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Bifunctional Enzyme (bifunctional + enzyme)
Selected AbstractsEnzymatic control of anhydrobiosis-related accumulation of trehalose in the sleeping chironomid, Polypedilum vanderplankiFEBS JOURNAL, Issue 20 2010Kanako Mitsumasu Larvae of an anhydrobiotic insect, Polypedilum vanderplanki, accumulate very large amounts of trehalose as a compatible solute on desiccation, but the molecular mechanisms underlying this accumulation are unclear. We therefore isolated the genes coding for trehalose metabolism enzymes, i.e. trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP) for the synthesis step, and trehalase (TREH) for the degradation step. Although computational prediction indicated that the alternative splicing variants (PvTps,/,) obtained encoded probable functional motifs consisting of a typical consensus domain of TPS and a conserved sequence of TPP, PvTps, did not exert activity as TPP, but only as TPS. Instead, a distinct gene (PvTpp) obtained expressed TPP activity. Previous reports have suggested that insect TPS is, exceptionally, a bifunctional enzyme governing both TPS and TPP. In this article, we propose that TPS and TPP activities in insects can be attributed to discrete genes. The translated product of the TREH ortholog (PvTreh) certainly degraded trehalose to glucose. Trehalose was synthesized abundantly, consistent with increased activities of TPS and TPP and suppressed TREH activity. These results show that trehalose accumulation observed during anhydrobiosis induction in desiccating larvae can be attributed to the activation of the trehalose synthetic pathway and to the depression of trehalose hydrolysis. [source] The domains carrying the opposing activities in adenylyltransferase are separated by a central regulatory domainFEBS JOURNAL, Issue 11 2007Paula Clancy Adenylyltransferase is a bifunctional enzyme that controls the enzymatic activity of dodecameric glutamine synthetase in Escherichia coli by reversible adenylylation and deadenylylation. Previous studies showed that the two similar but chemically distinct reactions are carried out by separate domains within adenylyltransferase. The N-terminal domain carries the deadenylylation activity, and the C-terminal domain carries the adenylylation activity [Jaggi R, van Heeswijk WC, Westerhoff HV, Ollis DL & Vasudevan SG (1997) EMBO J16, 5562,5571]. In this study, we further map the domain junctions of adenylyltransferase on the basis of solubility and enzymatic analysis of truncation constructs, and show for the first time that adenylyltransferase has three domains: the two activity domains and a central, probably regulatory (R), domain connected by interdomain Q-linkers (N-Q1-R-Q2-C). The various constructs, which have the opposing domain and or central domain removed, all retain their activity in the absence of their respective nitrogen status indicator, i.e. PII or PII-UMP. A panel of mAbs to adenylyltransferase was used to demonstrate that the cellular nitrogen status indicators, PII and PII-UMP, probably bind in the central regulatory domain to stimulate the adenylylation and deadenylylation reactions, respectively. In the light of these results, intramolecular signaling within adenylyltransferase is discussed. [source] Evolutionary analysis of fructose 2,6-bisphosphate metabolismIUBMB LIFE, Issue 3 2006Paul A. M. Michels Abstract Fructose 2,6-bisphosphate is a potent metabolic regulator in eukaryotic organisms; it affects the activity of key enzymes of the glycolytic and gluconeogenic pathways. The enzymes responsible for its synthesis and hydrolysis, 6-phosphofructo-2-kinase (PFK-2) and fructose-2,6-bisphosphatase (FBPase-2) are present in representatives of all major eukaryotic taxa. Results from a bioinformatics analysis of genome databases suggest that very early in evolution, in a common ancestor of all extant eukaryotes, distinct genes encoding PFK-2 and FBPase-2, or related enzymes with broader substrate specificity, fused resulting in a bifunctional enzyme both domains of which had, or later acquired, specificity for fructose 2,6-bisphosphate. Subsequently, in different phylogenetic lineages duplications of the gene of the bifunctional enzyme occurred, allowing the development of distinct isoenzymes for expression in different tissues, at specific developmental stages or under different nutritional conditions. Independently in different lineages of many unicellular eukaryotes one of the domains of the different PFK-2/FBPase-2 isoforms has undergone substitutions of critical catalytic residues, or deletions rendering some enzymes monofunctional. In a considerable number of other unicellular eukaryotes, mainly parasitic organisms, the enzyme seems to have been lost altogether. Besides the catalytic core, the PFK-2/FBPase-2 has often N- and C-terminal extensions which show little sequence conservation. The N-terminal extension in particular can vary considerably in length, and seems to have acquired motifs which, in a lineage-specific manner, may be responsible for regulation of catalytic activities, by phosphorylation or ligand binding, or for mediating protein-protein interactions. IUBMB Life, 58: 133 - 141, 2006 [source] Structure of the two-domain hexameric APS kinase from Thiobacillus denitrificans: structural basis for the absence of ATP sulfurylase activityACTA CRYSTALLOGRAPHICA SECTION D, Issue 10 2009Sean C. Gay The Tbd_0210 gene of the chemolithotrophic bacterium Thiobacillus denitrificans is annotated to encode a 60.5,kDa bifunctional enzyme with ATP sulfurylase and APS kinase activity. This putative bifunctional enzyme was cloned, expressed and structurally characterized. The 2.95,Å resolution X-ray crystal structure reported here revealed a hexameric assembly with D3 symmetry. Each subunit contains a large N-terminal sulfurylase-like domain and a C-terminal APS kinase domain reminiscent of the two-domain fungal ATP sulfurylases of Penicillium chrysogenum and Saccharomyces cerevisiae, which also exhibit a hexameric assembly. However, the T. denitrificans enzyme exhibits numerous structural and sequence differences in the N-terminal domain that render it inactive with respect to ATP sulfurylase activity. Surprisingly, the C-terminal domain does indeed display APS kinase activity, indicating that this gene product is a true APS kinase. Therefore, these results provide the first structural insights into a unique hexameric APS kinase that contains a nonfunctional ATP sulfurylase-like domain of unknown function. [source] Structures of dihydrofolate reductase-thymidylate synthase of Trypanosoma cruzi in the folate-free state and in complex with two antifolate drugs, trimetrexate and methotrexateACTA CRYSTALLOGRAPHICA SECTION D, Issue 7 2009Olga Senkovich The flagellate protozoan parasite Trypanosoma cruzi is the pathogenic agent of Chagas disease (also called American trypanosomiasis), which causes approximately 50,000 deaths annually. The disease is endemic in South and Central America. The parasite is usually transmitted by a blood-feeding insect vector, but can also be transmitted via blood transfusion. In the chronic form, Chagas disease causes severe damage to the heart and other organs. There is no satisfactory treatment for chronic Chagas disease and no vaccine is available. There is an urgent need for the development of chemotherapeutic agents for the treatment of T. cruzi infection and therefore for the identification of potential drug targets. The dihydrofolate reductase activity of T. cruzi, which is expressed as part of a bifunctional enzyme, dihydrofolate reductase,thymidylate synthase (DHFR-TS), is a potential target for drug development. In order to gain a detailed understanding of the structure,function relationship of T. cruzi DHFR, the three-dimensional structure of this protein in complex with various ligands is being studied. Here, the crystal structures of T. cruzi DHFR-TS with three different compositions of the DHFR domain are reported: the folate-free state, the complex with the lipophilic antifolate trimetrexate (TMQ) and the complex with the classical antifolate methotrexate (MTX). These structures reveal that the enzyme is a homodimer with substantial interactions between the two TS domains of neighboring subunits. In contrast to the enzymes from Cryptosporidium hominis and Plasmodium falciparum, the DHFR and TS active sites of T. cruzi lie on the same side of the monomer. As in other parasitic DHFR-TS proteins, the N-terminal extension of the T. cruzi enzyme is involved in extensive interactions between the two domains. The DHFR active site of the T. cruzi enzyme shows subtle differences compared with its human counterpart. These differences may be exploited for the development of antifolate-based therapeutic agents for the treatment of T. cruzi infection. [source] Crystallization and preliminary X-ray diffraction studies of FAD synthetase from Corynebacterium ammoniagenesACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 12 2009Beatriz Herguedas FAD synthetase from Corynebacterium ammoniagenes (CaFADS), a prokaryotic bifunctional enzyme that catalyses the phosphorylation of riboflavin as well as the adenylylation of FMN, has been crystallized using the hanging-drop vapour-diffusion method at 277,K. Diffraction-quality cubic crystals of native and selenomethionine-labelled (SeMet-CaFADS) protein belonged to the cubic space group P213, with unit-cell parameters a = b = c = 133.47,Å and a = b = c = 133.40,Å, respectively. Data sets for native and SeMet-containing crystals were collected to 1.95 and 2.42,Å resolution, respectively. [source] Crystallization and preliminary crystallographic analysis of bifunctional ,-glutamylcysteine synthetase,glutatione synthetase from Streptococcus agalactiaeACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2009Yasunori Nakashima ,-Glutamylcysteine synthetase,glutathione synthetase (,GCS-GS) is a bifunctional enzyme that catalyzes two consecutive steps of ATP-dependent peptide formation in glutathione biosynthesis. Streptococcus agalactiae,GCS-GS is a target for the development of potential therapeutic agents. ,GCS-GS was crystallized using the sitting-drop vapour-diffusion method. The crystals grew to dimensions of 0.3 × 0.2 × 0.2,mm under reducing conditions with 5,mM TCEP. X-ray data were collected to 2.8,Å resolution from a tetragonal crystal that belonged to space group I41. [source] Structure of N -acetylglucosamine-1-phosphate uridyltransferase (GlmU) from Mycobacterium tuberculosis in a cubic space groupACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 5 2009Sunil Kumar Verma GlmU is a bifunctional enzyme that catalyzes the final two steps in the biosynthesis of UDP-GlcNAc. Crystals of GlmU from Mycobacterium tuberculosis obtained using ammonium sulfate as a precipitant diffracted poorly (to 3.4,Å resolution) and displayed an unusually high solvent content (>80%) with sparse crystal packing that resulted in large solvent channels. With one molecule per asymmetric unit, the monomers from three neighbouring asymmetric units related by the crystal threefold formed a biological trimer. Although this is the first report of the structure of GlmU determined in a cubic crystal form, the trimeric arrangement here is similar to that observed for other GlmU structures determined in hexagonal (H3, H32, P6322) space groups. [source] Crystallization and preliminary X-ray analysis of a bifunctional catalase-phenol oxidase from Scytalidium thermophilumACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 5 2009Didem Sutay Kocabas Catalase-phenol oxidase from Scytalidium thermophilum is a bifunctional enzyme: its major activity is the catalase-mediated decomposition of hydrogen peroxide, but it also catalyzes phenol oxidation. To understand the structural basis of this dual functionality, the enzyme, which has been shown to be a tetramer in solution, has been purified by anion-exchange and gel-filtration chromatography and has been crystallized using the hanging-drop vapour-diffusion technique. Streak-seeding was used to obtain larger crystals suitable for X-ray analysis. Diffraction data were collected to 2.8,Å resolution at the Daresbury Synchrotron Radiation Source. The crystals belonged to space group P21 and contained one tetramer per asymmetric unit. [source] Structure of 3(17),-hydroxysteroid dehydrogenase (AKR1C21) holoenzyme from an orthorhombic crystal form: an insight into the bifunctionality of the enzymeACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 10 2007Urmi Dhagat Mouse 3(17),-hydroxysteroid dehydrogenase (AKR1C21) is a bifunctional enzyme that catalyses the oxidoreduction of the 3- and 17-hydroxy/keto groups of steroid substrates such as oestrogens, androgens and neurosteroids. The structure of the AKR1C21,NADPH binary complex was determined from an orthorhombic crystal belonging to space group P212121 at a resolution of 1.8,Å. In order to identify the factors responsible for the bifunctionality of AKR1C21, three steroid substrates including a 17-keto steroid, a 3-keto steroid and a 3,-hydroxysteroid were docked into the substrate-binding cavity. Models of the enzyme,coenzyme,substrate complexes suggest that Lys31, Gly225 and Gly226 are important for ligand recognition and orientation in the active site. [source] Expression, crystallization and preliminary crystallographic studies of a novel bifunctional N -acetylglutamate synthase/kinase from Xanthomonas campestris homologous to vertebrate N -acetylglutamate synthaseACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 12 2006Dashuang Shi A novel N -acetylglutamate synthase/kinase bifunctional enzyme of arginine biosynthesis that was homologous to vertebrate N -acetylglutamate synthases was identified in Xanthomonas campestris. The protein was overexpressed, purified and crystallized. The crystals belong to the hexagonal space group P6222, with unit-cell parameters a = b = 134.60, c = 192.11,Å, and diffract to about 3.0,Å resolution. Selenomethionine-substituted recombinant protein was produced and selenomethionine substitution was verified by mass spectroscopy. Multiple anomalous dispersion (MAD) data were collected at three wavelengths at SER-CAT, Advanced Photon Source, Argonne National Laboratory. Structure determination is under way using the MAD phasing method. [source] Investigation of the Substrate Specificity of Lacticin 481 Synthetase by Using Nonproteinogenic Amino AcidsCHEMBIOCHEM, Issue 5 2009Matthew R. Levengood Abstract One enzyme, many substrates. The substrate specificity of a lantibiotic biosynthetic enzyme, lacticin 481 synthetase, was probed by using synthetic prepeptides containing a variety of nonproteinogenic amino acids, including unnatural ,-amino acids, ,-amino acids, D -amino acids, and peptoids. Lantibiotics are peptide antimicrobial compounds that are characterized by the thioether-bridged amino acids lanthionine and methyllanthionine. For lacticin 481, these structures are installed in a two-step post-translational modification process by a bifunctional enzyme, lacticin 481 synthetase (LctM). LctM catalyzes the dehydration of Ser and Thr residues to generate dehydroalanine or dehydrobutyrine, respectively, and the subsequent intramolecular regio- and stereospecific Michael-type addition of cysteines onto the dehydroamino acids. In this study, semisynthetic substrates containing nonproteinogenic amino acids were prepared by expressed protein ligation and [3+2]-cycloaddition of azide and alkyne-functionalized peptides. LctM demonstrated broad substrate specificity toward substrates containing ,-amino acids, D -amino acids, and N -alkyl amino acids (peptoids) in certain regions of its peptide substrate. These findings showcase its promise for use in lantibiotic and peptide-engineering applications, whereby nonproteinogenic amino acids might impart improved stability or modulated biological activities. Furthermore, LctM permitted the incorporation of an alkyne-containing amino acid that can be utilized for the site-selective modification of mature lantibiotics and used in target identification. [source] | ||||||||||