Publications

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dynamic regulation of archaeal proteasome gate opening as studied by trosy nmr.the proteasome catalyzes the majority of protein degradation in the cell and plays an integral role in cellular homeostasis. control over proteolysis by the 20s core-particle (cp) proteasome is achieved by gated access of substrate; thus, an understanding of the molecular mechanism by which these gates regulate substrate entry is critical. we used methyl-transverse relaxation optimized nuclear magnetic resonance spectroscopy to show that the amino-terminal residues that compose the gates of the ...201020360109
x-ray structures of isopentenyl phosphate kinase.isoprenoid compounds are ubiquitous in nature, participating in important biological phenomena such as signal transduction, aerobic cellular respiration, photosynthesis, insect communication, and many others. they are derived from the 5-carbon isoprenoid substrates isopentenyl diphosphate (ipp) and its isomer dimethylallyl diphosphate (dmapp). in archaea and eukarya, these building blocks are synthesized via the mevalonate pathway. however, the genes required to convert mevalonate phosphate (mp) ...201020402538
molecular cloning and co-expression of thermoplasma volcanium proteasome subunit genes.in this study we describe, the construction of a co-expression vector allowing simultaneous production of thermoplasma volcanium 20s proteasome alpha- and beta-subunits in escherichia coli. this heterologous expression system provided high level production of fully active 20s proteasome that can be purified easily by using a conventional two-step chromatographic technique. the recombinant proteasome was purified to homogeneity 12-fold with a specific activity of 26.5 u/mg. sodium dodecyl sulfate ...201020460155
the xpd helicase: xpanding archaeal xpd structures to get a grip on human dna repair.xeroderma pigmentosum complementation group d protein (xpd) is an iron-sulfur cluster containing 5'-3' helicase and, in humans, part of the transcription factor tfiih. tfiih is involved in nucleotide excision repair as well as in transcription initiation. recently, three different groups have reported the structures of archaeal xpds. all structures revealed a four-domain organization with two reca-like domains, an arch domain and an iron-sulfur cluster domain. it was possible to rationalize seve ...201020482310
structural models for interactions between the 20s proteasome and its pan/19s activators.proteasome activity is regulated by sequestration of its proteolytic centers in a barrel-shaped structure that limits substrate access. substrates enter the proteasome by means of activator complexes that bind to the end rings of proteasome alpha subunits and induce opening of an axial entrance/exit pore. the pa26 activator binds in a pocket on the proteasome surface using main chain contacts of its c-terminal residues and uses an internal activation loop to trigger gate opening by repositioning ...201019889631
insights into substrate specificity of geranylgeranyl reductases revealed by the structure of digeranylgeranylglycerophospholipid reductase, an essential enzyme in the biosynthesis of archaeal membrane lipids.archaeal membrane lipids consist of branched, saturated hydrocarbons distinct from those found in bacteria and eukaryotes. digeranylgeranylglycerophospholipid reductase (dggr) catalyzes the hydrogenation process that converts unsaturated 2,3-di-o-geranylgeranylglyceryl phosphate to saturated 2,3-di-o-phytanylglyceryl phosphate as a critical step in the biosynthesis of archaeal membrane lipids. the saturation of hydrocarbon chains confers the ability to resist hydrolysis and oxidation and helps a ...201020869368
characterization of thermophilic archaeal isopentenyl phosphate kinases.archaea synthesize isopentenyl diphosphate (ipp) and dimethylallyl diphosphate (dmapp), the essential building blocks of isoprenoid compounds, from mevalonate (mva). however, an analysis of the genomes of several members of the archaea failed to identify genes for the enzymes required to convert phosphomevalonate (pm) to ipp in eukaryotes. the recent discovery of an isopentenyl kinase (ipk) in methanocaldococcus jannaschii (mj) suggests a new variation of the mva pathway where pm is decarboxylat ...201019928876
structure and filament dynamics of the psk41 actin-like parm protein: implications for plasmid dna segregation.type ii plasmid partition systems utilize parm ntpases in coordination with a centromere-binding protein called parr to mediate accurate dna segregation, a process critical for plasmid retention. the staphylococcus aureus psk41 plasmid is a medically important plasmid that confers resistance to multiple antibiotics, disinfectants, and antiseptics. in the first step of partition, the psk41 parr binds its dna centromere to form a superhelical partition complex that recruits parm, which then mediat ...201020106979
structures of three members of pfam pf02663 (fmde) implicated in microbial methanogenesis reveal a conserved α+β core domain and an auxiliary c-terminal treble-clef zinc finger.examination of the genomic context for members of the fmde pfam family (pf02663), such as the protein encoded by the fmde gene from the methanogenic archaeon methanobacterium thermoautotrophicum, indicates that 13 of them are co-transcribed with genes encoding subunits of molybdenum formylmethanofuran dehydrogenase (ec 1.2.99.5), an enzyme that is involved in microbial methane production. here, the first crystal structures from pf02663 are described, representing two bacterial and one archaeal s ...201020944230
the proteasome antechamber maintains substrates in an unfolded state.eukaryotes and archaea use a protease called the proteasome that has an integral role in maintaining cellular function through the selective degradation of proteins. proteolysis occurs in a barrel-shaped 20s core particle, which in thermoplasma acidophilum is built from four stacked homoheptameric rings of subunits, α and β, arranged α(7)β(7)β(7)α(7) (ref. 5). these rings form three interconnected cavities, including a pair of antechambers (formed by α(7)β(7)) through which substrates are passed ...201020944750
phylogenetic analysis of methanogens in the pig feces.in order to assess methanogen diversity in feces of pigs, archaeal 16s rrna gene clone libraries were constructed from feces of the pig. after the amplification by pcr using primers met86f and met1340r, equal quantities of pcr products from each of the five pigs were mixed together and used to construct the library. sequence analysis showed that the 74 clones were divided into ten phylotypes as defined by rflp analysis. phylogenetic analysis showed that three phylotypes were most closely affilia ...201121259005
crystal structure of bifunctional 5,10-methylenetetrahydrofolate dehydrogenase/cyclohydrolase from thermoplasma acidophilum.folate co-enzymes play a pivotal role in one-carbon transfer cellular processes. many eukaryotes encode the tri-functional tetrahydrofolate dehydrogenase/cyclohydrolase/synthetase (deh/cyc/syn) enzyme, which consists of a n-terminal bifunctional domain (deh/cyc) and a c-terminal monofunctional domain (syn). here, we report the first analogous archeal enzyme structures, for the bifunctional methylenetetrahydrofolate dehydrogenase/cyclohydrolase from thermoplasma acidophilum (tamthfdc) as the nati ...201121333632
atp binds to proteasomal atpases in pairs with distinct functional effects, implying an ordered reaction cycle.in the eukaryotic 26s proteasome, the 20s particle is regulated by six aaa atpase subunits and, in archaea, by a homologous ring complex, pan. to clarify the role of atp in proteolysis, we studied how nucleotides bind to pan. although pan has six identical subunits, it binds atps in pairs, and its subunits exhibit three conformational states with high, low, or no affinity for atp. when pan binds two atp?s molecules or two atp?s plus two adp molecules, it is maximally active in binding protein su ...201121335235
architectures of archaeal gins complexes, essential dna replication initiation factors.abstract: background: in the early stage of eukaryotic dna replication, the template dna is unwound by the mcm helicase, which is activated by forming a complex with the cdc45 and gins proteins. the eukaryotic gins forms a heterotetramer, comprising four types of subunits. on the other hand, the archaeal gins appears to be either a tetramer formed by two types of subunits in a 2:2 ratio (alpha2beta2) or a homotetramer of a single subunit (alpha4). due to the low sequence similarity between the a ...201121527023
glucose sensing by electrogenerated chemiluminescence of glucose-dehydrogenase produced nadh on electrodeposited redox hydrogel.in this work, we report a new sensing approach based on electrogenerated chemiluminescence (ecl) in an electrodeposited redox hydrogel using glucose dehydrogenase as a model system. the ecl-hydrogel films were electrodeposited by potential cycling of a pbs solution containing [poly(4-vinylpyridine)ru(2,2'-bipyridine)(2)cl(-)](+/2+). the film was easily prepared in a rapid, reproducible and well-controlled one-step procedure. the deposited hydrogel film is permeable to water-soluble chemicals and ...201121733762
The catalytic core of an archaeal 2-oxoacid dehydrogenase multienzyme complex is a 42-mer protein assembly.The acyl-transferase (E2) enzyme forms the structural and catalytic core of the tripartite 2-oxoacid dehydrogenase multienzyme complexes of the central metabolic pathways. Although this family of multienzyme complexes share a common architecture, their E2 cores form homo-trimers that, depending on the source, further associate into either octahedral (24-mer) or icosahedral (60-mer) assemblies, as predicted by the principles of quasi-equivalence. In the crystal structure of the E2 core from Therm ...201122188654
column bioleaching of low-grade mining ore containing high level of smithsonite, talc, sphaerocobaltite and azurite.present work describes the bioleaching potential of metals from low-grade mining ore containing smithsonite, sphaerocobaltite, azurite and talc as main gangue minerals with adapted consortium of sulfobacillus thermosulfidooxidans strain-rdb and thermoplasma acidophilum. bioleaching potential improved markedly by added energy source, acid preleaching and adaptation of microbial consortium with mixed metal ions. during whole leaching period including acid preleaching stage of 960 h and bioleaching ...201121947702
engineered thermoplasma acidophilum factor f3 mimics human aminopeptidase n (apn) as a target for anticancer drug development.human aminopeptidase n (hapn) is an appealing objective for the development of anti-cancer agents. the absence of mammalian apn experimental structure negatively impinges upon the progression of structure-based drug design. tricorn interacting factor f3 (factor f3) from thermoplasma acidophilum shares 33% sequence identity with hapn. engineered factor f3 with two point directed mutations resulted in a protein with an active site identical to hapn. in the present work, the engineered factor f3 ha ...201121493078
crystallization and preliminary x-ray diffraction analysis of transaldolase from thermoplasma acidophilum.the metabolic enzyme transaldolase from thermoplasma acidophilum was recombinantly expressed in escherichia coli and could be crystallized in two polymorphic forms. crystals were grown by the hanging-drop vapour-diffusion method using peg 6000 as precipitant. native data sets for crystal forms 1 and 2 were collected in-house to resolutions of 3.0 and 2.7 å, respectively. crystal form 1 belonged to the orthorhombic space group c222(1) with five monomers per asymmetric unit and crystal form 2 belo ...201121543867
site-directed methyl group labeling as an nmr probe of structure and dynamics in supramolecular protein systems: applications to the proteasome and to the clpp protease.methyl groups are powerful reporters of structure, motion, and function in nmr studies of supramolecular protein assemblies. their utility can be hindered, however, by spectral overlap, difficulties with assignment or lack of probes in biologically important regions of the molecule studied. here we show that (13)ch(3)-s- labeling of cys side chains using (13)c-methyl-methanethiosulfonate ((13)c-mmts) (iupac name: methylsulfonylsulfanylmethane) provides a convenient probe of molecular structure a ...201121557628
The structures of Thermoplasma volcanium phosphoribosyl pyrophosphate synthetase bound to ribose-5-phosphate and ATP analogs.Phosphoribosyl pyrophosphate (PRPP) synthetase catalyzes the transfer of the pyrophosphate group from ATP to ribose-5-phosphate (R5P) yielding PRPP and AMP. PRPP is an essential metabolite that plays a central role in cellular metabolism. The enzyme from a thermophilic archaeon Thermoplasma volcanium (Tv) was expressed in Escherichia coli, crystallized, and its X-ray molecular structure was determined in a complex with its substrate R5P and with substrate analogs ß,?-methylene ATP and ADP in two ...201121963988
twisted schiff base intermediates and substrate locale revise transaldolase mechanism.we examined the catalytic cycle of transaldolase (tal) from thermoplasma acidophilum by cryocrystallography and were able to structurally characterize--for the first time, to our knowledge--different genuine tal reaction intermediates. these include the schiff base adducts formed between the catalytic lysine and the donor ketose substrates fructose-6-phosphate and sedoheptulose-7-phosphate as well as the michaelis complex with acceptor aldose erythrose-4-phosphate. these structural snapshots nec ...201121857661
The Streptomyces-produced antibiotic fosfomycin is a promiscuous substrate for Archaeal isopentenyl phosphate kinase.Isopentenyl phosphate kinase (IPK) catalyzes the phosphorylation of isopentenyl phosphate to form the isoprenoid precursor isopentenyl diphosphate (IPP) in the archaeal mevalonate pathway. This enzyme is highly homologous to fosfomycin kinase (FomA), an antibiotic resistance enzyme found in a few strains of Streptomyces and Pseudomonas whose mode of action is inactivation by phosphorylation. Superposition of Thermoplasma acidophilum (THA) IPK and FomA structures aligns their respective substrate ...201122148590
uracil-dna glycosylase of thermoplasma acidophilumdirects long-patch base excision repair, which is promoted by deoxynucleoside triphosphates and atp/adp, into short-patch repair.hydrolytic deamination of cytosine to uracil in dna is increased in organisms adapted to high temperatures. hitherto, the uracil base excision repair (ber) pathway has only been described in two archaeons, the crenarchaeon pyrobaculum aerophilumand the euryarchaeon archaeoglobus fulgidus, which are hyperthermophiles and use single-nucleotide replacement. in the former the apurinic/apyrimidinic (ap) site intermediate is removed by the sequential action of a 5'-acting ap endonuclease and a 5'-deox ...201121665970
Functional and structural studies of the nucleotide excision repair helicase XPD suggest a polarity for DNA translocation.The XPD protein is a vital subunit of the general transcription factor TFIIH which is not only involved in transcription but is also an essential component of the eukaryotic nucleotide excision DNA repair (NER) pathway. XPD is a superfamily-2 5'-3' helicase containing an iron-sulphur cluster. Its helicase activity is indispensable for NER and it plays a role in the damage verification process. Here, we report the first structure of XPD from Thermoplasma acidophilum (taXPD) in complex with a shor ...201122081108
the gins complex from the thermophilic archaeon, thermoplasma acidophilum may function as a homotetramer in dna replication.the eukaryotic gins heterotetramer, consisting of sld5, psf1, psf2, and psf3, participates in "cmg complex" formation with mini-chromosome maintenance (mcm) and cdc45 as a key component of a replicative helicase. there are only two homologs of the gins proteins in archaea, and these proteins, gins51 and gins23, form a heterotetrameric gins with a 2:2 molar ratio. the pyrococcus furiosus gins stimulates the atpase and helicase activities of its cognate mcm, whereas the sulfolobus solfataricus gin ...201121656171
crystal structure of the novel paia n-acetyltransferase from thermoplasma acidophilum involved in the negative control of sporulation and degradative enzyme production.gcn5-related n-acetyltransferases (gnats) are the most widely distributed acetyltransferase systems among all three domains of life. gnats appear to be involved in several key processes, including microbial antibiotic resistance, compacting eukaryotic dna, controlling gene expression, and protein synthesis. here, we report the crystal structure of a putative gnat ta0374 from thermoplasma acidophilum, a hyperacidophilic bacterium, that has been determined in an apo-form, in complex with its natur ...201121633970
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