both 6-oxopurines and 6-aminopurines. bacillus cereus adenosine phosphorylase (adop) is a hexameric pnp; however, it is highly specific for 6-aminopurines. to investigate the structural basis ...201222349225
the dynamic stator stalk of rotary atpases.rotary atpases couple atp hydrolysis/synthesis with proton translocation across biological membranes and so are central components of the biological energy conversion machinery. their peripheral stalks are essential components that counteract torque generated by rotation of the central stalk during atp synthesis or hydrolysis. here we present a 2.25-å resolution crystal structure of the peripheral stalk from thermus thermophilus a-type atpase/synthase. we identify bending and twisting motions in ...201222353718
target- and resistance-based mechanistic studies with tp-434, a novel fluorocycline antibiotic.tp-434 is a novel, broad-spectrum fluorocycline antibiotic with activity against bacteria expressing major antibiotic resistance mechanisms, including tetracycline-specific efflux and ribosomal protection. the mechanism of action of tp-434 was assessed using both cell-based and in vitro assays. in escherichia coli cells expressing recombinant tetracycline resistance genes, the mic of tp-434 (0.063 μg/ml) was unaffected by tet(m), tet(k), and tet(b) and increased to 0.25 and 4 μg/ml in the presen ...201222354310
a genome-wide view of the expression and processing patterns of thermus thermophilus hb8 crispr rnas.the crispr-cas system represents an rna-based adaptive immune response system in prokaryotes and archaea. crisprs (clustered regularly interspaced short palindromic repeats) consist of arrays of short repeat-sequences interspaced by nonrepetitive short spacers, some of which show sequence similarity to foreign phage genetic elements. their cistronic transcripts are processed to produce the mature crispr rnas (crrnas), the elements that confer immunity by base-pairing with exogenous nucleic acids ...201222355165
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
cytosolic ni(ii) sensor in cyanobacterium: nickel detection follows nickel affinity across four families of metal sensors.efflux of surplus ni(ii) across the outer and inner membranes of synechocystis pcc 6803 is mediated by the nrs system under the control of a sensor of periplasmic ni(ii), nrss. here, we show that the product of orf sll0176, which encodes a csor/rcnr-like protein now designated inrs (for internal nickel-responsive sensor), represses nrsd (nrsd is deduced to efflux ni(ii) across the inner membrane) from a cryptic promoter between the final two orfs in the nrs operon. transcripts initiated from the ...201222356910
closely related archaeal haloarcula hispanica icosahedral viruses hhiv-2 and sh1 have nonhomologous genes encoding host recognition functions.studies on viral capsid architectures and coat protein folds have revealed the evolutionary lineages of viruses branching to all three domains of life. a widespread group of icosahedral tailless viruses, the prd1-adenovirus lineage, was the first to be established. a double β-barrel fold for a single major capsid protein is characteristic of these viruses. similar viruses carrying genes coding for two major capsid proteins with a more complex structure, such as thermus phage p23-77 and haloarcha ...201222357274
accommodation of profound sequence differences at the interfaces of eubacterial rna polymerase multi-protein assembly.evolutionarily divergent proteins have been shown to change their interacting partners. rna polymerase assembly is one of the rare cases which retain its component proteins in the course of evolution. this ubiquitous molecular assembly, involved in transcription, consists of four core subunits (alpha, beta, betaprime, and omega), which assemble to form the core enzyme. remarkably, the orientation of the four subunits in the complex is conserved from prokaryotes to eukaryotes although their seque ...201222359428
4,6-α-glucanotransferase activity occurs more widespread in lactobacillus strains and constitutes a separate gh70 subfamily.family 70 glycoside hydrolase glucansucrase enzymes exclusively occur in lactic acid bacteria and synthesize a wide range of α-d-glucan (abbreviated as α-glucan) oligo- and polysaccharides. of the 47 characterized gh70 enzymes, 46 use sucrose as glucose donor. a single gh70 enzyme was recently found to be inactive with sucrose and to utilize maltooligosaccharides [(1→4)-α-d-glucooligosaccharides] as glucose donor substrates for α-glucan synthesis, acting as a 4,6-α-glucanotransferase (4,6-αgt) e ...201222361861
4,6-α-glucanotransferase activity occurs more widespread in lactobacillus strains and constitutes a separate gh70 subfamily.family 70 glycoside hydrolase glucansucrase enzymes exclusively occur in lactic acid bacteria and synthesize a wide range of α-d-glucan (abbreviated as α-glucan) oligo- and polysaccharides. of the 47 characterized gh70 enzymes, 46 use sucrose as glucose donor. a single gh70 enzyme was recently found to be inactive with sucrose and to utilize maltooligosaccharides [(1→4)-α-d-glucooligosaccharides] as glucose donor substrates for α-glucan synthesis, acting as a 4,6-α-glucanotransferase (4,6-αgt) e ...201222361861
investigating the entire course of telithromycin binding to escherichia coli ribosomes.applying kinetics and footprinting analysis, we show that telithromycin, a ketolide antibiotic, binds to escherichia coli ribosomes in a two-step process. during the first, rapidly equilibrated step, telithromycin binds to a low-affinity site (k(t) = 500 nm), in which the lactone ring is positioned at the upper portion of the peptide exit tunnel, while the alkyl-aryl side chain of the drug inserts a groove formed by nucleotides a789 and u790 of 23s rrna. during the second step, telithromycin shi ...201222362747
crystal structures of the trna:m2g6 methyltransferase trm14/trmn from two domains of life.methyltransferases (mtases) form a major class of trna-modifying enzymes needed for the proper functioning of trna. recently, rna mtases from the trmn/trm14 family that are present in archaea, bacteria and eukaryota have been shown to specifically modify trna(phe) at guanosine 6 in the trna acceptor stem. here, we report the first x-ray crystal structures of the trna m(2)g6 (n(2)-methylguanosine) mtase (ttc)trmn from thermus thermophilus and its ortholog (pf)trm14 from pyrococcus furiosus. struc ...201222362751
the asparagine-transamidosome from helicobacter pylori: a dual-kinetic mode in non-discriminating aspartyl-trna synthetase safeguards the genetic code.helicobacter pylori catalyzes asn-trna(asn) formation by use of the indirect pathway that involves charging of asp onto trna(asn) by a non-discriminating aspartyl-trna synthetase (nd-asprs), followed by conversion of the mischarged asp into asn by the gatcab amidotransferase. we show that the partners of asparaginylation assemble into a dynamic asn-transamidosome, which uses a different strategy than the gln-transamidosome to prevent the release of the mischarged aminoacyl-trna intermediate. the ...201222362756
new insight into the transcarbamylase family: the structure of putrescine transcarbamylase, a key catalyst for fermentative utilization of agmatine.transcarbamylases reversibly transfer a carbamyl group from carbamylphosphate (cp) to an amine. although aspartate transcarbamylase and ornithine transcarbamylase (otc) are well characterized, little was known about putrescine transcarbamylase (ptc), the enzyme that generates cp for atp production in the fermentative catabolism of agmatine. we demonstrate that ptc (from enterococcus faecalis), in addition to using putrescine, can utilize l-ornithine as a poor substrate. crystal structures at 2.5 ...201222363663
subunit interactions at the v1-vo interface in yeast vacuolar atpase.eukaryotic vacuolar atpase (v-atpase) is regulated by a reversible dissociation mechanism that involves breaking and reforming of protein-protein interactions at the interface of the v(1)-atpase and v(o)-proton channel domains. we found previously that the head domain of the single copy c subunit (c(head)) binds one subunit eg heterodimer with high affinity (oot, r.a. and wilkens, s. (2010) j. biol. chem. 285, 24654-24664). here we generated a water-soluble construct of the n-terminal domain of ...201222367203
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
cross-talk between phosphorylation and lysine acetylation in a genome-reduced bacterium.protein post-translational modifications (ptms) represent important regulatory states that when combined have been hypothesized to act as molecular codes and to generate a functional diversity beyond genome and transcriptome. we systematically investigate the interplay of protein phosphorylation with other post-transcriptional regulatory mechanisms in the genome-reduced bacterium mycoplasma pneumoniae. systematic perturbations by deletion of its only two protein kinases and its unique protein ph ...201222373819
genetic recombination in bacillus subtilis: a division of labor between two single-strand dna-binding proteins.we have investigated the structural, biochemical and cellular roles of the two single-stranded (ss) dna-binding proteins from bacillus subtilis, ssba and ssbb. during transformation, ssbb localizes at the dna entry pole where it binds and protects internalized ssdna. the 2.8-å resolution structure of ssbb bound to ssdna reveals a similar overall protein architecture and ssdna-binding surface to that of escherichia coli ssb. ssba, which binds ssdna with higher affinity than ssbb, co-assembles ont ...201222373918
acyl carrier protein structural classification and normal mode analysis.all acyl carrier protein primary and tertiary structures were gathered into the thyme database. they are classified into 16 families by amino acid sequence similarity, with members of the different families having sequences with statistically highly significant differences. these classifications are supported by tertiary structure superposition analysis. tertiary structures from a number of families are very similar, suggesting that these families may come from a single distant ancestor. normal ...201222374859
mapping hidden potential identity elements by computing the average discriminating power of individual trna positions.the recently published discrete mathematical method, extended consensus partition (ecp), identifies nucleotide types at each position that are strictly absent from a given sequence set, while occur in other sets. these are defined as discriminating elements (des). in this study using the ecp approach, we mapped potential hidden identity elements that discriminate the 20 different trna identities. we filtered the tdna data set for the obligatory presence of well-established trna features, and the ...201222378766
interpreting protein structural dynamics from nmr chemical shifts.in this investigation, semiempirical nmr chemical shift prediction methods are used to evaluate the dynamically averaged values of backbone chemical shifts obtained from unbiased molecular dynamics (md) simulations of proteins. md-averaged chemical shift predictions generally improve agreement with experimental values when compared to predictions made from static x-ray structures. improved chemical shift predictions result from population-weighted sampling of multiple conformational states and f ...201222381384
structural insights into initial and intermediate steps of the ribosome-recycling process.the ribosome-recycling factor (rrf) and elongation factor-g (ef-g) disassemble the 70s post-termination complex (potc) into mrna, trna, and two ribosomal subunits. we have determined cryo-electron microscopic structures of the potc·rrf complex, with and without ef-g. we find that domain ii of rrf initially interacts with universally conserved residues of the 23s rrna helices 43 and 95, and protein l11 within the 50s ribosomal subunit. upon ef-g binding, both rrf and trna are driven towards the t ...201222388519
activity, stability, and structure of metagenome-derived lc11-rnase h1, a homolog of sulfolobus tokodaii rnase h1.metagenome-derived lc11-rnase h1 is a homolog of sulfolobus tokodaii rnase h1 (sto-rnase h1). it lacks a c-terminal tail, which is responsible for hyperstabilization of sto-rnase h1. sto-rnase h1 is characterized by its ability to cleave not only an rna/dna hybrid but also a double-stranded rna (dsrna). to examine whether lc11-rnase h1 also exhibits both rnase h and dsrnase activities, lc11-rnase h1 was overproduced in escherichia coli, purified, and characterized. lc11-rnase h1 exhibited rnase ...201222389131
the variable subdomain of escherichia coli seca functions to regulate seca atpase activity and adp release.bacterial seca proteins can be categorized by the presence or absence of a variable subdomain (var) located within nucleotide-binding domain ii of the seca dead motor. here we show that var is dispensable for seca function, since the var deletion mutant secaδ519-547 displayed a wild-type rate of cellular growth and protein export. loss or gain of var is extremely rare in the history of bacterial evolution, indicating that it appears to contribute to seca function within the relevant species in t ...201222389482
2011 william allan award: development and evolution. 201222405084
structural and enzymatic characterization of bacd, an l-amino acid dipeptide ligase from bacillus subtilis.bacd is an atp-dependent dipeptide ligase responsible for the biosynthesis of l-alanyl-l-anticapsin, a precursor of an antibiotic produced by bacillus spp. in contrast to the well-studied and phylogenetically related d-alanine: d-alanine ligase (ddl), bacd synthesizes dipeptides using l-amino acids as substrates and has a low substrate specificity in vitro. the enzyme is of great interest because of its potential application in industrial protein engineering for the environmentally friendly biol ...201222407814
pseudoscorpion mitochondria show rearranged genes and genome-wide reductions of rna gene sizes and inferred structures, yet typical nucleotide composition bias.pseudoscorpions are chelicerates and have historically been viewed as being most closely related to solifuges, harvestmen, and scorpions. no mitochondrial genomes of pseudoscorpions have been published, but the mitochondrial genomes of some lineages of chelicerata possess unusual features, including short rrna genes and trna genes that lack sequence to encode arms of the canonical cloverleaf-shaped trna. additionally, some chelicerates possess an atypical guanine-thymine nucleotide bias on the m ...201222409411
the bacterial sec-translocase: structure and mechanism.most bacterial secretory proteins pass across the cytoplasmic membrane via the translocase, which consists of a protein-conducting channel secyeg and an atp-dependent motor protein seca. the ancillary secdf membrane protein complex promotes the final stages of translocation. recent years have seen a major advance in our understanding of the structural and biochemical basis of protein translocation, and this has led to a detailed model of the translocation mechanism.201222411975
response to klyuyev and vassylyev: on the mechanism of tagetitoxin inhibition of transcription.in their commentary, klyuyev and vassylyev dispute a model of transcription inhibition by tagetitoxin (tgt) proposed by us based on biochemical analysis and computational docking. we maintain that, although an alternative explanation can be provided for any single observation reported by us, taken together our results support a model in which tgt acts by trapping the trigger loop (tl) in an inactive state (artsimovitch et al.). this model is consistent with all the data collected with a physiolo ...201222414748
the binding site and mechanism of the rna polymerase inhibitor tagetitoxin: an issue open to debate.in a recent paper in the journal of biological chemistry, artsimovitch et al. report a major revision of a crystallographic model and proposed mechanism of the rna polymerase inhibitor, tagetitoxin. this reassessment is based on theoretical modeling using molecular dynamics simulations. here, we argue that this theoretical model contradicts experimental results and a published crystal structure cannot exclude several mechanistically distinct alternative models and does not support some major con ...201222414754
decoding in the absence of a codon by tmrna and smpb in the ribosome.in bacteria, ribosomes stalled at the end of truncated messages are rescued by transfer-messenger rna (tmrna), a bifunctional molecule that acts as both a transfer rna (trna) and a messenger rna (mrna), and smpb, a small protein that works in concert with tmrna. here, we present the crystal structure of a tmrna fragment, smpb and elongation factor tu bound to the ribosome at 3.2 angstroms resolution. the structure shows how smpb plays the role of both the anticodon loop of trna and portions of m ...201222422985
structural basis for the rescue of stalled ribosomes: structure of yaej bound to the ribosome.in bacteria, the hybrid transfer-messenger rna (tmrna) rescues ribosomes stalled on defective messenger rnas (mrnas). however, certain gram-negative bacteria have evolved proteins that are capable of rescuing stalled ribosomes in a tmrna-independent manner. here, we report a 3.2 angstrom-resolution crystal structure of the rescue factor yaej bound to the thermus thermophilus 70s ribosome in complex with the initiator trna(i)(fmet) and a short mrna. the structure reveals that the c-terminal tail ...201222422986
exploring the proton pump and exit pathway for pumped protons in cytochrome ba3 from thermus thermophilus.the heme-copper oxygen reductases are redox-driven proton pumps. in the current work, the effects of mutations in a proposed exit pathway for pumped protons are examined in the ba(3)-type oxygen reductase from thermus thermophilus, leading from the propionates of heme a(3) to the interface between subunits i and ii. recent studies have proposed important roles for his376 and asp372, both of which are hydrogen-bonded to propionate-a of heme a(3), and for glu126(ii) (subunit ii), which is hydrogen ...201222431640
saccharomyces cerevisiae possesses a stress-inducible glycyl-trna synthetase gene.aminoacyl-trna synthetases are a large family of housekeeping enzymes that are pivotal in protein translation and other vital cellular processes. saccharomyces cerevisiae possesses two distinct nuclear glycyl-trna synthetase (glyrs) genes, grs1 and grs2. grs1 encodes both cytoplasmic and mitochondrial activities, while grs2 is essentially silent and dispensable under normal conditions. we herein present evidence that expression of grs2 was drastically induced upon heat shock, ethanol or hydrogen ...201222438917
structure and activity of the pseudomonas aeruginosa hotdog-fold thioesterases pa5202 and pa2801.the hotdog fold is one of the basic protein folds widely present in bacteria, archaea and eukaryotes. many of these proteins exhibit thioesterase activity against fatty acyl-coas and play important roles in lipid metabolism, cellular signalling and degradation of xenobiotics. the genome of the opportunistic pathogen pseudomonas aeruginosa contains over 20 genes encoding predicted hotdog-fold proteins, none of which have been experimentally characterized. we have found that two p. aeruginosa hotd ...201222439787
crystallization and preliminary x-ray crystallographic analysis of the catalytic domain of human dihydrouridine synthase.dihydrouridine synthases catalyse the reduction of uridine to dihydrouridine in the d-loop and variable loop of trna. the human dihydrouridine synthase hsdus2l has been implicated in the development of pulmonary carcinogenesis. here, the purification, crystallization and preliminary x-ray characterization of the hsdus2l catalytic domain are reported. the crystals belonged to space group p2(1) and contained a single molecule of hsdus2l in the asymmetric unit. a complete data set was collected to ...201222442237
cloning, purification, crystallization and preliminary x-ray analysis of the burkholderia pseudomallei l1 ribosomal protein.the gene encoding the l1 ribosomal protein from burkholderia pseudomallei strain d286 has been cloned into the petblue-1 vector system, overexpressed in escherichia coli and purified. crystals of the native protein were grown by the hanging-drop vapour-diffusion technique using peg 3350 as a precipitant and diffracted to beyond 1.65 å resolution. the crystals belonged to space group p2(1)2(1)2, with unit-cell parameters a = 53.6, b = 127.1, c = 31.8 å and with a single molecule in the asymmetric ...201222442241
production of l-ribose from l-ribulose by a triple-site variant of mannose-6-phosphate isomerase from geobacillus thermodenitrificans.a triple-site variant (w17q n90a l129f) of mannose-6-phosphate isomerase from geobacillus thermodenitrificans was obtained by combining variants with residue substitutions at different positions after random and site-directed mutagenesis. the specific activity and catalytic efficiency (k(cat)/k(m)) for l-ribulose isomerization of this variant were 3.1- and 7.1-fold higher, respectively, than those of the wild-type enzyme at ph 7.0 and 70°c in the presence of 1 mm co(2+). the triple-site variant ...201222447612
role of trehalose in salinity and temperature tolerance in the model halophilic bacterium chromohalobacter salexigens.the disaccharide trehalose is considered as a universal stress molecule, protecting cells and biomolecules from injuries imposed by high osmolarity, heat, oxidation, desiccation and freezing. chromohalobacter salexigens is a halophilic and extremely halotolerant γ-proteobacterium of the family halomonadaceae. in this work, we have investigated the role of trehalose as a protectant against salinity, temperature and desiccation in c. salexigens. a mutant deficient in the trehalose-6-phosphate synt ...201222448254
ultrafast excited-state deactivation of flavins bound to dodecin.dodecins, a group of flavin-binding proteins with a dodecameric quaternary structure, are able to incorporate two flavins within each of their six identical binding pockets building an aromatic tetrade with two tryptophan residues. dodecin from the archaeal halobacterium salinarum is a riboflavin storage device. we demonstrate that unwanted side reactions induced by reactive riboflavin species and degradation of riboflavin are avoided by ultrafast depopulation of the reactive excited state of ri ...201222451648
response to copper stress in streptomyces lividans extends beyond genes under direct control of a copper-sensitive operon repressor protein (csor).a copper-sensitive operon repressor protein (csor) has been identified in streptomyces lividans (csor(sl)) and found to regulate copper homeostasis with attomolar affinity for cu(i). solution studies reveal apo- and cu(i)-csor(sl) to be a tetramer assembly, and a 1.7-å resolution crystal structure of apo-csor(sl) reveals that a significant conformational change is necessary to enable cu(i) binding. in silico prediction of the csor regulon was confirmed in vitro (emsa) and in vivo (rna-seq), whic ...201222451651
folding without charges.surface charges of proteins have in several cases been found to function as "structural gatekeepers," which avoid unwanted interactions by negative design, for example, in the control of protein aggregation and binding. the question is then if side-chain charges, due to their desolvation penalties, play a corresponding role in protein folding by avoiding competing, misfolded traps? to find out, we removed all 32 side-chain charges from the 101-residue protein s6 from thermus thermophilus. the re ...201222454493
deep sequencing reveals minor protease resistance mutations in patients failing a protease inhibitor regimen.standard genotypic antiretroviral resistance testing, performed by bulk sequencing, does not readily detect variants that comprise <20% of the circulating hiv-1 rna population. nevertheless, it is valuable in selecting an antiretroviral regimen after antiretroviral failure. in patients with poor adherence, resistant variants may not reach this threshold. therefore, deep sequencing would be potentially valuable for detecting minority resistant variants. we compared bulk sequencing and deep sequen ...201222457522
substrate and enzyme functional groups contribute to translational quality control by bacterial prolyl-trna synthetase.aminoacyl-trna synthetases activate specific amino acid substrates and attach them via an ester linkage to cognate trna molecules. in addition to cognate proline, prolyl-trna synthetase (prors) can activate cysteine and alanine and misacylate trna(pro). editing of the misacylated aminoacyl-trna is required for error-free protein synthesis. an editing domain (ins) appended to bacterial prors selectively hydrolyzes ala-trna(pro), whereas cys-trna(pro) is cleared by a freestanding editing domain, y ...201222458656
the type ii secretion system: biogenesis, molecular architecture and mechanism.many gram-negative bacteria use the sophisticated type ii secretion system (t2ss) to translocate a wide range of proteins from the periplasm across the outer membrane. the inner-membrane platform of the t2ss is the nexus of the system and orchestrates the secretion process through its interactions with the periplasmic filamentous pseudopilus, the dodecameric outer-membrane complex and a cytoplasmic secretion atpase. here, recent structural and biochemical information is reviewed to describe our ...201222466878
posttranslational modification of cellular proteins by a ubiquitin-like protein in bacteria.posttranslational modification of proteins with ubiquitin and ubiquitin-like proteins plays important regulatory roles in eukaryotes. although a homologous conjugation system has recently been reported in archaea, there is no similar report in bacteria. this report describes the identification of a ubiquitin-like conjugation system in the bacterium thermus thermophilus. a series of in vivo analyses revealed that ttub, a bacterial ubiquitin-like protein that functions as a sulfur carrier in trna ...201222467871
solution nuclear magnetic resonance analyses of the anticodon arms of proteinogenic and nonproteinogenic trna(gly).although the fate of most trna molecules in the cell is aminoacylation and delivery to the ribosome, some trnas are destined to fulfill other functional roles. in addition to their central role in translation, trna molecules participate in processes such as regulation of gene expression, bacterial cell wall biosynthesis, viral replication, antibiotic biosynthesis, and suppression of alternative splicing. in bacteria, glycyl-trna molecules with anticodon sequences gcc and ucc exhibit multiple ext ...201222468768
chemoinformatic identification of novel inhibitors against mycobacterium tuberculosis l-aspartate α-decarboxylase.l-aspartate α-decarboxylase (adc) belongs to a class of pyruvoyl dependent enzymes and catalyzes the conversion of aspartate to β-alanine in the pantothenate pathway, which is critical for the growth of several micro-organisms, including mycobacterium tuberculosis (mtb). its presence only in micro-organisms, fungi and plants and its absence in animals, particularly human, make it a promising drug target. we have followed a chemoinformatics-based approach to identify potential drug-like inhibitor ...201222470451
genome sequence and transcriptome analysis of the radioresistant bacterium deinococcus gobiensis: insights into the extreme environmental adaptations.the desert is an excellent model for studying evolution under extreme environments. we present here the complete genome and ultraviolet (uv) radiation-induced transcriptome of deinococcus gobiensis i-0, which was isolated from the cold gobi desert and shows higher tolerance to gamma radiation and uv light than all other known microorganisms. nearly half of the genes in the genome encode proteins of unknown function, suggesting that the extreme resistance phenotype may be attributed to unknown ge ...201222470573
electron transfer in subunit nuoi (tyky) of escherichia coli nadh:quinone oxidoreductase (ndh-1).bacterial proton-translocating nadh:quinone oxidoreductase (ndh-1) consists of a peripheral and a membrane domain. the peripheral domain catalyzes the electron transfer from nadh to quinone through a chain of seven iron-sulfur (fe/s) clusters. subunit nuoi in the peripheral domain contains two [4fe-4s] clusters (n6a and n6b) and plays a role in bridging the electron transfer from cluster n5 to the terminal cluster n2. we constructed mutants for eight individual cys-coordinating fe/s clusters. wi ...201222474289
structural and mechanistic analysis of the membrane-embedded glycosyltransferase waaa required for lipopolysaccharide synthesis.waaa is a key enzyme in the biosynthesis of lps, a critical component of the outer envelope of gram-negative bacteria. embedded in the cytoplasmic face of the inner membrane, waaa catalyzes the transfer of 3-deoxy-d-manno-oct-2-ulosonic acid (kdo) to the lipid a precursor of lps. here we present crystal structures of the free and cmp-bound forms of waaa from aquifex aeolicus, an ancient gram-negative hyperthermophile. these structures reveal details of the cmp-binding site and implicate a unique ...201222474366
development of a shutterless continuous rotation method using an x-ray cmos detector for protein crystallography.a new shutterless continuous rotation method using an x-ray complementary metal-oxide semiconductor (cmos) detector has been developed for high-speed, precise data collection in protein crystallography. the principle of operation and the basic performance of the x-ray cmos detector (hamamatsu photonics kk c10158dk) have been shown to be appropriate to the shutterless continuous rotation method. the data quality of the continuous rotation method is comparable to that of the conventional oscillati ...200922477775
response of methicillin-resistant staphylococcus aureus to amicoumacin a.amicoumacin a exhibits strong antimicrobial activity against methicillin-resistant staphylococcus aureus (mrsa), hence we sought to uncover its mechanism of action. genome-wide transcriptome analysis of s. aureus col in response to amicoumacin a showed alteration in transcription of genes specifying several cellular processes including cell envelope turnover, cross-membrane transport, virulence, metabolism, and general stress response. the most highly induced gene was lrga, encoding an antiholin ...201222479511
structural and functional consequences of phosphate-arsenate substitutions in selected nucleotides: dna, rna, and atp.a recent finding of a bacterial strain (gfaj-1) that can rely on arsenic instead of phosphorus raised the questions of if and how arsenate can replace phosphate in biomolecules that are essential to sustain cell life. apart from questions related to chemical stability, there are those of the structural and functional consequences of phosphate-arsenate substitutions in vital nucleotides in gfaj1-like cells. in this study we selected three types of molecules (atp/adp as energy source and replicati ...201222480264
related bifunctional restriction endonuclease-methyltransferase triplets: tspdti, tth111ii/tthhb27i and tsoi with distinct specificities.we previously defined a family of restriction endonucleases (reases) from thermus sp., which share common biochemical and biophysical features, such as the fusion of both the nuclease and methyltransferase (mtase) activities in a single polypeptide, cleavage at a distance from the recognition site, large molecular size, modulation of activity by s-adenosylmethionine (sam), and incomplete cleavage of the substrate dna. members include related thermophilic reases with five distinct specificities: ...201222489904
life under multiple extreme conditions: diversity and physiology of the halophilic alkalithermophiles.around the world, there are numerous alkaline, hypersaline environments that are heated either geothermally or through intense solar radiation. it was once thought that such harsh environments were inhospitable and incapable of supporting a variety of life. however, numerous culture-dependent and -independent studies revealed the presence of an extensive diversity of aerobic and anaerobic bacteria and archaea that survive and grow under these multiple harsh conditions. this diversity includes th ...201222492435
rhodanese functions as sulfur supplier for key enzymes in sulfur energy metabolism.how microorganisms obtain energy is a challenging topic, and there have been numerous studies on the mechanisms involved. here, we focus on the energy substrate traffic in the hyperthermophilic bacterium aquifex aeolicus. this bacterium can use insoluble sulfur as an energy substrate and has an intricate sulfur energy metabolism involving several sulfur-reducing and -oxidizing supercomplexes and enzymes. we demonstrate that the cytoplasmic rhodanese sbdp participates in this sulfur energy metabo ...201222496367
para-like protein uses nonspecific chromosomal dna binding to partition protein complexes.recent data have shown that plasmid partitioning par-like systems are used by some bacterial cells to control localization of protein complexes. here we demonstrate that one of these homologs, ppfa, uses nonspecific chromosome binding to separate cytoplasmic clusters of chemotaxis proteins upon division. using fluorescent microscopy and point mutations, we show dynamic chromosome binding and walker-type atpase activity are essential for cluster segregation. the n-terminal domain of a cytoplasmic ...201222496588
genome signature difference between deinococcus radiodurans and thermus thermophilus.the extremely radioresistant bacteria of the genus deinococcus and the extremely thermophilic bacteria of the genus thermus belong to a common taxonomic group. considering the distinct living environments of deinococcus and thermus, different genes would have been acquired through horizontal gene transfer after their divergence from a common ancestor. their guanine-cytosine (gc) contents are similar; however, we hypothesized that their genomic signatures would be different. our findings indicate ...201222500246
structure of escherichia coli aspartate α-decarboxylase asn72ala: probing the role of asn72 in pyruvoyl cofactor formation.the crystal structure of the asn72ala site-directed mutant of escherichia coli aspartate α-decarboxylase (adc) has been determined at 1.7 å resolution. the refined structure is consistent with the presence of a hydrolysis product serine in the active site in place of the pyruvoyl group required for catalysis, which suggests that the role of asn72 is to protect the ester formed during adc activation from hydrolysis. in previously determined structures of activated adc, including the wild type and ...201222505409
substrate-induced change in the quaternary structure of type 2 isopentenyl diphosphate isomerase from sulfolobus shibatae.type 2 isopentenyl diphosphate isomerase catalyzes the interconversion between two active units for isoprenoid biosynthesis, i.e., isopentenyl diphosphate and dimethylallyl diphosphate, in almost all archaea and in some bacteria, including human pathogens. the enzyme is a good target for discovery of antibiotics because it is essential for the organisms that use only the mevalonate pathway to produce the active isoprene units and because humans possess a nonhomologous isozyme, type 1 isopentenyl ...201222505674
riboswitch (t-box)-mediated control of trna-dependent amidation in clostridium acetobutylicum rationalizes gene and pathway redundancy for asparagine and asparaginyl-trnaasn synthesis.analysis of the gram-positive clostridium acetobutylicum genome reveals an inexplicable level of redundancy for the genes putatively involved in asparagine (asn) and asn-trna(asn) synthesis. besides a duplicated set of gatcab trna-dependent amidotransferase genes, there is a triplication of aspartyl-trna synthetase genes and a duplication of asparagine synthetase b genes. this genomic landscape leads to the suspicion of the incoherent simultaneous use of the direct and indirect pathways of asn a ...201222505715
snapshot of virus evolution in hypersaline environments from the characterization of a membrane-containing salisaeta icosahedral phage 1.the multitude of archaea and bacteria inhabiting extreme environments has only become evident during the last decades. as viruses apply a significant evolutionary force to their hosts, there is an inherent value in learning about viruses infecting these extremophiles. in this study, we have focused on one such unique virus-host pair isolated from a hypersaline environment: an icosahedral, membrane-containing double-stranded dna virus--salisaeta icosahedral phage 1 (ssip-1) and its halophilic hos ...201222509017
bactquant: an enhanced broad-coverage bacterial quantitative real-time pcr assay.bacterial load quantification is a critical component of bacterial community analysis, but a culture-independent method capable of detecting and quantifying diverse bacteria is needed. based on our analysis of a diverse collection of 16 s rrna gene sequences, we designed a broad-coverage quantitative real-time pcr (qpcr) assay--bactquant--for quantifying 16 s rrna gene copy number and estimating bacterial load. we further utilized in silico evaluation to complement laboratory-based qpcr characte ...201222510143
dynamic, ligand-dependent conformational change triggers reaction of ribose-1,5-bisphosphate isomerase from thermococcus kodakarensis kod1.ribose-1,5-bisphosphate isomerase (r15pi) is a novel enzyme recently identified as a member of an amp metabolic pathway in archaea. the enzyme converts d-ribose 1,5-bisphosphate into ribulose 1,5-bisphosphate, providing the substrate for archaeal ribulose-1,5-bisphosphate carboxylase/oxygenases. we here report the crystal structures of r15pi from thermococcus kodakarensis kod1 (tk-r15pi) with and without its substrate or product. tk-r15pi is a hexameric enzyme formed by the trimerization of dime ...201222511789
the three-dimensional structural basis of type ii hyperprolinemia.type ii hyperprolinemia is an autosomal recessive disorder caused by a deficiency in δ(1)-pyrroline-5-carboxylate dehydrogenase (p5cdh; also known as aldh4a1), the aldehyde dehydrogenase that catalyzes the oxidation of glutamate semialdehyde to glutamate. here, we report the first structure of human p5cdh (hsp5cdh) and investigate the impact of the hyperprolinemia-associated mutation of ser352 to leu on the structure and catalytic properties of the enzyme. the 2. 5-å-resolution crystal structure ...201222516612
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