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yeast as a model of human mitochondrial trna base substitutions: investigation of the molecular basis of respiratory defects.we investigate the relationships between acylation defects and structure alterations due to base substitutions in yeast mitochondrial (mt) trna(uur)(leu). the studied substitutions are equivalent to the a3243g and t3250c human pathogenetic trna mutations. our data show that both mutations can produce trna(uur)(leu) acylation defects, although to a different extent. for mutant a14g (equivalent to melas a3243g base substitution), the presence of the trna and its defective aminoacylation could be o ...200818065717
5s rrna: structure and function from head to toe.5s rrna is uniquely positioned so as to link together all of the functional centers of the ribosome. previous studies have supported the hypothesis that 5s rrna acts as a physical transducer of information, facilitating communication between the different functional centers and coordinating of the multiple events catalyzed by the ribosome. here, we present a synthesis of both structural and genetic information to construct a more detailed picture of how 5s rrna may act to transmit and coordinate ...200518074004
dodecamer rotor ring defines h+/atp ratio for atp synthesis of prokaryotic v-atpase from thermus thermophilus.atp synthesis by v-atpase from the thermophilic bacterium thermus thermophilus driven by the acid-base transition was investigated. the rate of atp synthesis increased in parallel with the increase in proton motive force (pmf) >110 mv, which is composed of a difference in proton concentration (deltaph) and the electrical potential differences (deltapsi) across membranes. the optimum rate of synthesis reached 85 s(-1), and the h(+)/atp ratio of 4.0 +/- 0.1 was obtained. atp was synthesized at a c ...200718077374
soj (para) dna binding is mediated by conserved arginines and is essential for plasmid segregation.soj is a member of the para family of atpases involved in plasmid and chromosomal segregation. it binds nonspecifically and cooperatively to dna although the function of this binding is unknown. here, we show that mutation of conserved arginine residues that map to the surface of bacillus subtilis soj caused only minimal effects on nucleotide-dependent dimerization but had dramatic effects on dna binding. using a model plasmid partitioning system in escherichia coli, we find that soj dna-binding ...200718077387
akap18 contains a phosphoesterase domain that binds amp.protein kinase a anchoring proteins (akaps), defined by their capacity to target the camp-dependent protein kinase to distinct subcellular locations, function as molecular scaffolds mediating the assembly of multicomponent complexes to integrate and organise multiple signalling events. despite their central importance in regulating cellular processes, little is known regarding their diverse structures and molecular mechanisms. here, using bioinformatics and x-ray crystallography, we define a cen ...200818082768
akap18 contains a phosphoesterase domain that binds amp.protein kinase a anchoring proteins (akaps), defined by their capacity to target the camp-dependent protein kinase to distinct subcellular locations, function as molecular scaffolds mediating the assembly of multicomponent complexes to integrate and organise multiple signalling events. despite their central importance in regulating cellular processes, little is known regarding their diverse structures and molecular mechanisms. here, using bioinformatics and x-ray crystallography, we define a cen ...200818082768
escherichia coli cytosolic glycerophosphodiester phosphodiesterase (ugpq) requires mg2+, co2+, or mn2+ for its enzyme activity.escherichia coli cytosolic glycerophosphodiester phosphodiesterase, ugpq, functions in the absence of other proteins encoded by the ugp operon and requires mg2+, mn2+, or co2+, in contrast to ca2+-dependent periplasmic glycerophosphodiester phosphodiesterase, glpq. ugpq has broad substrate specificity toward various glycerophosphodiesters, producing sn-glycerol-3-phosphate and the corresponding alcohols. ugpq accumulates under conditions of phosphate starvation, suggesting that it allows the uti ...200818083802
escherichia coli cytosolic glycerophosphodiester phosphodiesterase (ugpq) requires mg2+, co2+, or mn2+ for its enzyme activity.escherichia coli cytosolic glycerophosphodiester phosphodiesterase, ugpq, functions in the absence of other proteins encoded by the ugp operon and requires mg2+, mn2+, or co2+, in contrast to ca2+-dependent periplasmic glycerophosphodiester phosphodiesterase, glpq. ugpq has broad substrate specificity toward various glycerophosphodiesters, producing sn-glycerol-3-phosphate and the corresponding alcohols. ugpq accumulates under conditions of phosphate starvation, suggesting that it allows the uti ...200818083802
structure of the minimized alpha/beta-hydrolase fold protein from thermus thermophilus hb8.the gene encoding ttha1544 is a singleton found in the thermus thermophilus hb8 genome and encodes a 131-amino-acid protein. the crystal structure of ttha1544 has been determined at 2.0 a resolution by the single-wavelength anomalous dispersion method in order to elucidate its function. there are two molecules in the asymmetric unit. each molecule consists of four alpha-helices and six beta-strands, with the beta-strands composing a central beta-sheet. a structural homology search revealed that ...200718084077
structure of bacillus subtilis superoxide dismutase.the soda gene of bacillus subtilis was expressed in escherichia coli, purified and crystallized. the crystal structure of mnsod was solved by molecular replacement with four dimers per asymmetric unit and refined to an r factor of 21.1% at 1.8 a resolution. the dimer structure is very similar to that of the related enzyme from b. anthracis. larger structural differences were observed with the human mnsod, which has one less helix in the helical domain and a longer loop between two beta-strands a ...200718084079
an unexpected outcome of surface engineering an integral membrane protein: improved crystallization of cytochrome ba(3) from thermus thermophilus.past work has shown that it is feasible to mutate surface residues of soluble proteins and to a lesser extent membrane proteins in order to improve their crystallization behavior. described here is a successful application of this approach to the integral membrane protein thermus thermophilus cytochrome ba(3) oxidase. two mutant forms of this enzyme (i-k258r and i-k258r/ii-e4q) were created in which symmetrical crystal contacts within crystals of wild-type enzyme were modified. these mutant prot ...200718084085
the ins and outs of translation. 200718086326
mycobacterium tuberculosis cyp130: crystal structure, biophysical characterization, and interactions with antifungal azole drugs.cyp130 is one of the 20 mycobacterium tuberculosis cytochrome p450 enzymes, only two of which, cyp51 and cyp121, have so far been studied as individually expressed proteins. here we characterize a third heterologously expressed m. tuberculosis cytochrome p450, cyp130, by uv-visible spectroscopy, isothermal titration calorimetry, and x-ray crystallography, including determination of the crystal structures of ligand-free and econazole-bound cyp130 at a resolution of 1.46 and 3.0a(,) respectively. ...200818089574
mycobacterium tuberculosis cyp130: crystal structure, biophysical characterization, and interactions with antifungal azole drugs.cyp130 is one of the 20 mycobacterium tuberculosis cytochrome p450 enzymes, only two of which, cyp51 and cyp121, have so far been studied as individually expressed proteins. here we characterize a third heterologously expressed m. tuberculosis cytochrome p450, cyp130, by uv-visible spectroscopy, isothermal titration calorimetry, and x-ray crystallography, including determination of the crystal structures of ligand-free and econazole-bound cyp130 at a resolution of 1.46 and 3.0a(,) respectively. ...200818089574
mammalian 2',3' cyclic nucleotide phosphodiesterase (cnp) can function as a trna splicing enzyme in vivo.yeast and plant trna splicing entails discrete healing and sealing steps catalyzed by a trna ligase that converts the 2',3' cyclic phosphate and 5'-oh termini of the broken trna exons to 3'-oh/2'-po4 and 5'-po4 ends, respectively, then joins the ends to yield a 2'-po4, 3'-5' phosphodiester splice junction. the junction 2'-po4 is removed by a trna phosphotransferase, tpt1. animal cells have two potential trna repair pathways: a yeast-like system plus a distinctive mechanism, also present in archa ...200818094118
assembly of the 5' and 3' minor domains of 16s ribosomal rna as monitored by tethered probing from ribosomal protein s20.the ribosomal protein (r-protein) s20 is a primary binding protein. as such, it interacts directly and independently with the 5' domain as well as the 3' minor domain of 16s ribosomal rna (rrna) in minimal particles and the fully assembled 30s subunit. the interactions observed between r-protein s20 and the 5' domain of 16s rrna are quite extensive, while those between r-protein s20 and the 3' minor domain are significantly more limited. in this study, directed hydroxyl radical probing mediated ...200818155048
assembly of the 5' and 3' minor domains of 16s ribosomal rna as monitored by tethered probing from ribosomal protein s20.the ribosomal protein (r-protein) s20 is a primary binding protein. as such, it interacts directly and independently with the 5' domain as well as the 3' minor domain of 16s ribosomal rna (rrna) in minimal particles and the fully assembled 30s subunit. the interactions observed between r-protein s20 and the 5' domain of 16s rrna are quite extensive, while those between r-protein s20 and the 3' minor domain are significantly more limited. in this study, directed hydroxyl radical probing mediated ...200818155048
a flexible peptide tether controls accessibility of a unique c-terminal rna-binding domain in leucyl-trna synthetases.a unique c-terminal domain extension is required by most leucyl-trna synthetases (leurs) for aminoacylation. in one exception, the enzymatic activity of yeast mitochondrial leurs is actually impeded by its own c-terminal domain. it was proposed that the yeast mitochondrial leurs has compromised its aminoacylation activity to some extent and adapted its c terminus for a second role in rna splicing, which is also essential. x-ray crystal structures of the leurs-trna complex show that the 60 residu ...200818155724
a flexible peptide tether controls accessibility of a unique c-terminal rna-binding domain in leucyl-trna synthetases.a unique c-terminal domain extension is required by most leucyl-trna synthetases (leurs) for aminoacylation. in one exception, the enzymatic activity of yeast mitochondrial leurs is actually impeded by its own c-terminal domain. it was proposed that the yeast mitochondrial leurs has compromised its aminoacylation activity to some extent and adapted its c terminus for a second role in rna splicing, which is also essential. x-ray crystal structures of the leurs-trna complex show that the 60 residu ...200818155724
crystal structure of bacillus stearothermophilus uvra provides insight into atp-modulated dimerization, uvrb interaction, and dna binding.the nucleotide excision repair pathway corrects many structurally unrelated dna lesions. damage recognition in bacteria is performed by uvra, a member of the abc atpase superfamily whose functional form is a dimer with four nucleotide-binding domains (nbds), two per protomer. in the 3.2 a structure of uvra from bacillus stearothermophilus, we observe that the nucleotide-binding sites are formed in an intramolecular fashion and are not at the dimer interface as is typically found in other abc atp ...200818158267
crystal structure of bacillus stearothermophilus uvra provides insight into atp-modulated dimerization, uvrb interaction, and dna binding.the nucleotide excision repair pathway corrects many structurally unrelated dna lesions. damage recognition in bacteria is performed by uvra, a member of the abc atpase superfamily whose functional form is a dimer with four nucleotide-binding domains (nbds), two per protomer. in the 3.2 a structure of uvra from bacillus stearothermophilus, we observe that the nucleotide-binding sites are formed in an intramolecular fashion and are not at the dimer interface as is typically found in other abc atp ...200818158267
protein factors in pre-mrna 3'-end processing.most eukaryotic mrna precursors (premrnas) must undergo extensive processing, including cleavage and polyadenylation at the 3'-end. processing at the 3'-end is controlled by sequence elements in the pre-mrna (cis elements) as well as protein factors. despite the seeming biochemical simplicity of the processing reactions, more than 14 proteins have been identified for the mammalian complex, and more than 20 proteins have been identified for the yeast complex. the 3'-end processing machinery also ...200818158581
genome evolution and the emergence of fruiting body development in myxococcus xanthus.lateral gene transfer (lgt) is thought to promote speciation in bacteria, though well-defined examples have not been put forward.200718159227
fitness of streptococcus pneumoniae fluoroquinolone-resistant strains with topoisomerase iv recombinant genes.the low prevalence of ciprofloxacin-resistant (cp r) streptococcus pneumoniae isolates carrying recombinant topoisomerase iv genes could be attributed to a fitness cost imposed by the horizontal transfer, which often implies the acquisition of larger-than-normal pare-parc intergenic regions. a study of the transcription of these genes and of the fitness cost for 24 isogenic cp r strains was performed. six first-level transformants were obtained either with pcr products containing the parc quinol ...200818160515
fitness of streptococcus pneumoniae fluoroquinolone-resistant strains with topoisomerase iv recombinant genes.the low prevalence of ciprofloxacin-resistant (cp r) streptococcus pneumoniae isolates carrying recombinant topoisomerase iv genes could be attributed to a fitness cost imposed by the horizontal transfer, which often implies the acquisition of larger-than-normal pare-parc intergenic regions. a study of the transcription of these genes and of the fitness cost for 24 isogenic cp r strains was performed. six first-level transformants were obtained either with pcr products containing the parc quinol ...200818160515
early steps in the dna base excision/single-strand interruption repair pathway in mammalian cells.base excision repair (ber) is an evolutionarily conserved process for maintaining genomic integrity by eliminating several dozen damaged (oxidized or alkylated) or inappropriate bases that are generated endogenously or induced by genotoxicants, predominantly, reactive oxygen species (ros). ber involves 4-5 steps starting with base excision by a dna glycosylase, followed by a common pathway usually involving an ap-endonuclease (ape) to generate 3' oh terminus at the damage site, followed by repai ...200818166975
kinetic pathway of pyrophosphorolysis by a retrotransposon reverse transcriptase.dna and rna polymerases use a common phosphoryl transfer mechanism for base addition that requires two or three acidic amino acid residues at their active sites. we previously showed, for the reverse transcriptase (rt) encoded by the yeast retrotransposon ty1, that one of the three conserved active site aspartates (d(211)) can be substituted by asparagine and still retain in vitro polymerase activity, although in vivo transposition is lost. transposition is partially restored by second site supp ...200818167548
structures of open (r) and close (t) states of prephenate dehydratase (pdt)--implication of allosteric regulation by l-phenylalanine.the enzyme prephenate dehydratase (pdt) converts prephenate to phenylpyruvate in l-phenylalanine biosynthesis. pdt is allosterically regulated by l-phe and other amino acids. we report the first crystal structures of pdt from staphylococcus aureus in a relaxed (r) state and pdt from chlorobium tepidum in a tense (t) state. the two enzymes show low sequence identity (27.3%) but the same prototypic architecture and domain organization. both enzymes are tetramers (dimer of dimers) in crystal and so ...200818171624
structures of open (r) and close (t) states of prephenate dehydratase (pdt)--implication of allosteric regulation by l-phenylalanine.the enzyme prephenate dehydratase (pdt) converts prephenate to phenylpyruvate in l-phenylalanine biosynthesis. pdt is allosterically regulated by l-phe and other amino acids. we report the first crystal structures of pdt from staphylococcus aureus in a relaxed (r) state and pdt from chlorobium tepidum in a tense (t) state. the two enzymes show low sequence identity (27.3%) but the same prototypic architecture and domain organization. both enzymes are tetramers (dimer of dimers) in crystal and so ...200818171624
identification and role of functionally important motifs in the 970 loop of escherichia coli 16s ribosomal rna.the 970 loop (helix 31) of escherichia coli 16s ribosomal rna contains two modified nucleotides, m(2)g966 and m(5)c967. positions a964, a969, and c970 are conserved among the bacteria, archaea, and eukarya. the nucleotides present at positions 965, 966, 967, 968, and 971, however, are only conserved and unique within each domain. all organisms contain a modified nucleoside at position 966, but the type of the modification is domain specific. biochemical and structure studies have placed this loo ...200818177894
identification and role of functionally important motifs in the 970 loop of escherichia coli 16s ribosomal rna.the 970 loop (helix 31) of escherichia coli 16s ribosomal rna contains two modified nucleotides, m(2)g966 and m(5)c967. positions a964, a969, and c970 are conserved among the bacteria, archaea, and eukarya. the nucleotides present at positions 965, 966, 967, 968, and 971, however, are only conserved and unique within each domain. all organisms contain a modified nucleoside at position 966, but the type of the modification is domain specific. biochemical and structure studies have placed this loo ...200818177894
structural biophysics of the nusb:nuse antitermination complex.in prokaryotic transcription regulation, several host factors form a complex with rna polymerase and the nascent mrna. as part of a process known as antitermination, two of these host factors, nusb and nuse, bind to form a heterodimer, which interacts with a specific boxa site on the rna. the nusb/nuse/boxa rna ternary complex interacts with the rna polymerase transcription complex, stabilizing it and allowing transcription past premature termination points. the nusb protein also binds boxa rna ...200818177898
structural biophysics of the nusb:nuse antitermination complex.in prokaryotic transcription regulation, several host factors form a complex with rna polymerase and the nascent mrna. as part of a process known as antitermination, two of these host factors, nusb and nuse, bind to form a heterodimer, which interacts with a specific boxa site on the rna. the nusb/nuse/boxa rna ternary complex interacts with the rna polymerase transcription complex, stabilizing it and allowing transcription past premature termination points. the nusb protein also binds boxa rna ...200818177898
structural insight into the mechanism of substrate specificity of aedes kynurenine aminotransferase.aedes aegypti kynurenine aminotransferase (aekat) is a multifunctional aminotransferase. it catalyzes the transamination of a number of amino acids and uses many biologically relevant alpha-keto acids as amino group acceptors. aekat also is a cysteine s-conjugate beta-lyase. the most important function of aekat is the biosynthesis of kynurenic acid, a natural antagonist of nmda and alpha7-nicotinic acetylcholine receptors. here, we report the crystal structures of aekat in complex with its best ...200818186649
cross-crystal averaging reveals that the structure of the peptidyl-transferase center is the same in the 70s ribosome and the 50s subunit.recently, two crystal structures of the thermus thermophilus 70s ribosome in the same functional state were determined at 2.8 and 3.7 a resolution but were different throughout. the most functionally significant structural differences are in the conformation of the peptidyl-transferase center (ptc) and the interface between the ptc and the cca end of the p-site trna. likewise, the 3.7 a ptc differed from the functionally equivalent structure of the haloarcula marismortui 50s subunit. to ascertai ...200818187576
the elongation factor rfah and the initiation factor sigma bind to the same site on the transcription elongation complex.rna polymerase is a target for numerous regulatory events in all living cells. recent studies identified a few "hot spots" on the surface of bacterial rna polymerase that mediate its interactions with diverse accessory proteins. prominent among these hot spots, the beta' subunit clamp helices serve as a major binding site for the initiation factor sigma and for the elongation factor rfah. furthermore, the two proteins interact with the nontemplate dna strand in transcription complexes and thus m ...200818195372
evolution of mal abc transporter operons in the thermococcales and thermotogales.the mal genes that encode maltose transporters have undergone extensive lateral transfer among ancestors of the archaea thermococcus litoralis and pyrococcus furiosus. bacterial hyperthermophiles of the order thermotogales live among these archaea and so may have shared in these transfers. the genome sequence of thermotoga maritima bears evidence of extensive acquisition of archaeal genes, so its ancestors clearly had the capacity to do so. we examined deep phylogenetic relationships among the m ...200818197971
nondecarboxylating and decarboxylating isocitrate dehydrogenases: oxalosuccinate reductase as an ancestral form of isocitrate dehydrogenase.isocitrate dehydrogenase (icdh) from hydrogenobacter thermophilus catalyzes the reduction of oxalosuccinate, which corresponds to the second step of the reductive carboxylation of 2-oxoglutarate in the reductive tricarboxylic acid cycle. in this study, the oxidation reaction catalyzed by h. thermophilus icdh was kinetically analyzed. as a result, a rapid equilibrium random-order mechanism was suggested. the affinities of both substrates (isocitrate and nad+) toward the enzyme were extremely low ...200818203822
chaperones in control of protein disaggregation.the chaperone protein network controls both initial protein folding and subsequent maintenance of proteins in the cell. although the native structure of a protein is principally encoded in its amino-acid sequence, the process of folding in vivo very often requires the assistance of molecular chaperones. chaperones also play a role in a post-translational quality control system and thus are required to maintain the proper conformation of proteins under changing environmental conditions. many fact ...200818216875
solution structure of ribosomal protein l40e, a unique c4 zinc finger protein encoded by archaeon sulfolobus solfataricus.the ribosomal protein l40e from archaeon sulfolobus solfataricus is a component of the 50s ribosomal subunit. l40e is a 56-residue, highly basic protein that contains a c4 zinc finger motif, crkc_x(10)_crrc. homologs are found in both archaea and eukaryotes but are not present in bacteria. eukaryotic genomes encode l40e as a ubiquitin-fusion protein. l40e was absent from the crystal structure of euryarchaeota 50s ribosomal subunit. here we report the three-dimensional solution structure of l40e ...200818218710
the solution structure of ribosomal protein s17e from methanobacterium thermoautotrophicum: a structural homolog of the ff domain.the ribosomal protein s17e from the archaeon methanobacterium thermoautotrophicum is a component of the 30s ribosomal subunit. s17e is a 62-residue protein conserved in archaea and eukaryotes and has no counterparts in bacteria. mammalian s17e is a phosphoprotein component of eukaryotic ribosomes. archaeal s17e proteins range from 59 to 79 amino acids, and are about half the length of the eukaryotic homologs which have an additional c-terminal region. here we report the three-dimensional solutio ...200818218711
molecular adaptation and expression evolution following duplication of genes for organellar ribosomal protein s13 in rosids.gene duplication has been a fundamental process in the evolution of eukaryotic genomes. after duplication one copy (or both) can undergo divergence in sequence, expression pattern, and function. two divergent copies of the ribosomal protein s13 gene (rps13) of chloroplast origin are found in the nucleus of the rosids arabidopsis, gossypium, and glycine. one encodes chloroplast-imported rps13 (nucp rps13), while the other encodes mitochondria-imported rps13 (numit rps13). the rps13 gene has been ...200818221556
molecular and physiological role of the trehalose-hydrolyzing alpha-glucosidase from thermus thermophilus hb27.trehalose supports the growth of thermus thermophilus strain hb27, but the absence of obvious genes for the hydrolysis of this disaccharide in the genome led us to search for enzymes for such a purpose. we expressed a putative alpha-glucosidase gene (ttc0107), characterized the recombinant enzyme, and found that the preferred substrate was alpha,alpha-1,1-trehalose, a new feature among alpha-glucosidases. the enzyme could also hydrolyze the disaccharides kojibiose and sucrose (alpha-1,2 linkage) ...200818223075
novel monofunctional histidinol-phosphate phosphatase of the dddd superfamily of phosphohydrolases.the ton_0887 gene was identified as the missing histidinol-phosphate phosphatase (holpase) in the hyperthermophilic archaeon "thermococcus onnurineus" na1. the protein contained conserved motifs of the dddd superfamily of phosphohydrolase, and the recombinantly expressed protein exhibited strong holpase activity. in this study, we functionally assessed for the first time the monofunctional dddd-type holpase, which is organized in the gene cluster.200818223080
novel members of glycoside hydrolase family 13 derived from environmental dna.starch and pullulan-modifying enzymes of the alpha-amylase family (glycoside hydrolase family 13) have several industrial applications. to date, most of these enzymes have been derived from isolated organisms. to increase the number of members of this enzyme family, in particular of the thermophilic representatives, we have applied a consensus primer-based approach using dna from enrichments from geothermal habitats. with this approach, we succeeded in isolating three new enzymes: a neopullulana ...200818223106
functional site profiling and electrostatic analysis of cysteines modifiable to cysteine sulfenic acid.cysteine sulfenic acid (cys-soh), a reversible modification, is a catalytic intermediate at enzyme active sites, a sensor for oxidative stress, a regulator of some transcription factors, and a redox-signaling intermediate. this post-translational modification is not random: specific features near the cysteine control its reactivity. to identify features responsible for the propensity of cysteines to be modified to sulfenic acid, a list of 47 proteins (containing 49 known cys-soh sites) was compi ...200818227433
evidence for the involvement of acid/base chemistry in the reaction catalyzed by the type ii isopentenyl diphosphate/dimethylallyl diphosphate isomerase from staphylococcus aureus.the type ii isopentenyl diphosphate/dimethylallyl diphosphate isomerase (idi-2) is a flavin mononucleotide (fmn)-dependent enzyme that catalyzes the reversible isomerization of isopentenyl pyrophosphate (ipp) to dimethylallyl pyrophosphate (dmapp), a reaction with no net change in redox state of the coenzyme or substrate. here, uv-vis spectral analysis of the idi-2 reaction revealed the accumulation of a reduced neutral dihydroflavin intermediate when the reduced enzyme was incubated with ipp or ...200818229948
structural insights into ribosome recycling factor interactions with the 70s ribosome.at the end of translation in bacteria, ribosome recycling factor (rrf) is used together with elongation factor g to recycle the 30s and 50s ribosomal subunits for the next round of translation. in x-ray crystal structures of rrf with the escherichia coli 70s ribosome, rrf binds to the large ribosomal subunit in the cleft that contains the peptidyl transferase center. upon binding of either e. coli or thermus thermophilus rrf to the e. coli ribosome, the tip of ribosomal rna helix 69 in the large ...200818234219
the native cyclobutane pyrimidine dimer photolyase of rice is phosphorylated.the cyclobutane pyrimidine dimer (cpd) is a major type of dna damage induced by ultraviolet b (uvb) radiation. cpd photolyase, which absorbs blue/uva light as an energy source to monomerize dimers, is a crucial factor for determining the sensitivity of rice (oryza sativa) to uvb radiation. here, we purified native class ii cpd photolyase from rice leaves. as the final purification step, cpd photolyase was bound to cpd-containing dna conjugated to magnetic beads and then released by blue-light ir ...200818235036
functional role of the additional domains in inulosucrase (isla) from leuconostoc citreum cw28.inulosucrase (isla) from leuconostoc citreum cw28 belongs to a new subfamily of multidomain fructosyltransferases (ftfs), containing additional domains from glucosyltransferases. it is not known what the function of the additional domains in this subfamily is.200818237396
assays for transfer rna-dependent amino acid biosynthesis.selenocysteinyl-trna(sec), cysteinyl-trna(cys), glutaminyl-trna(gln), and asparaginyl-trna(asn) in many organisms are formed in an indirect pathway in which a non-cognate amino acid is first attached to the trna. this non-cognate amino acid is then converted to the cognate amino acid by a trna-dependent modifying enzyme. the in vitro characterization of these modifying enzymes is challenging due to the fact the substrate, aminoacyl-trna, is labile and requires a prior enzymatic step to be synthe ...200818241795
a single mammalian mitochondrial translation initiation factor functionally replaces two bacterial factors.the mechanism of translation in eubacteria and organelles is thought to be similar. in eubacteria, the three initiation factors if1, if2, and if3 are vital. although the homologs of if2 and if3 are found in mammalian mitochondria, an if1 homolog has never been detected. here, we show that bovine mitochondrial if2 (if2(mt)) complements e. coli containing a deletion of the if2 gene (e. coli deltainfb). we find that if1 is no longer essential in an if2(mt)-supported e. coli deltainfb strain. furthe ...200818243113
cooperative damage recognition by uvra and uvrb: identification of uvra residues that mediate dna binding.nucleotide excision repair (ner) is responsible for the recognition and removal of numerous structurally unrelated dna lesions. in prokaryotes, the proteins uvra, uvrb and uvrc orchestrate the recognition and excision of aberrant lesions from dna. despite the progress we have made in understanding the ner pathway, it remains unclear how the uvra dimer interacts with dna to facilitate dna damage recognition. the purpose of this study was to define amino acid residues in uvra that provide binding ...200818248777
from one amino acid to another: trna-dependent amino acid biosynthesis.aminoacyl-trnas (aa-trnas) are the essential substrates for translation. most aa-trnas are formed by direct aminoacylation of trna catalyzed by aminoacyl-trna synthetases. however, a smaller number of aa-trnas (asn-trna, gln-trna, cys-trna and sec-trna) are made by synthesizing the amino acid on the trna by first attaching a non-cognate amino acid to the trna, which is then converted to the cognate one catalyzed by trna-dependent modifying enzymes. asn-trna or gln-trna formation in most prokaryo ...200818252769
functional interaction between bases c1049 in domain ii and g2751 in domain vi of 23s rrna in escherichia coli ribosomes.the factor-binding center within the escherichia coli ribosome is comprised of two discrete domains of 23s rrna: the gtpase-associated region (gar) in domain ii and the sarcin-ricin loop in domain vi. these two regions appear to collaborate in the factor-dependent events that occur during protein synthesis. current x-ray crystallography of the ribosome shows an interaction between c1049 in the gar and g2751 in domain vi. we have confirmed this interaction by site-directed mutagenesis and chemica ...200818252772
rimo, a miab-like enzyme, methylthiolates the universally conserved asp88 residue of ribosomal protein s12 in escherichia coli.ribosomal protein s12 undergoes a unique posttranslational modification, methylthiolation of residue d88, in escherichia coli and several other bacteria. using mass spectrometry, we have identified the enzyme responsible for this modification in e. coli, the ylig gene product. this enzyme, which we propose be called rimo, is a radical-s-adenosylmethionine protein that bears strong sequence similarity to miab, which methylthiolates trna. we show that rimo and miab represent two of four subgroups ...200818252828
functional importance of individual rrna 2'-o-ribose methylations revealed by high-resolution phenotyping.ribosomal rnas contain numerous modifications at specific nucleotides. despite their evolutionary conservation, the functional role of individual 2'-o-ribose methylations in rrna is not known. a distinct family of small nucleolar rnas, box c/d snornas, guides the methylating complex to specific rrna sites. using a high-resolution phenotyping approach, we characterized 20 box c/d snorna gene deletions for altered growth dynamics under a wide array of environmental perturbations, encompassing intr ...200818256246
crystallization and preliminary x-ray characterization of qued from bacillus subtilis, an enzyme involved in queuosine biosynthesis.qued (previously named ykvk) is one of several enzymes involved in the biosynthesis of the hypermodified nucleoside queuosine. queuosine is incorporated into trna at position 34 of four trnas: trna(his), trna(asp), trna(asn) and trna(tyr). the crystallization and preliminary x-ray crystallographic studies of qued are described here. the recombinant protein from bacillus subtilis was overproduced in escherichia coli and crystallized using the hanging-drop vapor-diffusion method from 25% peg 600, ...200818259064
structure/function analysis of yeast ribosomal protein l2.ribosomal protein l2 is a core element of the large subunit that is highly conserved among all three kingdoms. l2 contacts almost every domain of the large subunit rrna and participates in an intersubunit bridge with the small subunit rrna. it contains a solvent-accessible globular domain that interfaces with the solvent accessible side of the large subunit that is linked through a bridge to an extension domain that approaches the peptidyltransferase center. here, screening of randomly generated ...200818263608
a novel chromate reductase from thermus scotoductus sa-01 related to old yellow enzyme.bacteria can reduce toxic and carcinogenic cr(vi) to insoluble and less toxic cr(iii). thermus scotoductus sa-01, a south african gold mine isolate, has been shown to be able to reduce a variety of metals, including cr(vi). here we report the purification to homogeneity and characterization of a novel chromate reductase. the oxidoreductase is a homodimeric protein, with a monomer molecular mass of approximately 36 kda, containing a noncovalently bound flavin mononucleotide cofactor. the chromate ...200818263719
decreased aminoacylation in pathology-related mutants of mitochondrial trnatyr is associated with structural perturbations in trna architecture.a growing number of human pathologies are ascribed to mutations in mitochondrial trna genes. here, we report biochemical investigations on three mt-trna(tyr) molecules with point substitutions associated with diseases. the mutations occur in the atypical t- and d-loops at positions homologous to those involved in the tertiary interaction network of canonical trnas. they do not correspond to tyrosine identity positions and likely do not contact the mitochondrial tyrosyl-trna synthetase during the ...200818268021
ph-dependent structural changes of helix 69 from escherichia coli 23s ribosomal rna.helix 69 in 23s rrna is a region in the ribosome that participates in a considerable number of rna-rna and rna-protein interactions. conformational flexibility is essential for such a region to interact and accommodate protein factors at different stages of protein biosynthesis. in this study, ph-dependent structural and stability changes were observed for helix 69 through a variety of spectroscopic techniques, such as circular dichroism spectroscopy, uv melting, and nuclear magnetic resonance s ...200818268024
genetic and chemical modifiers of a cug toxicity model in drosophila.non-coding cug repeat expansions interfere with the activity of human muscleblind-like (mbnl) proteins contributing to myotonic dystrophy 1 (dm1). to understand this toxic rna gain-of-function mechanism we developed a drosophila model expressing 60 pure and 480 interrupted cug repeats in the context of a non-translatable rna. these flies reproduced aspects of the dm1 pathology, most notably nuclear accumulation of cug transcripts, muscle degeneration, splicing misregulation, and diminished muscl ...200818270582
still looking for the magic spot: the crystallographically defined binding site for ppgpp on rna polymerase is unlikely to be responsible for rrna transcription regulation.identification of the rna polymerase (rnap) binding site for ppgpp, a central regulator of bacterial transcription, is crucial for understanding its mechanism of action. a recent high-resolution x-ray structure defined a ppgpp binding site on thermus thermophilus rnap. we report here effects of ppgpp on 10 mutant escherichia coli rnaps with substitutions for the analogous residues within 3-4 a of the ppgpp binding site in the t. thermophilus cocrystal. none of the substitutions in e. coli rnap s ...200818272182
expression of recombinant rhipicephalus (boophilus) microplus, r. annulatus and r. decoloratus bm86 orthologs as secreted proteins in pichia pastoris.rhipicephalus (boophilus) spp. ticks economically impact on cattle production in africa and other tropical and subtropical regions of the world. tick vaccines constitute a cost-effective and environmentally friendly alternative to tick control. the r. microplus bm86 protective antigen has been produced by recombinant dna technology and shown to protect cattle against tick infestations.200818275601
bifunctional nmn adenylyltransferase/adp-ribose pyrophosphatase: structure and function in bacterial nad metabolism.bacterial nadm-nudix is a bifunctional enzyme containing a nicotinamide mononucleotide (nmn) adenylyltransferase and an adp-ribose (adpr) pyrophosphatase domain. while most members of this enzyme family, such as that from a model cyanobacterium synechocystis sp., are involved primarily in nicotinamide adenine dinucleotide (nad) salvage/recycling pathways, its close homolog in a category-a biodefense pathogen, francisella tularensis, likely plays a central role in a recently discovered novel path ...200818275811
protein structure fitting and refinement guided by cryo-em density.for many macromolecular assemblies, both a cryo-electron microscopy map and atomic structures of its component proteins are available. here we describe a method for fitting and refining a component structure within its map at intermediate resolution (<15 a). the atomic positions are optimized with respect to a scoring function that includes the crosscorrelation coefficient between the structure and the map as well as stereochemical and nonbonded interaction terms. a heuristic optimization that r ...200818275820
the application of fast-nmr for the identification of novel drug discovery targets.the continued success of genome sequencing projects has resulted in a wealth of information, but 40-50% of identified genes correspond to hypothetical proteins or proteins of unknown function. the functional annotation screening technology by nmr (fast-nmr) screen was developed to assign a biological function for these unannotated proteins with a structure solved by the protein structure initiative. fast-nmr is based on the premise that a biological function can be described by a similarity in b ...200818275915
on the evolution of the trna-dependent amidotransferases, gatcab and gatde.glutaminyl-trna synthetase and asparaginyl-trna synthetase evolved from glutamyl-trna synthetase and aspartyl-trna synthetase, respectively, after the split in the last universal communal ancestor (luca). glutaminyl-trna(gln) and asparaginyl-trna(asn) were likely formed in luca by amidation of the mischarged species, glutamyl-trna(gln) and aspartyl-trna(asn), by trna-dependent amidotransferases, as is still the case in most bacteria and all known archaea. the amidotransferase gatcab is found in ...200818279892
drosophila muscleblind is involved in troponin t alternative splicing and apoptosis.muscleblind-like proteins (mbnl) have been involved in a developmental switch in the use of defined cassette exons. such transition fails in the ctg repeat expansion disease myotonic dystrophy due, in part, to sequestration of mbnl proteins by cug repeat rna. four protein isoforms (mbla-d) are coded by the unique drosophila muscleblind gene.200818286170
fine structure of the promoter-sigma region 1.2 interaction.we recently proposed that a nontemplate strand base in the discriminator region of bacterial promoters, the region between the -10 element and the transcription start site, makes sequence-specific contacts to region 1.2 of the sigma subunit of escherichia coli rna polymerase (rnap). because rrna promoters contain sequences within the discriminator region that are suboptimal for interaction with sigma1.2, these promoters have the kinetic properties required for regulation by the rnap-binding fact ...200818287032
role of 16s ribosomal rna methylations in translation initiation in escherichia coli.translation initiation from the ribosomal p-site is the specialty of the initiator trnas (trna(fmet)). presence of the three consecutive g-c base pairs (g29-c41, g30-c40 and g31-c39) in their anticodon stems, a highly conserved feature of the initiator trnas across the three kingdoms of life, has been implicated in their preferential binding to the p-site. how this feature is exploited by ribosomes has remained unclear. using a genetic screen, we have isolated an escherichia coli strain, carryin ...200818288206
conversion of beta-carotene into astaxanthin: two separate enzymes or a bifunctional hydroxylase-ketolase protein?abstract: astaxanthin is a xanthophyll of great interest in animal nutrition and human health. the market prospect in the nutraceutics industries for this health-protective molecule is very promising. astaxanthin is synthesized by several bacteria, algae and plants from beta-carotene by the sequential action of two enzymes: a beta-carotene, 3,3'-hydroxylase that introduces an hydroxyl group at the 3 (and 3') positions of each of the two beta-ionone rings of beta-carotene, and a beta-carotene ket ...200818289382
yeast cytosine deaminase mutants with increased thermostability impart sensitivity to 5-fluorocytosine.prodrug gene therapy (pgt) is a treatment strategy in which tumor cells are transfected with a 'suicide' gene that encodes a metabolic enzyme capable of converting a nontoxic prodrug into a potent cytotoxin. one of the most promising pgt enzymes is cytosine deaminase (cd), a microbial salvage enzyme that converts cytosine to uracil. cd also converts 5-fluorocytosine (5fc) to 5-fluorouracil, an inhibitor of dna synthesis and rna function. over 150 studies of cd-mediated pgt applications have been ...200818291415
methanothermobacter thermautotrophicus trna gln confines the amidotransferase gatcab to asparaginyl-trna asn formation.many prokaryotes form the amide aminoacyl-trnas glutaminyl-trna and asparaginyl-trna by trna-dependent amidation of the mischarged trna species, glutamyl-trna(gln) or aspartyl-trna(asn). archaea employ two such amidotransferases, gatcab and gatde, while bacteria possess only one, gatcab. the methanothermobacter thermautotrophicus gatde is slightly more efficient using asn as an amide donor than gln (k(cat)/k(m) of 5.4 s(-1)/mm and 1.2 s(-1)/mm, respectively). unlike the bacterial gatcab enzymes ...200818291416
genomic and proteomic analysis of phieco32, a novel escherichia coli bacteriophage.a novel bacteriophage infecting escherichia coli was isolated during a large-scale screen for bacteriophages that may be used for therapy of mastitis in cattle. the 77,554-bp genome of the bacteriophage, named phieco32, was sequenced and annotated, and its virions were characterized by electron microscopy and proteomics. two phieco32-encoded proteins that interact with host rna polymerase were identified. one of them is an ecf family sigma factor that may be responsible for transcription of some ...200818294652
murine leukemia virus reverse transcriptase: structural comparison with hiv-1 reverse transcriptase.recent x-ray crystal structure determinations of moloney murine leukemia virus reverse transcriptase (momlv rt) have allowed for more accurate structure/function comparisons to hiv-1 rt than were formerly possible. previous biochemical studies of momlv rt in conjunction with knowledge of sequence homologies to hiv-1 rt and overall fold similarities to rts in general, provided a foundation upon which to build. in addition, numerous crystal structures of the momlv rt fingers/palm subdomain had als ...200818294720
translational attenuation and mrna stabilization as mechanisms of erm(b) induction by erythromycin.translational attenuation has been proposed to be the mechanism by which the erm(b) gene is induced. here, we report genetic and biochemical evidence, obtained by using erythromycin as the inducing antibiotic, that supports this hypothesis. we also show that erythromycin increases the level of the erm(b) transcript by stalling the ribosome on the leader mrna and thereby facilitating the stabilization and processing of the mrna. erythromycin-induced mrna stabilization and processing were observed ...200818299414
structural insights into an equilibrium folding intermediate of an archaeal ankyrin repeat protein.repeat proteins are widespread in nature, with many of them functioning as binding molecules in protein-protein recognition. their simple structural architecture is used in biotechnology for generating proteins with high affinities to target proteins. recent folding studies of ankyrin repeat (ar) proteins revealed a new mechanism of protein folding. the formation of an intermediate state is rate limiting in the folding reaction, suggesting a scaffold function of this transient state for intrinsi ...200818305166
mitochondrial deafness alleles confer misreading of the genetic code.despite the fact that important genetic diseases are caused by mutant mitochondrial ribosomes, the molecular mechanisms by which such ribosomes result in a clinical phenotype remain largely unknown. the absence of experimental models for mitochondrial diseases has also prevented the rational search for therapeutic interventions. here, we report on the construction of bacterial hybrid ribosomes that contain various versions of the mitochondrial decoding region of ribosomal rna. we show that the p ...200818308926
evolution of acceptor stem trna recognition by class ii prolyl-trna synthetase.aminoacyl-trna synthetases (aars) are an essential family of enzymes that catalyze the attachment of amino acids to specific trnas during translation. previously, we showed that base-specific recognition of the trna(pro) acceptor stem is critical for recognition by escherichia coli prolyl-trna synthetase (prors), but not for human prors. to further delineate species-specific differences in acceptor stem recognition, atomic group mutagenesis was used to probe the role of sugar-phosphate backbone ...200818310681
"hot cores" in proteins: comparative analysis of the apolar contact area in structures from hyper/thermophilic and mesophilic organisms.a wide variety of stabilizing factors have been invoked so far to elucidate the structural basis of protein thermostability. these include, amongst the others, a higher number of ion-pairs interactions and hydrogen bonds, together with a better packing of hydrophobic residues. it has been frequently observed that packing of hydrophobic side chains is improved in hyperthermophilic proteins, when compared to their mesophilic counterparts. in this work, protein crystal structures from hyper/thermop ...200818312638
real-time electron transfer in respiratory complex i.electron transfer in complex i from escherichia coli was investigated by an ultrafast freeze-quench approach. the reaction of complex i with nadh was stopped in the time domain from 90 mus to 8 ms and analyzed by electron paramagnetic resonance (epr) spectroscopy at low temperatures. the data show that after binding of the first molecule of nadh, two electrons move via the fmn cofactor to the iron-sulfur (fe/s) centers n1a and n2 with an apparent time constant of approximately 90 mus, implying t ...200818316732
structure of mouse adp-ribosylhydrolase 3 (marh3).adp-ribosylation is a reversible and covalent post-translational modification in which the attachment of adp-ribose is catalyzed by adp-ribosyltransferases and the removal of adp-ribose is catalyzed by adp-ribosylhydrolases. adp-ribosylhydrolase 3 from mouse, consisting of 347 amino-acid residues, has been cloned, purified and crystallized. the three-dimensional structure has been resolved at a resolution of 1.8 a. the structure constitutes a compact all-alpha-helical protein with two mg(2+) ion ...200818323597
purification, crystallization and preliminary x-ray diffraction analysis of aspartate semialdehyde dehydrogenase (rv3708c) from mycobacterium tuberculosis.aspartate semialdehyde dehydrogenase from mycobacterium tuberculosis (asd, asadh, rv3708c), which is the second enzyme in the lysine/homoserine-biosynthetic pathways, has been expressed heterologously in escherichia coli. the enzyme was purified using affinity and gel-filtration chromatographic techniques and crystallized in two different crystal forms. preliminary diffraction data analysis suggested the presence of up to four monomers in the asymmetric unit of the orthorhombic crystal form a an ...200818323599
expression, purification, crystallization and preliminary x-ray characterization of two crystal forms of stationary-phase survival e protein from campylobacter jejuni.survival e (sure) protein from campylobacter jejuni, a gram-negative mesophile, has been overexpressed in escherichia coli as a soluble protein, successfully purified and crystallized in two distinct crystal forms. the first form belongs to space group p2(1)2(1)2(1), with a tetramer in the asymmetric unit and unit-cell parameters a = 80.5, b = 119.0, c = 135.3 a. the second form belongs to space group c2, with unit-cell parameters a = 121.4, b = 47.1, c = 97.8 a, and contains a dimer in the asym ...200818323612
on helicases and other motor proteins.helicases are molecular machines that utilize energy derived from atp hydrolysis to move along nucleic acids and to separate base-paired nucleotides. the movement of the helicase can also be described as a stationary helicase that pumps nucleic acid. recent structural data for the hexameric e1 helicase of papillomavirus in complex with single-stranded dna and mgadp has provided a detailed atomic and mechanistic picture of its atp-driven dna translocation. the structural and mechanistic features ...200818329872
refining homology models by combining replica-exchange molecular dynamics and statistical potentials.a protocol is presented for the global refinement of homology models of proteins. it combines the advantages of temperature-based replica-exchange molecular dynamics (remd) for conformational sampling and the use of statistical potentials for model selection. the protocol was tested using 21 models. of these 14 were models of 10 small proteins for which high-resolution crystal structures were available, the remainder were targets of the recent caspr exercise. it was found that remd in combinatio ...200818338384
quality control of bacterial mrna decoding and decay.studies in eukaryotes and prokaryotes have revealed that gene expression is not only controlled through altering the rate of transcription but also through varying rates of translation and mrna decay. indeed, the expression level of a protein is strongly affected by the steady state level of its mrna. rna decay can, along with transcription, play an important role in regulating gene expression by fine-tuning the steady state level of a given transcript and affecting its subsequent decoding durin ...200818342642
altering the coenzyme preference of xylose reductase to favor utilization of nadh enhances ethanol yield from xylose in a metabolically engineered strain of saccharomyces cerevisiae.abstract:200818346277
the bacterial and mitochondrial ribosomal a-site molecular switches possess different conformational substates.the a site of the small ribosomal subunit participates in the fidelity of decoding by switching between two states, a resting 'off' state and an active decoding 'on' state. eight crystal structures of rna duplexes containing two minimal decoding a sites of the homo sapiens mitochondrial wild-type, the a1555g mutant or bacteria have been solved. the resting 'off' state of the mitochondrial wild-type a site is surprisingly different from that of the bacterial a site. the mitochondrial a1555g mutan ...200818346970
structural insight into the mechanism of substrate specificity and catalytic activity of an hd-domain phosphohydrolase: the 5'-deoxyribonucleotidase yfbr from escherichia coli.hd-domain phosphohydrolases have nucleotidase and phosphodiesterase activities and play important roles in the metabolism of nucleotides and in signaling. we present three 2.1-a-resolution crystal structures (one in the free state and two complexed with natural substrates) of an hd-domain phosphohydrolase, the escherichia coli 5'-nucleotidase yfbr. the free-state structure of yfbr contains a large cavity accommodating the metal-coordinating hd motif (h33, h68, d69, and d137) and other conserved ...200818353368
genome comparison and proteomic characterization of thermus thermophilus bacteriophages p23-45 and p74-26: siphoviruses with triplex-forming sequences and the longest known tails.the genomes of two closely related lytic thermus thermophilus siphoviruses with exceptionally long (approximately 800 nm) tails, bacteriophages p23-45 and p74-26, were sequenced completely. the p23-45 genome consists of 84,201 bp with 117 putative open reading frames (orfs), and the p74-26 genome has 83,319 bp and 116 putative orfs. the two genomes are 92% identical with 113 orfs shared. only 25% of phage gene product functions can be predicted from similarities to proteins and protein domains w ...200818355836
roles of pgaabcd genes in synthesis, modification, and export of the escherichia coli biofilm adhesin poly-beta-1,6-n-acetyl-d-glucosamine.the linear homopolymer poly-beta-1,6-n-acetyl-d-glucosamine (beta-1,6-glcnac; pga) serves as an adhesin for the maintenance of biofilm structural stability in diverse eubacteria. its function in escherichia coli k-12 requires the gene products of the pgaabcd operon, all of which are necessary for biofilm formation. pgac is an apparent glycosyltransferase that is required for pga synthesis. using a monoclonal antibody directed against e. coli pga, we now demonstrate that pgad is also needed for p ...200818359807
evolution of the arginase fold and functional diversity.novel structural superfamilies can be identified among the large number of protein structures deposited in the protein data bank based on conservation of fold in addition to conservation of amino acid sequence. since sequence diverges more rapidly than fold in protein evolution, proteins with little or no significant sequence identity are occasionally observed to adopt similar folds, thereby reflecting unanticipated evolutionary relationships. here, we review the unique alpha/beta fold first obs ...200818360740
the structure of lepa, the ribosomal back translocase.lepa is a highly conserved elongation factor that promotes the back translocation of trnas on the ribosome during the elongation cycle. we have determined the crystal structure of lepa from escherichia coli at 2.8-a resolution. the high degree of sequence identity between lepa and ef-g is reflected in the structural similarity between the individual homologous domains of lepa and ef-g. however, the orientation of domains iii and v in lepa differs from their orientations in ef-g. lepa also contai ...200818362332
functional segregation of a predicted "hinge" site within the beta-strand linkers of escherichia coli leucyl-trna synthetase.some aminoacyl-trna synthetases (aarss) employ an editing mechanism to ensure the fidelity of protein synthesis. leucyl-trna synthetase (leurs), isoleucyl-trna synthetase (ilers), and valyl-trna synthetase (valrs) share a common insertion, called the cp1 domain, which is responsible for clearing misformed products. this discrete domain is connected to the main body of the enzyme via two beta-strand tethers. the cp1 hydrolytic editing active site is located approximately 30 a from the aminoacylat ...200818363380
crystal structure of human spermine synthase: implications of substrate binding and catalytic mechanism.the crystal structures of two ternary complexes of human spermine synthase (ec 2.5.1.22), one with 5'-methylthioadenosine and spermidine and the other with 5'-methylthioadenosine and spermine, have been solved. they show that the enzyme is a dimer of two identical subunits. each monomer has three domains: a c-terminal domain, which contains the active site and is similar in structure to spermidine synthase; a central domain made up of four beta-strands; and an n-terminal domain with remarkable s ...200818367445
structural biology of proline catabolism.the proline catabolic enzymes proline dehydrogenase and delta(1)-pyrroline-5-carboxylate dehydrogenase catalyze the 4-electron oxidation of proline to glutamate. these enzymes play important roles in cellular redox control, superoxide generation, apoptosis and cancer. in some bacteria, the two enzymes are fused into the bifunctional enzyme, proline utilization a. here we review the three-dimensional structural information that is currently available for proline catabolic enzymes. crystal structu ...200818369526
whep domains direct noncanonical function of glutamyl-prolyl trna synthetase in translational control of gene expression.the heterotetrameric gait complex suppresses translation of selected mrnas in interferon-gamma-activated monocytic cells. specificity is dictated by glutamyl-prolyl trna synthetase (eprs) binding to a 3'utr element in target mrnas. eprs consists of two synthetase cores joined by a linker containing three whep domains of unknown function. here we show the critical role of eprs whep domains in targeting and regulating gait complex binding to rna. the upstream whep pair directs high-affinity bindin ...200818374644
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