| conformational coupling, bridge helix dynamics and active site dehydration in catalysis by rna polymerase. | molecular dynamics simulation of thermus thermophilus (tt) rna polymerase (rnap) in a catalytic conformation demonstrates that the active site dnmp-ntp base pair must be substantially dehydrated to support full active site closing and optimum conditions for phosphodiester bond synthesis. in silico mutant beta r428a rnap, which was designed based on substitutions at the homologous position (rpb2 r512) of saccharomyces cerevisiae (sc) rnap ii, was used as a reference structure to compare to tt rna ... | 2010 | 20478425 |
| binding of a small molecule at a protein-protein interface regulates the chaperone activity of hsp70-hsp40. | heat shock protein 70 (hsp70) is a highly conserved molecular chaperone that plays multiple roles in protein homeostasis. in these various tasks, the activity of hsp70 is shaped by interactions with co-chaperones, such as hsp40. the hsp40 family of co-chaperones binds to hsp70 through a conserved j-domain, and these factors stimulate atpase and protein-folding activity. using chemical screens, we identified a compound, 115-7c, which acts as an artificial co-chaperone for hsp70. specifically, the ... | 2010 | 20481474 |
| thermal stability, ph dependence and inhibition of four murine kynurenine aminotransferases. | kynurenine aminotransferase (kat) catalyzes the transamination of kynunrenine to kynurenic acid (kyna). kyna is a neuroactive compound and functions as an antagonist of alpha7-nicotinic acetylcholine receptors and is the only known endogenous antagonist of n-methyl-d-aspartate receptors. four kat enzymes, kat i/glutamine transaminase k/cysteine conjugate beta-lyase 1, kat ii/aminoadipate aminotransferase, kat iii/cysteine conjugate beta-lyase 2, and kat iv/glutamic-oxaloacetic transaminase 2/mit ... | 2010 | 20482848 |
| insights into the hyperthermostability and unusual region-specificity of archaeal pyrococcus abyssi trna m1a57/58 methyltransferase. | the s-adenosyl-l-methionine dependent methylation of adenine 58 in the t-loop of trnas is essential for cell growth in yeast or for adaptation to high temperatures in thermophilic organisms. in contrast to bacterial and eukaryotic trna m(1)a58 methyltransferases that are site-specific, the homologous archaeal enzyme from pyrococcus abyssi catalyzes the formation of m(1)a also at the adjacent position 57, m(1)a57 being a precursor of 1-methylinosine. we report here the crystal structure of p. aby ... | 2010 | 20483913 |
| identification of residual structure in the unfolded state of ribonuclease h1 from the moderately thermophilic chlorobium tepidum: comparison with thermophilic and mesophilic homologues. | ribonucleases h from organisms that grow at different temperatures demonstrate a variable change in heat capacity upon unfolding (deltac degrees (p)) [ratcliff, k., et al. (2009) biochemistry 48, 5890-5898]. this deltac degrees (p) has been shown to correlate with a tolerance to higher temperatures and residual structure in the unfolded state of the thermophilic proteins. in the rnase h from thermus thermophilus, the low deltac degrees (p) has been shown to arise from the same region as the fold ... | 2010 | 20491485 |
| nmr investigations of the rieske protein from thermus thermophilus support a coupled proton and electron transfer mechanism. | the rieske protein component of the cytochrome bc complex contains a [2fe-2s] cluster ligated by two cysteines and two histidines. we report here the pk(a) values of each of the imidazole rings of the two ligating histidines (his134 and his154) in the oxidized and reduced states of the rieske protein from thermus thermophilus (ttrp) as determined by nmr spectroscopy. knowledge of these pk(a) values is of critical interest because of their pertinence to the mechanism of electron and proton transf ... | 2010 | 20496909 |
| rna polymerase mutations that facilitate replication progression in the rep uvrd recf mutant lacking two accessory replicative helicases. | we observed that cells lacking rep and uvrd, two replication accessory helicases, and the recombination protein recf are cryo-sensitive on rich medium. we isolated five mutations that suppress this luria-bertani (lb)-cryo-sensitivity and show that they map in the genes encoding the rna polymerase subunits rpob and rpoc. these rpob (d444g, h447r and n518d) and rpoc mutants (h113r and p451l) were characterized. rpob(h447r) and rpob(d444g) prevent activation of the prrn core promoter in rich medium ... | 2010 | 20497334 |
| the qrc membrane complex, related to the alternative complex iii, is a menaquinone reductase involved in sulfate respiration. | biological sulfate reduction is a process with high environmental significance due to its major contribution to the carbon and sulfur cycles in anaerobic environments. however, the respiratory chain of sulfate-reducing bacteria is still poorly understood. here we describe a new respiratory complex that was isolated as a major protein present in the membranes of desulfovibrio vulgaris hildenborough. the complex, which was named qrc, is the first representative of a new family of redox complexes. ... | 2010 | 20498375 |
| thioesterases: a new perspective based on their primary and tertiary structures. | thioesterases (tes) are classified into ec 3.1.2.1 through ec 3.1.2.27 based on their activities on different substrates, with many remaining unclassified (ec 3.1.2.-). analysis of primary and tertiary structures of known tes casts a new light on this enzyme group. we used strong primary sequence conservation based on experimentally proved proteins as the main criterion, followed by verification with tertiary structure superpositions, mechanisms, and catalytic residue positions, to accurately de ... | 2010 | 20506386 |
| molecular dynamics simulations suggest that rna three-way junctions can act as flexible rna structural elements in the ribosome. | we present extensive explicit solvent molecular dynamics analysis of three rna three-way junctions (3wjs) from the large ribosomal subunit: the 3wj formed by helices 90-92 (h90-h92) of 23s rrna; the 3wj formed by h42-h44 organizing the gtpase associated center (gac) of 23s rrna; and the 3wj of 5s rrna. h92 near the peptidyl transferase center binds the 3'-cca end of amino-acylated trna. the gac binds protein factors and stimulates gtp hydrolysis driving protein synthesis. the 5s rrna binds the c ... | 2010 | 20507916 |
| essential biological processes of an emerging pathogen: dna replication, transcription, and cell division in acinetobacter spp. | within the last 15 years, members of the bacterial genus acinetobacter have risen from relative obscurity to be among the most important sources of hospital-acquired infections. the driving force for this has been the remarkable ability of these organisms to acquire antibiotic resistance determinants, with some strains now showing resistance to every antibiotic in clinical use. there is an urgent need for new antibacterial compounds to combat the threat imposed by acinetobacter spp. and other in ... | 2010 | 20508250 |
| a cytochrome c fusion protein domain for convenient detection, quantification, and enhanced production of membrane proteins in escherichia coli--expression and characterization of cytochrome-tagged complex i subunits. | overproduction of membrane proteins can be a cumbersome task, particularly if high yields are desirable. nadh:quinone oxidoreductase (complex i) contains several very large membrane-spanning protein subunits that hitherto have been impossible to express individually in any appreciable amounts in escherichia coli. the polypeptides contain no prosthetic groups and are poorly antigenic, making optimization of protein production a challenging task. in this work, the c-terminal ends of the complex i ... | 2010 | 20509166 |
| transcriptional control by two leucine-responsive regulatory proteins in halobacterium salinarum r1. | archaea combine bacterial-as well as eukaryotic-like features to regulate cellular processes. halobacterium salinarum r1 encodes eight leucine-responsive regulatory protein (lrp)-homologues. the function of two of them, irp (oe3923f) and lrpa1 (oe2621r), were analyzed by gene deletion and overexpression, including genome scale impacts using microarrays. | 2010 | 20509863 |
| deletion of switch 3 results in an archaeal rna polymerase that is defective in transcript elongation. | switch 3 is a polypeptide loop conserved in all multisubunit dna-dependent rna polymerases (rnaps) that extends into the main cleft of the rnap and contacts each base in a nascent transcript as that base is released from the internal dna-rna hybrid. plasmids have been constructed and transformed into thermococcus kodakaraensis, which direct the constitutive synthesis of the archaeal rnap subunit rpob with an n-terminal his(6) tag and the switch 3 loop either intact (wild-type) or deleted (deltas ... | 2010 | 20511223 |
| remembering malcolm j. casadaban. | malcolm j. casadaban died on 13 september 2009 from an infection and was found to have a weakened strain of the bacterium yersinia pestis in his blood. this tragic event took the life of one of the most creative and influential geneticists of our time. in the late 1970s and '80s, malcolm invented novel approaches which changed the way many of us did science. jon beckwith, tom silhavy, and olaf schneewind have chronicled his scientific life from graduate school to his death and give us insight in ... | 2010 | 20511498 |
| free zinc ions outside a narrow concentration range are toxic to a variety of cells in vitro. | the zinc(ii) ion has recently been implicated in a number of novel functions and pathologies in loci as diverse as the brain, retina, small intestine, prostate, heart, pancreas, and immune system. zinc ions are a required nutrient but elevated concentrations are known to kill cells in vitro. paradoxical observations regarding zinc's effects have appeared frequently in the literature, and often their physiological relevance is unclear. we found that for pc-12, hela and ht-29 cell lines as well as ... | 2010 | 20511678 |
| cell surface display of chimeric glycoproteins via the s-layer of paenibacillus alvei. | the gram-positive, mesophilic bacterium paenibacillus alvei ccm 2051(t) possesses a two-dimensional crystalline protein surface layer (s-layer) with oblique lattice symmetry composed of a single type of o-glycoprotein species. herein, we describe a strategy for nanopatterned in vivo cell surface co-display of peptide and glycan epitopes based on this s-layer glycoprotein self-assembly system. the open reading frame of the corresponding structural gene spaa codes for a protein of 983 amino acids, ... | 2010 | 20513375 |
| the structure of phaz7 at atomic (1.2 a) resolution reveals details of the active site and suggests a substrate-binding mode. | poly-(r)-hydroxyalkanoates (phas) are bacterial polyesters that are degraded by a group of enzymes known as pha depolymerases. paucimonas lemoignei phaz7 depolymerase is the only extracellular depolymerase that has been described as being active towards amorphous phas. a previously determined crystal structure of phaz7 revealed an alpha/beta-hydrolase fold and a ser-his-asp catalytic triad. in order to address questions regarding the catalytic mechanism and substrate binding, the atomic resoluti ... | 2010 | 20516591 |
| crystallization and preliminary x-ray diffraction analysis of various enzyme-substrate complexes of isopropylmalate dehydrogenase from thermus thermophilus. | the thermus thermophilus 3-isopropylmalate dehydrogenase (tt-ipmdh) enzyme catalyses the penultimate step of the leucine-biosynthesis pathway. it converts (2r,3s)-3-isopropylmalate to (2s)-2-isopropyl-3-oxosuccinate in the presence of divalent mg(2+) or mn(2+) and with the help of nad(+). in order to elucidate the detailed structural and functional mode of the enzymatic reaction, crystals of tt-ipmdh were grown in the presence of various combinations of substrate and/or cofactors. here, the crys ... | 2010 | 20516614 |
| structural characterization of tartrate dehydrogenase: a versatile enzyme catalyzing multiple reactions. | the first structure of an nad-dependent tartrate dehydrogenase (tdh) has been solved to 2 a resolution by single anomalous diffraction (sad) phasing as a complex with the intermediate analog oxalate, mg(2+) and nadh. this tdh structure from pseudomonas putida has a similar overall fold and domain organization to other structurally characterized members of the hydroxy-acid dehydrogenase family. however, there are considerable differences between tdh and these functionally related enzymes in the r ... | 2010 | 20516620 |
| crystal structures of the apo and atp bound mycobacterium tuberculosis nitrogen regulatory pii protein. | pii constitutes a family of signal transduction proteins that act as nitrogen sensors in microorganisms and plants. mycobacterium tuberculosis (mtb) has a single homologue of pii whose precise role has as yet not been explored. we have solved the crystal structures of the mtb pii protein in its apo and atp bound forms to 1.4 and 2.4 a resolutions, respectively. the protein forms a trimeric assembly in the crystal lattice and folds similarly to the other pii family proteins. the mtb pii:atp binar ... | 2010 | 20521335 |
| three-dimensional structure of beta-cell-specific zinc transporter, znt-8, predicted from the type 2 diabetes-associated gene variant slc30a8 r325w. | abstract: | 2010 | 20525392 |
| redesign, reconstruction, and directed extension of the brevibacterium linens c40 carotenoid pathway in escherichia coli. | in this study, the carotenoid biosynthetic pathways of brevibacterium linens dsmz 20426 were reconstructed, redesigned, and extended with additional carotenoid-modifying enzymes of other sources in a heterologous host escherichia coli. the modular lycopene pathway synthesized an unexpected carotenoid structure, 3,4-didehydrolycopene, as well as lycopene. extension of the novel 3,4-didehydrolycopene pathway with the mutant pantoea lycopene cyclase crty(2) and the rhodobacter spheroidene monooxyge ... | 2010 | 20525861 |
| crystal structure of arginase from plasmodium falciparum and implications for l-arginine depletion in malarial infection . | the 2.15 a resolution crystal structure of arginase from plasmodium falciparum, the parasite that causes cerebral malaria, is reported in complex with the boronic acid inhibitor 2(s)-amino-6-boronohexanoic acid (abh) (k(d) = 11 microm). this is the first crystal structure of a parasitic arginase. various protein constructs were explored to identify an optimally active enzyme form for inhibition and structural studies and to probe the structure and function of two polypeptide insertions unique to ... | 2010 | 20527960 |
| domain characterization and interaction of the yeast vacuolar atpase subunit c with the peripheral stator stalk subunits e and g. | the proton pumping activity of the eukaryotic vacuolar atpase (v-atpase) is regulated by a unique mechanism that involves reversible enzyme dissociation. in yeast, under conditions of nutrient depletion, the soluble catalytic v(1) sector disengages from the membrane integral v(o), and at the same time, both functional units are silenced. notably, during enzyme dissociation, a single v(1) subunit, c, is released into the cytosol. the affinities of the other v(1) and v(o) subunits for subunit c ar ... | 2010 | 20529855 |
| central role of the rna polymerase trigger loop in intrinsic rna hydrolysis. | the active center of rna polymerase can hydrolyze phosphodiester bonds in nascent rna, a reaction thought to be important for proofreading of transcription. the reaction proceeds via a general two mg(2+) mechanism and is assisted by the 3' end nucleotide of the transcript. here, by using thermus aquaticus rna polymerase, we show that the reaction also requires the flexible domain of the active center, the trigger loop (tl). we show that the invariant histidine (beta' his1242) of the tl is essent ... | 2010 | 20534498 |
| organometallic mechanism of action and inhibition of the 4fe-4s isoprenoid biosynthesis protein gcpe (ispg). | we report the results of a series of chemical, epr, endor, and hyscore spectroscopic investigations of the mechanism of action (and inhibition) of gcpe, e-1-hydroxy-2-methyl-but-2-enyl-4-diphosphate (hmbpp) synthase, also known as ispg, an fe(4)s(4) cluster-containing protein. we find that the epoxide of hmbpp when reduced by gcpe generates the same transient epr species as observed on addition of the substrate, 2-c-methyl-d-erythritol-2, 4-cyclo-diphosphate. endor and hyscore spectra of these t ... | 2010 | 20534554 |
| redox status affects the catalytic activity of glutamyl-trna synthetase. | glutamyl-trna synthetases (glurs) provide glu-trna for different processes including protein synthesis, glutamine transamidation and tetrapyrrole biosynthesis. many organisms contain multiple glurss, but whether these duplications solely broaden trna specificity or also play additional roles in tetrapyrrole biosynthesis is not known. previous studies have shown that glurs1, one of two glurss from the extremophile acidithiobacillus ferrooxidans, is inactivated when intracellular heme is elevated ... | 2010 | 20541532 |
| discovery and characterization of hemq: an essential heme biosynthetic pathway component. | here we identify a previously undescribed protein, hemq, that is required for heme synthesis in gram-positive bacteria. we have characterized hemq from bacillus subtilis and a number of actinobacteria. hemq is a multimeric heme-binding protein. spectroscopic studies indicate that this heme is high spin ferric iron and is ligated by a conserved histidine with the sixth coordination site available for binding a small molecule. the presence of hemq along with the terminal two pathway enzymes, proto ... | 2010 | 20543190 |
| regulation of dynamic polarity switching in bacteria by a ras-like g-protein and its cognate gap. | the rod-shaped cells of the bacterium myxococcus xanthus move uni-directionally and occasionally undergo reversals during which the leading/lagging polarity axis is inverted. cellular reversals depend on pole-to-pole relocation of motility proteins that localize to the cell poles between reversals. we show that mgla is a ras-like g-protein and acts as a nucleotide-dependent molecular switch to regulate motility and that mglb represents a novel gtpase-activating protein (gap) family and is the co ... | 2010 | 20543819 |
| multi-site-specific 16s rrna methyltransferase rsmf from thermus thermophilus. | cells devote a significant effort toward the production of multiple modified nucleotides in rrnas, which fine tune the ribosome function. here, we report that two methyltransferases, rsmb and rsmf, are responsible for all four 5-methylcytidine (m(5)c) modifications in 16s rrna of thermus thermophilus. like escherichia coli rsmb, t. thermophilus rsmb produces m(5)c967. in contrast to e. coli rsmf, which introduces a single m(5)c1407 modification, t. thermophilus rsmf modifies three positions, gen ... | 2010 | 20558545 |
| the multifunctional pe_pgrs11 protein from mycobacterium tuberculosis plays a role in regulating resistance to oxidative stress. | mycobacterium tuberculosis utilizes unique strategies to survive amid the hostile environment of infected host cells. infection-specific expression of a unique mycobacterial cell surface antigen that could modulate key signaling cascades can act as a key survival strategy in curtailing host effector responses like oxidative stress. we demonstrate here that hypothetical pe_pgrs11 orf encodes a functional phosphoglycerate mutase. the transcriptional analysis revealed that pe_pgrs11 is a hypoxia-re ... | 2010 | 20558725 |
| type ii isopentenyl diphosphate isomerase: probing the mechanism with alkyne/allene diphosphate substrate analogues. | isopentenyl diphosphate isomerase (idi) catalyzes the interconversion of isopentenyl diphosphate (ipp) and dimethylallyl diphosphate (dmapp), the basic five-carbon building blocks of isoprenoid molecules. two structurally unrelated classes of idis are known. type i ipp isomerase (idi-1) utilizes a divalent metal in a protonation-deprotonation reaction. in contrast, the type ii enzyme (idi-2) requires reduced flavin, raising the possibility that the reaction catalyzed by idi-2 involves the net ad ... | 2010 | 20560533 |
| design and use of peptide-based antibodies decreasing superoxide production by mitochondrial complex i and complex ii. | mitochondria are the major source of reactive oxygen species. both complex i and complex ii mediate o(2) (*-) production in mitochondria and host reactive protein thiols. to explore the functions of the specific domains involved in the redox modifications of complexes i and ii, various peptide-based antibodies were generated against these complexes, and their inhibitory effects were subsequently measured. the redox domains involved in s-glutathionylation and nitration, as well as the binding mot ... | 2010 | 20564035 |
| cation selectivity by the cora mg2+ channel requires a fully hydrated cation. | the cora mg(2+) channel is the primary uptake system in about half of all bacteria and archaea. however, the basis for its mg(2+) selectivity is unknown. previous data suggested that cora binds a fully hydrated mg(2+) ion, unlike other ion channels. the crystal structure of thermotoga maritima cora shows a homopentamer with two transmembrane segments per monomer connected by a short periplasmic loop. this highly conserved loop, (281)efmpelkws(289) in salmonella enterica serovar typhimurium cora, ... | 2010 | 20568735 |
| single-stranded dna binding protein from human malarial parasite plasmodium falciparum is encoded in the nucleus and targeted to the apicoplast. | apicoplast, an essential organelle of human malaria parasite plasmodium falciparum contains a ∼35 kb circular genome and is a possible target for therapy. proteins required for the replication and maintenance of the apicoplast dna are not clearly known. here we report the presence of single-stranded dna binding protein (ssb) in p falciparum. pfssb is targeted to the apicoplast and it binds to apicoplast dna. a strong ssdna binding activity specific to ssb was also detected in p. falciparum lysat ... | 2010 | 20571080 |
| mechanism of concerted inhibition of alpha2beta2-type hetero-oligomeric aspartate kinase from corynebacterium glutamicum. | aspartate kinase (ak) is the first and committed enzyme of the biosynthetic pathway producing aspartate family amino acids, lysine, threonine, and methionine. ak from corynebacterium glutamicum (cgak), a bacterium used for industrial fermentation of amino acids, including glutamate and lysine, is inhibited by lysine and threonine in a concerted manner. to elucidate the mechanism of this unique regulation in cgak, we determined the crystal structures in several forms: an inhibitory form complexed ... | 2010 | 20573952 |
| structural insights into the catalytic mechanism of bacterial guanosine-diphospho-d-mannose pyrophosphorylase and its regulation by divalent ions. | gmp catalyzes the formation of gdp-man, a fundamental precursor for protein glycosylation and bacterial cell wall and capsular polysaccharide biosynthesis. crystal structures of gmp from the thermophilic bacterium thermotoga maritima in the apo form, in complex with the substrates mannose-1-phosphate or gtp and bound with the end product gdp-man in the presence of the essential divalent cation mg(2+), were solved in the 2.1-2.8 a resolution range. the t. maritima gmp molecule is organized in two ... | 2010 | 20573954 |
| functional and structural impact of target uridine substitutions on the h/aca ribonucleoprotein particle pseudouridine synthase. | box h/aca ribonucleoprotein protein particles catalyze the majority of pseudouridylation in functional rna. different from stand alone pseudouridine synthases, the rnp pseudouridine synthase comprises multiple protein subunits and an rna subunit. previous studies showed that each subunit, regardless its location, is sensitive to the step of subunit placement at the catalytic center and potentially to the reaction status of the substrate. here we describe the impact of chemical substitutions of t ... | 2010 | 20575532 |
| the genome sequence of the crenarchaeon acidilobus saccharovorans supports a new order, acidilobales, and suggests an important ecological role in terrestrial acidic hot springs. | acidilobus saccharovorans is an anaerobic, organotrophic, thermoacidophilic crenarchaeon isolated from a terrestrial hot spring. we report the complete genome sequence of a. saccharovorans, which has permitted the prediction of genes for embden-meyerhof and entner-doudoroff pathways and genes associated with the oxidative tricarboxylic acid cycle. the electron transfer chain is branched with two sites of proton translocation and is linked to the reduction of elemental sulfur and thiosulfate. the ... | 2010 | 20581186 |
| recognition of the amber uag stop codon by release factor rf1. | we report the crystal structure of a termination complex containing release factor rf1 bound to the 70s ribosome in response to an amber (uag) codon at 3.6-a resolution. the amber codon is recognized in the 30s subunit-decoding centre directly by conserved elements of domain 2 of rf1, including t186 of the pvt motif. together with earlier structures, the mechanisms of recognition of all three stop codons by release factors rf1 and rf2 can now be described. our structure confirms that the backbon ... | 2010 | 20588254 |
| using molecular dynamics to probe the structural basis for enhanced stability in thermal stable cytochromes p450. | high-temperature molecular dynamics (md) has been used to assess if md can be employed as a useful tool for probing the structural basis for enhanced stability in thermal stable cytochromes p450. cyp119, the most thermal stable p450 known, unfolds more slowly during 500 k md simulations than p450s that melt at lower temperatures, p450cam and p450cin. a comparison of the 500 k md trajectories shows that the cys ligand loop, a critically important structural feature just under the heme, in both p4 ... | 2010 | 20593793 |
| p. aeruginosa pilt structures with and without nucleotide reveal a dynamic type iv pilus retraction motor. | type iv pili are bacterial extracellular filaments that can be retracted to create force and motility. retraction is accomplished by the motor protein pilt. crystal structures of pseudomonas aeruginosa pilt with and without bound beta,gamma-methyleneadenosine-5'-triphosphate have been solved at 2.6 a and 3.1 a resolution, respectively, revealing an interlocking hexamer formed by the action of a crystallographic 2-fold symmetry operator on three subunits in the asymmetric unit and held together b ... | 2010 | 20595000 |
| the ccmc:heme:ccme complex in heme trafficking and cytochrome c biosynthesis. | a superfamily of integral membrane proteins is characterized by a conserved tryptophan-rich region (called the wwd domain) in an external loop at the inner membrane surface. the three major members of this family (ccmc, ccmf, and ccsba) are each involved in cytochrome c biosynthesis, yet the function of the wwd domain is unknown. it has been hypothesized that the wwd domain binds heme to present it to an acceptor protein (apoccme for ccmc or apocytochrome c for ccmf and ccsba) such that the heme ... | 2010 | 20599545 |
| properties of the c-terminal tail of human mitochondrial inner membrane protein oxa1l and its interactions with mammalian mitochondrial ribosomes. | in humans the mitochondrial inner membrane protein oxa1l is involved in the biogenesis of membrane proteins and facilitates the insertion of both mitochondrial- and nuclear-encoded proteins from the mitochondrial matrix into the inner membrane. the c-terminal approximately 100-amino acid tail of oxa1l (oxa1l-ctt) binds to mitochondrial ribosomes and plays a role in the co-translational insertion of mitochondria-synthesized proteins into the inner membrane. contrary to suggestions made for yeast ... | 2010 | 20601428 |
| structure of an archaeal non-discriminating glutamyl-trna synthetase: a missing link in the evolution of gln-trnagln formation. | the molecular basis of the genetic code relies on the specific ligation of amino acids to their cognate trna molecules. however, two pathways exist for the formation of gln-trna(gln). the evolutionarily older indirect route utilizes a non-discriminating glutamyl-trna synthetase (nd-glurs) that can form both glu-trna(glu) and glu-trna(gln). the glu-trna(gln) is then converted to gln-trna(gln) by an amidotransferase. since the well-characterized bacterial nd-glurs enzymes recognize trna(glu) and t ... | 2010 | 20601684 |
| bacillus anthracis surface-layer proteins assemble by binding to the secondary cell wall polysaccharide in a manner that requires csab and tago. | bacillus anthracis, the causative agent of anthrax, requires surface (s)-layer proteins for the pathogenesis of infection. previous work characterized s-layer protein binding via the surface layer homology domain to a pyruvylated carbohydrate in the envelope of vegetative forms. the molecular identity of this carbohydrate and the mechanism of its display in the bacterial envelope are still unknown. analyzing acid-solubilized, purified carbohydrates by mass spectrometry and nmr spectroscopy, we i ... | 2010 | 20603129 |
| the subunit composition of mitochondrial nadh:ubiquinone oxidoreductase (complex i) from pichia pastoris. | respiratory complex i (nadh:quinone oxidoreductase) is an entry point to the electron transport chain in the mitochondria of many eukaryotes. it is a large, multisubunit enzyme with a hydrophilic domain in the matrix and a hydrophobic domain in the mitochondrial inner membrane. here we present a comprehensive analysis of the protein composition and post-translational modifications of complex i from pichia pastoris, using a combination of proteomic and bioinformatic approaches. forty-one subunits ... | 2010 | 20610779 |
| evolution and thermodynamics of the slow unfolding of hyperstable monomeric proteins. | the unfolding speed of some hyperthermophilic proteins is dramatically lower than that of their mesostable homologs. ribonuclease hii from the hyperthermophilic archaeon thermococcus kodakaraensis (tk-rnase hii) is stabilized by its remarkably slow unfolding rate, whereas rnase hi from the thermophilic bacterium thermus thermophilus (tt-rnase hi) unfolds rapidly, comparable with to that of rnase hi from escherichia coli (ec-rnase hi). | 2010 | 20615256 |
| promoter melting triggered by bacterial rna polymerase occurs in three steps. | rna synthesis, carried out by dna-dependent rna polymerase (rnap) in a process called transcription, involves several stages. in bacteria, transcription initiation starts with promoter recognition and binding of rnap holoenzyme, resulting in the formation of the closed (r.p(c)) rnap-promoter dna complex. subsequently, a transition to the open r.p(o) complex occurs, characterized by separation of the promoter dna strands in an approximately 12 base-pair region to form the transcription bubble. us ... | 2010 | 20615963 |
| synthesis of glu-trna(gln) by engineered and natural aminoacyl-trna synthetases. | a protein engineering approach to delineating which distinct elements of homologous trna synthetase architectures are responsible for divergent rna-amino acid pairing specificities is described. previously, we constructed a hybrid enzyme in which 23 amino acids from the catalytic domain of escherichia coli glutaminyl-trna synthetase (glnrs) were replaced with the corresponding residues of human glutamyl-trna synthetase (glurs). the engineered hybrid (glnrs s1/l1/l2) synthesizes glu-trna(gln) mor ... | 2010 | 20617848 |
| a novel heme a insertion factor gene cotranscribes with the thermus thermophilus cytochrome ba3 oxidase locus. | studying the biogenesis of the thermus thermophilus cytochrome ba(3) oxidase, we analyze heme a cofactor insertion into this membrane protein complex. only three proteins linked to oxidase maturation have been described for this extreme thermophile, and in particular, no evidence for a canonical surf1 homologue, required for heme a insertion, is available from genome sequence data. here, we characterize the product of an open reading frame, cbax, in the operon encoding subunits of the ba(3)-type ... | 2010 | 20622059 |
| diversity of glycosyl hydrolases from cellulose-depleting communities enriched from casts of two earthworm species. | the guts and casts of earthworms contain microbial assemblages that process large amounts of organic polymeric substrates from plant litter and soil; however, the enzymatic potential of these microbial communities remains largely unexplored. in the present work, we retrieved carbohydrate-modifying enzymes through the activity screening of metagenomic fosmid libraries from cellulose-depleting microbial communities established with the fresh casts of two earthworm species, aporrectodea caliginosa ... | 2010 | 20622123 |
| structural studies of tri-functional human gart. | human purine de novo synthesis pathway contains several multi-functional enzymes, one of which, tri-functional gart, contains three enzymatic activities in a single polypeptide chain. we have solved structures of two domains bearing separate catalytic functions: glycinamide ribonucleotide synthetase and aminoimidazole ribonucleotide synthetase. structures are compared with those of homologous enzymes from prokaryotes and analyzed in terms of the catalytic mechanism. we also report small angle x- ... | 2010 | 20631005 |
| biosynthesis of compatible solutes in rhizobial strains isolated from phaseolus vulgaris nodules in tunisian fields. | associated with appropriate crop and soil management, inoculation of legumes with microbial biofertilizers can improve food legume yield and soil fertility and reduce pollution by inorganic fertilizers. rhizospheric bacteria are subjected to osmotic stress imposed by drought and/or nacl, two abiotic constraints frequently found in semi-arid lands. osmostress response in bacteria involves the accumulation of small organic compounds called compatible solutes. whereas most studies on rhizobial osmo ... | 2010 | 20633304 |
| dynamics of secy translocons with translocation-defective mutations. | the secy/sec61 translocon complex, located in the endoplasmic reticulum membrane of eukaryotes (sec61) or the plasma membrane of prokaryotes (secy), mediates the transmembrane secretion or insertion of nascent proteins. mutations that permit the secretion of nascent proteins with defective signal sequences (prl-phenotype), or interfere with the transmembrane orientation of newly synthesized protein segments, can affect protein topogenesis. the crystallographic structure of secyebeta from methano ... | 2010 | 20637421 |
| functional analysis of thermus thermophilus transcription factor nusg. | transcription elongation factors from the nusg family are ubiquitous from bacteria to humans and play diverse roles in the regulation of gene expression. these proteins consist of at least two domains. the n-terminal domains directly bind to the largest, β' in bacteria, subunit of rna polymerase (rnap), whereas the c-terminal domains interact with other cellular components and serve as platforms for the assembly of large nucleoprotein complexes. escherichia coli nusg and its paralog rfah modify ... | 2010 | 20639538 |
| rosr (cg1324), a hydrogen peroxide-sensitive marr-type transcriptional regulator of corynebacterium glutamicum. | the cg1324 gene (rosr) of corynebacterium glutamicum encodes a marr-type transcriptional regulator. by a comparative transcriptome analysis with dna microarrays of a δrosr mutant and the wild type and subsequent emsas with purified rosr protein, direct target genes of rosr were identified. the narkghji operon, which encodes a nitrate/nitrite transporter and the dissimilatory nitrate reductase complex, was activated by rosr. all other target genes were repressed by rosr. they encode four putative ... | 2010 | 20643656 |
| mitochondrial enzyme rhodanese is essential for 5 s ribosomal rna import into human mitochondria. | 5 s rrna is an essential component of ribosomes. in eukaryotic cells, it is distinguished by particularly complex intracellular traffic, including nuclear export and re-import. the finding that in mammalian cells 5 s rrna can eventually escape its usual circuit toward nascent ribosomes to get imported into mitochondria has made the scheme more complex, and it has raised questions about both the mechanism of 5 s rrna mitochondrial targeting and its function inside the organelle. previously, we sh ... | 2010 | 20663881 |
| linkage isomerization in heme-nox compounds: understanding no, nitrite, and hyponitrite interactions with iron porphyrins. | nitric oxide (no) and its derivatives such as nitrite and hyponitrite are biologically important species of relevance to human health. much of their physiological relevance stems from their interactions with the iron centers in heme proteins. the chemical reactivities displayed by the heme-nox species (nox = no, nitrite, hyponitrite) are a function of the binding modes of the nox ligands. hence, an understanding of the types of binding modes extant in heme-nox compounds is important if we are to ... | 2010 | 20666385 |
| interference with histidyl-trna synthetase by a crispr spacer sequence as a factor in the evolution of pelobacter carbinolicus. | pelobacter carbinolicus, a bacterium of the family geobacteraceae, cannot reduce fe(iii) directly or produce electricity like its relatives. how p. carbinolicus evolved is an intriguing problem. the genome of p. carbinolicus contains clustered regularly interspaced short palindromic repeats (crispr) separated by unique spacer sequences, which recent studies have shown to produce rna molecules that interfere with genes containing identical sequences. | 2010 | 20667132 |
| the crystal structure of human transketolase and new insights into its mode of action. | the crystal structure of human transketolase (tkt), a thiamine diphosphate (thdp) and ca(2+)-dependent enzyme that catalyzes the interketol transfer between ketoses and aldoses as part of the pentose phosphate pathway, has been determined to 1.75 å resolution. the recombinantly produced protein crystallized in space group c2 containing one monomer in the asymmetric unit. two monomers form the homodimeric biological assembly with two identical active sites at the dimer interface. although the pro ... | 2010 | 20667822 |
| structural and functional characterization of salmonella enterica serovar typhimurium ycbl: an unusual type ii glyoxalase. | ycbl has been annotated as either a metallo-β-lactamase or glyoxalase ii (glx2), both members of the zinc metallohydrolase superfamily, that contains many enzymes with a diverse range of activities. here, we report crystallographic and biochemical data for salmonella enterica serovar typhimurium ycbl that establishes it as glx2, which differs in certain structural and functional properties compared with previously known examples. these features include the insertion of an α-helix after residue 8 ... | 2010 | 20669241 |
| insights into the nitric oxide reductase mechanism of flavodiiron proteins from a flavin-free enzyme. | flavodiiron proteins (fdps) catalyze reductive scavenging of dioxygen and nitric oxide in air-sensitive microorganisms. fdps contain a distinctive non-heme diiron/flavin mononucleotide (fmn) active site. alternative mechanisms for the nitric oxide reductase (nor) activity consisting of either protonation of a diiron-bridging hyponitrite or "super-reduction" of a diferrous-dinitrosyl by the proximal fmnh(2) in the rate-determining step have been proposed. to test these alternative mechanisms, we ... | 2010 | 20669924 |
| poxa, yjek, and elongation factor p coordinately modulate virulence and drug resistance in salmonella enterica. | we report an interaction between poxa, encoding a paralog of lysyl trna-synthetase, and the closely linked yjek gene, encoding a putative 2,3-beta-lysine aminomutase, that is critical for virulence and stress resistance in salmonella enterica. salmonella poxa and yjek mutants share extensive phenotypic pleiotropy, including attenuated virulence in mice, an increased ability to respire under nutrient-limiting conditions, hypersusceptibility to a variety of diverse growth inhibitors, and altered e ... | 2010 | 20670890 |
| dual biosynthesis pathway for longer-chain polyamines in the hyperthermophilic archaeon thermococcus kodakarensis. | long-chain and/or branched-chain polyamines are unique polycations found in thermophiles. cytoplasmic polyamines were analyzed for cells cultivated at various growth temperatures in the hyperthermophilic archaeon thermococcus kodakarensis. spermidine [34] and n4-aminopropylspermine [3(3)43] were identified as major polyamines at 60°c, and the amounts of n4-aminopropylspermine [3(3)43] increased as the growth temperature rose. to identify genes involved in polyamine biosynthesis, a gene disruptio ... | 2010 | 20675472 |
| functional role of thr-312 and thr-315 in the proton-transfer pathway in ba3 cytochrome c oxidase from thermus thermophilus. | cytochrome ba(3) from thermus thermophilus is a member of the family of b-type heme-copper oxidases, which have a low degree of sequence homology to the well-studied mitochondrial-like a-type enzymes. recently, it was suggested that the ba(3) oxidase has only one pathway for the delivery of protons to the active site and that this pathway is spatially analogous to the k-pathway in the a-type oxidases [chang, h.-y., et al. (2009) proc. natl. acad. sci. u.s.a. 106, 16169-16173]. this suggested pat ... | 2010 | 20677778 |
| origin of light-induced spin-correlated radical pairs in cryptochrome. | blue-light excitation of cryptochromes and homologues uniformly triggers electron transfer (et) from the protein surface to the flavin adenine dinucleotide (fad) cofactor. a cascade of three conserved tryptophan residues has been considered to be critically involved in this photoreaction. if the fad is initially in its fully oxidized (diamagnetic) redox state, light-induced et via the tryptophan triad generates a series of short-lived spin-correlated radical pairs comprising an fad radical and a ... | 2010 | 20684534 |
| mechanism of microrna-target interaction: molecular dynamics simulations and thermodynamics analysis. | micrornas (mirnas) are endogenously produced approximately 21-nt riboregulators that associate with argonaute (ago) proteins to direct mrna cleavage or repress the translation of complementary rnas. capturing the molecular mechanisms of mirna interacting with its target will not only reinforce the understanding of underlying rna interference but also fuel the design of more effective small-interfering rna strands. to address this, in the present work the rna-bound (ago-mirna, ago-mirna-target) a ... | 2010 | 20686687 |
| three distinct peptides from the n domain of translation termination factor erf1 surround stop codon in the ribosome. | to study positioning of the polypeptide release factor erf1 toward a stop signal in the ribosomal decoding site, we applied photoactivatable mrna analogs, derivatives of oligoribonucleotides. the human erf1 peptides cross-linked to these short mrnas were identified. cross-linkers on the guanines at the second, third, and fourth stop signal positions modified fragment 31-33, and to lesser extent amino acids within region 121-131 (the "yxcxxxf loop") in the n domain. hence, both regions are involv ... | 2010 | 20688868 |
| structural and functional studies of wlba: a dehydrogenase involved in the biosynthesis of 2,3-diacetamido-2,3-dideoxy-d-mannuronic acid . | 2,3-diacetamido-2,3-dideoxy-d-mannuronic acid (mannac3naca) is an unusual dideoxy sugar first identified nearly 30 years ago in the lipopolysaccharide of pseudomonas aeruginosa o:3a,d. it has since been observed in other organisms, including bordetella pertussis, the causative agent of whooping cough. five enzymes are required for the biosynthesis of udp-mannac3naca starting from udp-n-acetyl-d-glucosamine. here we describe a structural study of wlba, the nad-dependent dehydrogenase that catalyz ... | 2010 | 20690587 |
| structures of hypoxanthine-guanine phosphoribosyltransferase (ttha0220) from thermus thermophilus hb8. | hypoxanthine-guanine phosphoribosyltransferase (hgprtase), which is a key enzyme in the purine-salvage pathway, catalyzes the synthesis of imp or gmp from alpha-d-phosphoribosyl-1-pyrophosphate and hypoxanthine or guanine, respectively. structures of hgprtase from thermus thermophilus hb8 in the unliganded form, in complex with imp and in complex with gmp have been determined at 2.1, 1.9 and 2.2 a resolution, respectively. the overall fold of the imp complex was similar to that of the unliganded ... | 2010 | 20693661 |
| mutations in 23s rrna at the peptidyl transferase center and their relationship to linezolid binding and cross-resistance. | the oxazolidinone antibiotic linezolid targets the peptidyl transferase center (ptc) on the bacterial ribosome. thirteen single and four double 23s rrna mutations were introduced into a mycobacterium smegmatis strain with a single rrna operon. converting bacterial base identity by single mutations at positions 2032, 2453, and 2499 to human cytosolic base identity did not confer significantly reduced susceptibility to linezolid. the largest decrease in linezolid susceptibility for any of the intr ... | 2010 | 20696869 |
| yeast 18 s rrna is directly involved in the ribosomal response to stringent aug selection during translation initiation. | in eukaryotes, the 40 s ribosomal subunit serves as the platform of initiation factor assembly, to place itself precisely on the aug start codon. structural arrangement of the 18 s rrna determines the overall shape of the 40 s subunit. here, we present genetic evaluation of yeast 18 s rrna function using 10 point mutations altering the polysome profile. all the mutants reduce the abundance of the mutant 40 s, making it limiting for translation initiation. two of the isolated mutations, g875a, al ... | 2010 | 20699223 |
| structural basis for the bacterial transcription-repair coupling factor/rna polymerase interaction. | the transcription-repair coupling factor (trcf, the product of the mfd gene) is a widely conserved bacterial protein that mediates transcription-coupled dna repair. trcf uses its atp-dependent dna translocase activity to remove transcription complexes stalled at sites of dna damage, and stimulates repair by recruiting components of the nucleotide excision repair pathway to the site. a protein/protein interaction between trcf and the β-subunit of rna polymerase (rnap) is essential for trcf functi ... | 2010 | 20702425 |
| a flexible loop in yeast ribosomal protein l11 coordinates p-site trna binding. | high-resolution structures reveal that yeast ribosomal protein l11 and its bacterial/archael homologs called l5 contain a highly conserved, basically charged internal loop that interacts with the peptidyl-transfer rna (trna) t-loop. we call this the l11 'p-site loop'. chemical protection of wild-type ribosome shows that that the p-site loop is inherently flexible, i.e. it is extended into the ribosomal p-site when this is unoccupied by trna, while it is retracted into the terminal loop of 25s rr ... | 2010 | 20705654 |
| the diheme cytochrome c(4) from vibrio cholerae is a natural electron donor to the respiratory cbb(3) oxygen reductase. | the respiratory chain of vibrio cholerae contains three bd-type quinol oxygen reductases as well as one cbb(3) oxygen reductase. the cbb(3) oxygen reductase has been previously isolated and characterized; however, the natural mobile electron donor(s) that shuttles electrons between the bc(1) complex and the cbb(3) oxygen reductase is not known. the most likely candidates are the diheme cytochrome c(4) and monoheme cytochrome c(5), which have been previously shown to be present in the periplasm o ... | 2010 | 20715760 |
| crystal structure of a transfer-ribonucleoprotein particle that promotes asparagine formation. | four out of the 22 aminoacyl-trnas (aa-trnas) are systematically or alternatively synthesized by an indirect, two-step route requiring an initial mischarging of the trna followed by trna-dependent conversion of the non-cognate amino acid. during trna-dependent asparagine formation, trna(asn) promotes assembly of a ribonucleoprotein particle called transamidosome that allows channelling of the aa-trna from non-discriminating aspartyl-trna synthetase active site to the gatcab amidotransferase site ... | 2010 | 20717102 |
| dna mismatch repair in eukaryotes and bacteria. | dna mismatch repair (mmr) corrects mismatched base pairs mainly caused by dna replication errors. the fundamental mechanisms and proteins involved in the early reactions of mmr are highly conserved in almost all organisms ranging from bacteria to human. the significance of this repair system is also indicated by the fact that defects in mmr cause human hereditary nonpolyposis colon cancers as well as sporadic tumors. to date, 2 types of mmrs are known: the human type and escherichia coli type. t ... | 2010 | 20725617 |
| multifactorial determinants of protein expression in prokaryotic open reading frames. | a quantitative description of the relationship between protein expression levels and open reading frame (orf) nucleotide sequences is important for understanding natural systems, designing synthetic systems, and optimizing heterologous expression. codon identity, mrna secondary structure, and nucleotide composition within orfs markedly influence expression levels. bioinformatic analysis of orf sequences in 816 bacterial genomes revealed that these features show distinct regional trends. to inves ... | 2010 | 20727358 |
| a mutation within the β subunit of escherichia coli rna polymerase impairs transcription from bacteriophage t4 middle promoters. | during infection of escherichia coli, bacteriophage t4 usurps the host transcriptional machinery, redirecting it to the expression of early, middle, and late phage genes. middle genes, whose expression begins about 1 min postinfection, are transcribed both from the extension of early rna into middle genes and by the activation of t4 middle promoters. middle-promoter activation requires the t4 transcriptional activator mota and coactivator asia, which are known to interact with σ(70), the specifi ... | 2010 | 20729353 |
| an extreme thermophile, thermus thermophilus, is a polyploid bacterium. | an extremely thermophilic bacterium, thermus thermophilus hb8, is one of the model organisms for systems biology. its genome consists of a chromosome (1.85 mb), a megaplasmid (0.26 mb) designated ptt27, and a plasmid (9.3 kb) designated ptt8, and the complete sequence is available. we show here that t. thermophilus is a polyploid organism, harboring multiple genomic copies in a cell. in the case of the hb8 strain, the copy number of the chromosome was estimated to be four or five, and the copy n ... | 2010 | 20729360 |
| molecular basis of vancomycin dependence in vana-type staphylococcus aureus vrsa-9. | the vancomycin-resistant staphylococcus aureus vrsa-9 clinical isolate was partially dependent on glycopeptide for growth. the responsible vana operon had the same organization as that of tn1546 and was located on a plasmid. the chromosomal d-ala:d-ala ligase (ddl) gene had two point mutations that led to q260k and a283e substitutions, resulting in a 200-fold decrease in enzymatic activity compared to that of the wild-type strain vrsa-6. to gain insight into the mechanism of enzyme impairment, w ... | 2010 | 20729361 |
| n-terminal fusion of a hyperthermophilic chitin-binding domain to xylose isomerase from thermotoga neapolitana enhances kinetics and thermostability of both free and immobilized enzymes. | immobilization of a thermostable d-xylose isomerase (ec 5.3.1.5) from thermotoga neapolitana 5068 (tnxi) on chitin beads was accomplished via a n-terminal fusion with a chitin-binding domain (cbd) from a hyperthermophilic chitinase produced by pyrococcus furiosus (pf1233) to create a fusion protein (cbd-tnxi). the turnover numbers for glucose to fructose conversion for both unbound and immobilized cbd-tnxi were greater than the wild-type enzyme: k(cat) (min(-1)) was approximately 1,000, 3,800, a ... | 2010 | 20730758 |
| binding of the dimeric deinococcus radiodurans single-stranded dna binding protein to single-stranded dna. | deinococcus radiodurans single-stranded (ss) dna binding protein (drssb) originates from a radiation-resistant bacterium and participates in dna recombination, replication, and repair. although it functions as a homodimer, it contains four dna binding domains (ob-folds) and thus is structurally similar to the escherichia coli ssb (ecossb) homotetramer. we examined the equilibrium binding of drssb to ssdna for comparison with that of ecossb. we find that the occluded site size of drssb on poly(dt ... | 2010 | 20795631 |
| modular pathways for editing non-cognate amino acids by human cytoplasmic leucyl-trna synthetase. | to prevent potential errors in protein synthesis, some aminoacyl-transfer rna (trna) synthetases have evolved editing mechanisms to hydrolyze misactivated amino acids (pre-transfer editing) or misacylated trnas (post-transfer editing). class ia leucyl-trna synthetase (leurs) may misactivate various natural and non-protein amino acids and then mischarge trna(leu). it is known that the fidelity of prokaryotic leurs depends on multiple editing pathways to clear the incorrect intermediates and produ ... | 2011 | 20805241 |
| modular pathways for editing non-cognate amino acids by human cytoplasmic leucyl-trna synthetase. | to prevent potential errors in protein synthesis, some aminoacyl-transfer rna (trna) synthetases have evolved editing mechanisms to hydrolyze misactivated amino acids (pre-transfer editing) or misacylated trnas (post-transfer editing). class ia leucyl-trna synthetase (leurs) may misactivate various natural and non-protein amino acids and then mischarge trna(leu). it is known that the fidelity of prokaryotic leurs depends on multiple editing pathways to clear the incorrect intermediates and produ ... | 2011 | 20805241 |
| targeting the chromosome partitioning protein para in tuberculosis drug discovery. | to identify inhibitors of the essential chromosome partitioning protein para that are active against mycobacterium tuberculosis. | 2010 | 20810423 |
| template-directed ligation of tethered mononucleotides by t4 dna ligase for kinase ribozyme selection. | in vitro selection of kinase ribozymes for small molecule metabolites, such as free nucleosides, will require partition systems that discriminate active from inactive rna species. while nucleic acid catalysis of phosphoryl transfer is well established for phosphorylation of 5' or 2' oh of oligonucleotide substrates, phosphorylation of diffusible small molecules has not been demonstrated. | 2010 | 20811490 |
| structural biology of redox partner interactions in p450cam monooxygenase: a fresh look at an old system. | the p450cam monooxygenase system consists of three separate proteins: the fad-containing, nadh-dependent oxidoreductase (putidaredoxin reductase or pdr), cytochrome p450cam and the 2fe2s ferredoxin (putidaredoxin or pdx), which transfers electrons from pdr to p450cam. over the past few years our lab has focused on the interaction between these redox components. it has been known for some time that pdx can serve as an effector in addition to its electron shuttle role. the binding of pdx to p450ca ... | 2011 | 20816746 |
| structural biology of redox partner interactions in p450cam monooxygenase: a fresh look at an old system. | the p450cam monooxygenase system consists of three separate proteins: the fad-containing, nadh-dependent oxidoreductase (putidaredoxin reductase or pdr), cytochrome p450cam and the 2fe2s ferredoxin (putidaredoxin or pdx), which transfers electrons from pdr to p450cam. over the past few years our lab has focused on the interaction between these redox components. it has been known for some time that pdx can serve as an effector in addition to its electron shuttle role. the binding of pdx to p450ca ... | 2011 | 20816746 |
| a new hypothesis on the simultaneous direct and indirect proton pump mechanisms in nadh-quinone oxidoreductase (complex i). | recently, sazanov's group reported the x-ray structure of whole complex i [nature, 465, 441 (2010)], which presented a strong clue for a "piston-like" structure as a key element in an "indirect" proton pump. we have studied the nuol subunit which has a high sequence similarity to na(+)/h(+) antiporters, as do the nuom and n subunits. we constructed 27 site-directed nuol mutants. our data suggest that the h(+)/e(-) stoichiometry seems to have decreased from (4h(+)/2e(-)) in the wild-type to appro ... | 2010 | 20816962 |
| bacteria, yeast, worms, and flies: exploiting simple model organisms to investigate human mitochondrial diseases. | the extensive conservation of mitochondrial structure, composition, and function across evolution offers a unique opportunity to expand our understanding of human mitochondrial biology and disease. by investigating the biology of much simpler model organisms, it is often possible to answer questions that are unreachable at the clinical level. here, we review the relative utility of four different model organisms, namely the bacterium escherichia coli, the yeast saccharomyces cerevisiae, the nema ... | 2010 | 20818735 |
| crystallization and preliminary x-ray crystallographic study of genx, a lysyl-trna synthetase paralogue from escherichia coli, in complex with translation elongation factor p. | genx, a lysyl-trna synthetase paralogue from escherichia coli, was overexpressed in e. coli, purified by three chromatographic steps and cocrystallized with a lysyl adenylate analogue (lysams) by the hanging-drop vapour-diffusion method using peg 4000 as a precipitant. the genx-lysams crystals belonged to the triclinic space group p1, with unit-cell parameters a=54.80, b=69.15, c=94.08 a, alpha=95.47, beta=106.51, gamma=90.46 degrees, and diffracted to 1.9 a resolution. furthermore, genx was coc ... | 2010 | 20823541 |
| the membrane subunit nuol(nd5) is involved in the indirect proton pumping mechanism of escherichia coli complex i. | complex i pumps protons across the membrane by using downhill redox energy. here, to investigate the proton pumping mechanism by complex i, we focused on the largest transmembrane subunit nuol (escherichia coli nd5 homolog). nuol/nd5 is believed to have h(+) translocation site(s), because of a high sequence similarity to multi-subunit na(+)/h(+) antiporters. we mutated thirteen highly conserved residues between nuol/nd5 and mrpa of na(+)/h(+) antiporters in the chromosomal nuol gene. the dnadh o ... | 2010 | 20826797 |
| sequence- and structure-specific rna processing by a crispr endonuclease. | many bacteria and archaea contain clustered regularly interspaced short palindromic repeats (crisprs) that confer resistance to invasive genetic elements. central to this immune system is the production of crispr-derived rnas (crrnas) after transcription of the crispr locus. here, we identify the endoribonuclease (csy4) responsible for crispr transcript (pre-crrna) processing in pseudomonas aeruginosa. a 1.8 angstrom crystal structure of csy4 bound to its cognate rna reveals that csy4 makes sequ ... | 2010 | 20829488 |
| molecular modeling study for interaction between bacillus subtilis obg and nucleotides. | the bacterial obg proteins (spo0b-associated gtp-binding protein) belong to the subfamily of p-loop gtpase proteins that contain two equally and highly conserved domains, a c-terminal gtp binding domain and an n-terminal glycine-rich domain which is referred as the "obg fold" and now it is considered as one of the new targets for antibacterial drug. when the obg protein is associated with gtp, it becomes activated, because conformation of obg fold changes due to the structural changes of gtpase ... | 2010 | 20830302 |
| structural and biochemical studies of a fluoroacetyl-coa-specific thioesterase reveal a molecular basis for fluorine selectivity. | we have initiated a broad-based program aimed at understanding the molecular basis of fluorine specificity in enzymatic systems, and in this context, we report crystallographic and biochemical studies on a fluoroacetyl-coenzyme a (coa) specific thioesterase (flk) from streptomyces cattleya. our data establish that flk is competent to protect its host from fluoroacetate toxicity in vivo and demonstrate a 10(6)-fold discrimination between fluoroacetyl-coa (k(cat)/k(m) = 5 × 10⁷ m⁻¹ s⁻¹) and acetyl ... | 2010 | 20836570 |
| inhibition of osteoclast bone resorption by disrupting vacuolar h+-atpase a3-b2 subunit interaction. | vacuolar h(+)-atpases (v-atpases) are highly expressed in ruffled borders of bone-resorbing osteoclasts, where they play a crucial role in skeletal remodeling. to discover protein-protein interactions with the a subunit in mammalian v-atpases, a gal4 activation domain fusion library was constructed from an in vitro osteoclast model, receptor activator of nf-κb ligand-differentiated raw 264.7 cells. this library was screened with a bait construct consisting of a gal4 binding domain fused to the n ... | 2010 | 20837476 |
| small-molecule antioxidant proteome-shields in deinococcus radiodurans. | for deinococcus radiodurans and other bacteria which are extremely resistant to ionizing radiation, ultraviolet radiation, and desiccation, a mechanistic link exists between resistance, manganese accumulation, and protein protection. we show that ultrafiltered, protein-free preparations of d. radiodurans cell extracts prevent protein oxidation at massive doses of ionizing radiation. in contrast, ultrafiltrates from ionizing radiation-sensitive bacteria were not protective. the d. radiodurans ult ... | 2010 | 20838443 |