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Cofactor
Kinds of Cofactor Terms modified by Cofactor Selected AbstractsObservation of Terahertz Vibrations in the Nitrogenase FeMo Cofactor by Femtosecond Pump,Probe Spectroscopy,ANGEWANDTE CHEMIE, Issue 23 2010Ines Delfino Dr. Hochgepumpt: Die Dynamik des durch Resonanz-Raman-Spektroskopie nicht charakterisierbaren FeMo-Cofaktors (siehe Bild) wurde nun mit kohärenter Puls-Schwingungsspektroskopie als Sonde untersucht: Ein Laserpuls mit sichtbarem Licht (15,fs) pumpte die Probe in einen angeregten elektronischen Zustand, und ein zweiter Puls (<10,fs) untersuchte die Änderung in der Transmission als Funktion des Zeitabstands. [source] Eosin Y-Sensitized Artificial Photosynthesis by Highly Efficient Visible-Light-Driven Regeneration of Nicotinamide CofactorCHEMBIOCHEM, Issue 10 2009Sahng Ha Lee Abstract Dye-sensitized photosynthesis: Eosin Y (EY), a dye photosensitizer, works efficiently as a molecular photoelectrode by catalyzing the visible-light-driven electron-transfer reaction for efficient regeneration of NADH through a photosensitizer,electron relay dyad. Injection of the photosensitized electron resulted in highly accelerated regeneration of NADH, which can be used by glutamate dehydrogenase for the photosynthesis of L -glutamate. [source] ChemInform Abstract: Aurophilicity as a Cofactor in Crystal Engineering.CHEMINFORM, Issue 23 2002Au(I) Bimetallic Assembly., Dicyanoaurate(I) Anion as a Building Block in a Novel Co(II) Abstract ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a "Full Text" option. The original article is trackable via the "References" option. [source] Noncovalent Modulation of pH-Dependent Reactivity of a Mn,Salen Cofactor in Myoglobin with Hydrogen PeroxideCHEMISTRY - A EUROPEAN JOURNAL, Issue 30 2009Jun-Long Zhang Dr. Abstract To demonstrate protein modulation of metal-cofactor reactivity through noncovalent interactions, pH-dependent sulfoxidation and 2,2,-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) oxidation reactivity of a designed myoglobin (Mb) containing non-native Mn,salen complex (1) was investigated using H2O2 as the oxidant. Incorporation of 1 inside the Mb resulted in an increase in the turnover numbers through exclusion of water from the metal complex and prevention of Mn,salen dimer formation. Interestingly, the presence of protein in itself is not enough to confer the increase activity as mutation of the distal His64 in Mb to Phe to remove hydrogen-bonding interactions resulted in no increase in the turnover numbers, while mutation His64 to Arg, another residue with ability to hydrogen-bond interactions, resulted in an increase in reactivity. These results strongly suggest that the distal ligand His64, through its hydrogen-bonding interaction, plays important roles in enhancing and fine-tuning reactivity of the Mn,salen complex. Nonlinear least-squares fitting of rate versus pH plots demonstrates that 1,Mb(H64X) (X=H, R and F) and the control Mn,salen 1 exhibit pKa values varying from pH,6.4 to 8.3, and that the lower pKa of the distal ligand in 1,Mb(H64X), the higher the reactivity it achieves. Moreover, in addition to the pKa at high pH, 1,Mb displays another pKa at low pH, with pKa of 5.0±0.08. A comparison of the effect of different pH on sulfoxidation and ABTS oxidation indicates that, while the intermediate produced at low pH conditions could only perform sulfoxidation, the intermediate at high pH could oxidize both sulfoxides and ABTS. Such a fine-control of reactivity through hydrogen-bonding interactions by the distal ligand to bind, orient and activate H2O2 is very important for designing artificial enzymes with dramatic different and tunable reactivity from catalysts without protein scaffolds. [source] A New Regeneration System for Oxidized Nicotinamide CofactorsADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 9 2009Seda Aksu Abstract A novel regeneration system for oxidized nicotinamide cofactors (NAD+ and NADP+) is presented. By combining 2,2,-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid (ABTS)-catalyzed oxidation of NAD(P)H with laccase-catalyzed utilization of molecular oxygen as terminal oxidant, a simple chemo-enzymatic NAD(P)+ regeneration method is achieved. Thus, the advantages of both worlds, chemical oxidation of reduced nicotinamide cofactors and laccase-catalyzed utilization of oxygen from air are combined in a simple and generally applicable new approach for biooxidation catalysis. This new application of the well-known laccase-mediator system (LMS) is successfully used to promote alcohol dehydrogenase-catalyzed oxidation reactions of primary and secondary alcohols. Already under non-optimized conditions, high turnover numbers of >300 and >16000 were obtained for the nicotinamide cofactor and ABTS, respectively. In this communication, we present the proof-of-principle and initial characterization of the proposed new regeneration system. [source] Removal of the PsaF Polypeptide Biases Electron Transfer in Favor of the PsaB Branch of Cofactors in Triton X-100 Photosystem I Complexes from Synechococcus sp.PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 6 2008PCC 700 Continuous wave (CW) and transient electron paramagnetic resonance studies have implied that when PsaF is removed genetically, the double reduction of A1A is facile, the lifetime of A1A, is shorter and the ratio of fast to slow kinetic phases increases in PS I complexes isolated with Triton X-100 (Van der Est, A., A. I. Valieva, Y. E. Kandrashkin, G. Shen, D. A. Bryant and J. H. Golbeck [2004] Biochemistry43, 1264,1275). Changes in the lifetimes of A1A, and A1B, are characteristic of mutants involving the quinone binding sites, but changes in the relative amplitudes of A1A, and A1B, are characteristic of mutants involving the primary electron acceptors, A0A and A0B. Here, we measured the fast and slow phases of electron transfer from A1B, and A1A, to FX in psaF and psaE psaF null mutants using time-resolved CW and pump-probe optical absorption spectroscopy. The lifetime of the fast kinetic phase was found to be unaltered, but the lifetime of the slow kinetic phase was shorter in the psaF null mutant and even more so in the psaE psaF null mutant. Concomitantly, the amplitude of the fast kinetic phase increased by a factor of 1.8 and 2.0 in the psaF and psaE psaF null mutants, respectively, at the expense of the slow kinetic phase. The change in ratio of the fast to slow kinetic phases is explained as either a redirection of electron transfer through A1B at the expense of A1A, or a shortening of the lifetime of A1A, to become identical to that of A1B,. The constant lifetime and the characteristics of the near-UV spectrum of the fast kinetic phase favor the former explanation. A unified hypothesis is presented of a displacement of the A-jk(1) ,-helix and switchback loop, which would weaken the H-bond from Leu722 to A1A, accounting for the acceleration of the slow kinetic phase, as well as weaken the H-bond from Tyr696 to A0A, accounting for the bias of electron transfer in favor of the PsaB branch of cofactors. [source] Biocatalytic Friedel,Crafts Alkylation Using Non-natural Cofactors,ANGEWANDTE CHEMIE, Issue 50 2009Harald Stecher Dipl.-Ing. Eine neuartige biokatalytische C-C-Verknüpfung, die äquivalent zur Friedel-Crafts-Alkylierung ist, wird vorgestellt. S -Adenosyl- L -methionin (SAM), der Haupt-Methyldonor bei Methyltransferase(Mtase)-katalysierten biologischen Methylierungen, kann Alkylierungen bewirken (siehe Schema). Diese Enzyme akzeptieren nichtnatürliche Cofaktoren und können andere Funktionalitäten als Me auf aromatische Substrate übertragen. [source] Computational Design of Four-Helix Bundle Proteins That Bind Nonbiological CofactorsBIOTECHNOLOGY PROGRESS, Issue 1 2008Andreas Lehmann Recent work is discussed concerning the computational design of four-helix bundle proteins that form complexes with nonbiological cofactors. Given that often there are no suitable natural proteins to provide starting points in the creation of such nonbiological systems, computational design is well suited for the design and study of new protein-cofactor complexes. Recent design efforts are presented in the context of prior work on the de novo design and engineering of porphyrin-binding four-helix bundle proteins and current developments in nonlinear optical materials. Such protein-nonbiological cofactor complexes stand to enable new applications in protein science and materials research. [source] Replacement of Natural Cofactors by Selected Hydrogen Peroxide Donors or Organic Peroxides Results in Improved Activity for CYP3A4 and CYP2D6CHEMBIOCHEM, Issue 6 2006Amandine Chefson P450 enzymes made easier to use in synthesis. The use of cumene hydroperoxide with CYP2D6 or CYP3A4 leads to as much as a twofold increase in the rate of product formation compared to the use of the natural cofactors. [source] Precursors of Biological Cofactors from Ultraviolet Irradiation of Circumstellar/Interstellar Ice AnaloguesCHEMISTRY - A EUROPEAN JOURNAL, Issue 17 2005Uwe J. Meierhenrich Priv.-Doz. Abstract Biological cofactors include functionalized derivatives of cyclic tetrapyrrole structures that incorporate different metal ions. They build up structural partnerships with proteins, which play a crucial role in biochemical reactions. Porphyrin, chlorin, bacteriochlorin, and corrin are the basic structures of cofactors (heme, chlorophyll, bacteriochlorophyll, siroheme, F 430, and vitamin B12). Laboratory and theoretical work suggest that the molecular building blocks of proteins (,-amino acids) and nucleic acids (carbohydrates, purines, and pyrimidines) were generated under prebiotic conditions. On the other hand, experimental data on the prebiotic chemistry of cofactors are rare. We propose to search directly for the pathways of the formation of cofactors in the laboratory. Herein we report on the detection of N-heterocycles and amines in the room-temperature residue obtained after photo- and thermal processing of an interstellar ice analogue under high vacuum at 12 K. Among them, hexahydro-1,3,5-triazine and its derivatives, together with monopyrrolic molecules, are precursors of porphinoid cofactors. Hexahydropyrimidine was also detected. This is the first detection of these compounds in experiments simulating circumstellar/interstellar conditions. Except for 2-aminopyrrole and 2,4-diaminofuran, which were only found in 13C-labeled experiments, all the reported species were detected in both 12C- and 13C-labeled experiments, excluding contamination. The molecules reported here might be present in circumstellar/interstellar grains and cometary dust and could be detected by the Stardust and Rosetta missions. [source] Maternal uniparental isodisomy is responsible for serious molybdenum cofactor deficiencyDEVELOPMENTAL MEDICINE & CHILD NEUROLOGY, Issue 9 2010HAKAN GÜMÜ Molybdenum cofactor (MoCo) deficiency is a rare autosomal recessive inherited metabolic disorder resulting in the combined deficiency of aldehyde oxidase, xanthine dehydrogenase, and sulfite oxidase. We report a male infant with MoCo deficiency whose clinical findings consisted of microcephaly, intractable seizures soon after birth, feeding difficulties, and developmental delay. Sequencing of MOCS1, MOCS2, and GEPH genes, and single nucleotide polymorphism genotyping array analysis showed, to our knowledge, unusual inheritance of MoCo deficiency/maternal uniparental isodisomy for the first time in the literature. At 10 months of age, he now has microcephaly and developmental delay, and his seizures are controlled with phenobarbital, clonozepam, and vigabatrin therapy. [source] Does gastroesophageal reflux contribute to the development of chronic sinusitis?DISEASES OF THE ESOPHAGUS, Issue 6 2006A review of the evidence SUMMARY., Although recent studies suggest that gastroesophageal reflux disease (GERD) may contribute to a variety of ear, nose and throat and pulmonary diseases, the cause-and-effect relationship for the vast majority remains far from proven. In this article, the evidence supporting a possible causal association between GERD and chronic sinusitis has been reviewed. The evidence would suggest that: (i) a higher prevalence of GERD and a different esophagopharyngeal distribution of the gastric refluxate occurs in patients with chronic sinusitis unresponsive to conventional medical and surgical therapy compared to the general population; (ii) a biologically plausible pathogenetic mechanism exists whereby GERD may result in chronic sinusitis; and (iii) clinical manifestations of chronic sinusitis respond variably to antireflux therapy. While these findings suggest that GERD may contribute to the pathogenesis of chronic sinusitis in some patients, it is apparent that the quality of the evidence supporting each of these three lines of evidence is low and therefore does not conclusively establish a cause-and-effect relationship. A number of unresolved issues regarding prevalence, pathophysiological mechanism, diagnosis and treatment exist that deserve further investigation in order to solidify the relationship between GERD and chronic sinusitis. In conclusion, given the possible relationship between GERD and chronic sinusitis, until more convincing data are available, it may be prudent to investigate for GERD as a potential cofactor or initiating factor in patients with chronic sinusitis when no other etiology exists, or in those whose symptoms are unresponsive to conventional therapies. [source] Disposable Amperometric Sensors for Thiols with Special Reference to GlutathioneELECTROANALYSIS, Issue 18 2008Dipankar Bhattacharyay Abstract The antioxidant ,reduced glutathione' tripeptide is conventionally called glutathione (GSH). The oxidized form is a sulfur-sulfur linked compound, known as glutathione disulfide (GSSG). Glutathione is an essential cofactor for antioxidant enzymes; it provides protection also for the mitochondria against endogenous oxygen radicals. The ratio of these two forms can act as a marker for oxidative stress. The majority of the methods available for estimation of both the forms of glutathione are based on colorimetric and electrochemical assays. In this study, electrochemical sensors were developed for the estimation of both GSH and GSSG. Two different types of transducers were used: i) screen-printed three-electrode disposable sensor (SPE) containing carbon working electrode, carbon counter electrode and silver/silver chloride reference electrode; ii) three-electrode disposable system (CDE) consisting of three copper electrodes. 5,5,-dithiobis(2-nitrobenzoic acid) (DTNB) was used as detector element for estimation of total reduced thiol content. The enzyme glutathione reductase along with a co-enzyme reduced nicotinamide adenine dinucleotide phosphate was used to estimate GSSG. By combining the two methods GSH can also be estimated. The detector elements were immobilized on the working electrodes of the sensors by bulk polymerization of acrylamide. The responses were observed amperometrically. The detection limit for thiol (GSH) was less than 0.6,ppm when DTNB was used, whereas for GSSG it was less than 0.1,ppm. [source] Comparative Study of Cyanobacteria as Biocatalysts for the Asymmetric Synthesis of Chiral Building BlocksENGINEERING IN LIFE SCIENCES (ELECTRONIC), Issue 2 2006J. Havel Abstract The three representative cyanobacteria, Synechococcus PCC7942, Anabaena variabilis, and Nostoc muscorum, were studied for their ability to asymmetrically reduce the prochiral ketones 2,-3,-4,-5,-6,-pentafluoroacetophenone, ethyl 4-chloroacetate, 4-chloroacetophenone, and ethylbenzoylacetate to the corresponding chiral alcohols. Photosynthesis as well as respiration was applied for intracellular regeneration of the NAD(P)H cofactor. It was shown for the first time that all cyanobacteria were able to reduce the prochiral ketones asymmetrically without light for cofactor regeneration. By comparison of the cell specific product formation capacities of cyanobacteria with typical heterotrophic whole cell biocatalysts in batch processes, it is shown that comparable or, in some cases, better performances at high enantiomeric excess (ee > 99.8,%) are obtained. As a consequence of a generally strong product inhibition, in situ product removal must be applied in order to restore process efficiency when using cyanobacteria as biocatalysts. [source] Folate, colorectal carcinogenesis, and DNA methylation: Lessons from animal studiesENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 1 2004Young-In Kim Abstract Folate, a water-soluble B vitamin and cofactor in one-carbon transfer, is an important nutritional factor that may modulate the development of colorectal cancer (CRC). Epidemiologic and clinical studies indicate that dietary folate intake and blood folate levels are inversely associated with CRC risk. Collectively, these studies suggest an , 40% reduction in the risk of CRC in individuals with the highest dietary folate intake compared with those with the lowest intake. Animal studies using chemical and genetically predisposed rodent models have provided considerable support for a causal relationship between folate depletion and colorectal carcinogenesis as well as a dose-dependent protective effect of folate supplementation. However, animal studies also have shown that the dose and timing of folate intervention are critical in providing safe and effective chemoprevention; exceptionally high supplemental folate levels and folate intervention after microscopic neoplastic foci are established in the colorectal mucosa promote, rather than suppress, colorectal carcinogenesis. These animal studies, in conjunction with clinical observations, suggest that folate possesses dual modulatory effects on carcinogenesis depending on the timing and dose of folate intervention. Folate deficiency has an inhibitory effect, whereas folate supplementation has a promoting effect on the progression of established neoplasms. In contrast, folate deficiency in normal epithelial tissues appears to predispose them to neoplastic transformation, and modest levels of folate supplementation suppress the development of tumors in normal tissues. Notwithstanding the limitations associated with animal models, these studies suggest that the optimal timing and dose of folate intervention must be established for safe and effective chemoprevention in humans. Folate is an important factor in DNA synthesis, stability, and integrity, the repair aberrations of which have been implicated in colorectal carcinogenesis. Folate may also modulate DNA methylation, which is an important epigenetic determinant in gene expression (an inverse relationship), in the maintenance of DNA integrity and stability, in chromosomal modifications, and in the development of mutations. A mechanistic understanding of how folate status modulates colorectal carcinogenesis further strengthens the case for a causal relationship and provides insight into a possible chemopreventive role of folate. Environ. Mol. Mutagen. 44:10,25, 2004. © 2004 Wiley-Liss, Inc. [source] C5a anaphylatoxin as a product of complement activation up-regulates the complement inhibitory factor H in rat Kupffer cellsEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 11 2004Gerald Schlaf Abstract The 155-kDa complement regulator factor H (FH) is the predominant soluble regulatory protein of the complement system. It acts as a cofactor for the factor I-mediated conversion of the component C3b to iC3b, competes with factor B for a binding site on C3b and C3(H2O) and promotes the dissociation of the C3bBb complex. The primary site of synthesis is the liver, i.e. FH-specific mRNA and protein were identified in both hepatocytes (HC) and Kupffer cells (KC). Previous studies in rat primary HC and KC had shown that the proinflammatory cytokine IFN-, influences the balance between activation and inhibition of the complement system through up-regulation of the inhibitory FH. In this study we show that C5a, as a product of complement activation, stimulates the expression of FH-specific mRNA and protein in KC and thus induces a negative feedback. Quantitative-competitive RT-PCR showed an approximate threefold C5a-induced up-regulation of FH. ELISA analyses revealed a corresponding increase in FH protein in the supernatants of KC. The up-regulation of FH was completely inhibited by the C5a-blocking monoclonal antibody 6-9F. Furthermore, an involvement of LPS and IFN-, was excluded, which strongly indicates a direct effect of C5a on the expression of FH in KC. [source] Functional characterization of an orphan cupin protein from Burkholderia xenovorans reveals a mononuclear nonheme Fe2+ -dependent oxygenase that cleaves ,-diketonesFEBS JOURNAL, Issue 20 2009Stefan Leitgeb Cupins constitute a large and widespread superfamily of ,-barrel proteins in which a mononuclear metal site is both a conserved feature of the structure and a source of functional diversity. Metal-binding residues are contributed from two core motifs that provide the signature for the superfamily. On the basis of conservation of this two-motif structure, we have identified an ORF in the genome of Burkholderia xenovorans that encodes a novel cupin protein (Bxe_A2876) of unknown function. Recombinant Bxe_A2876, as isolated from Escherichia coli cell extract, was a homotetramer in solution, and showed mixed fractional occupancy of its 16.1 kDa subunit with metal ligands (0.06 copper; 0.11 iron; 0.17 zinc). Our quest for possible catalytic functions of Bxe_A2876 focused on Cu2+ and Fe2+ oxygenase activities known from related cupin enzymes. Fe2+ elicited enzymatic catalysis of O2 -dependent conversion of various ,-diketone substrates via a nucleophilic mechanism of carbon,carbon bond cleavage. Data from X-ray absorption spectroscopy (XAS) support a five-coordinate or six-coordinate Fe2+ center where the metal is bound by three imidazole nitrogen atoms at 1.98 Å. Results of structure modeling studies suggest that His60, His62 and His102 are the coordinating residues. In the ,best-fit' model, one or two oxygens from water and a carboxylate oxygen (presumably from Glu96) are further ligands of Fe2+ at estimated distances of 2.04 Å and 2.08 Å, respectively. The three-histidine Fe2+ site of Bxe_A2876 is compared to the mononuclear nonheme Fe2+ centers of the structurally related cysteine dioxygenase and acireductone dioxygenase, which also use a facial triad of histidines for binding of their metal cofactor but promote entirely different substrate transformations. [source] A zinc finger HIT domain-containing protein, ZNHIT-1, interacts with orphan nuclear hormone receptor Rev-erb, and removes Rev-erb,-induced inhibition of apoCIII transcriptionFEBS JOURNAL, Issue 20 2007Jiadong Wang The orphan receptors, Rev-erb, and Rev-erb,, are members of the nuclear receptor superfamily and specifically repress apolipoprotein CIII (apoCIII) gene expression in rats and humans. Moreover, Rev-erb, null mutant mice have elevated very low density lipoprotein triacylglycerol and apoCIII levels. However, ligands for Rev-erb are unknown and the regulatory mechanism of Rev-erb is poorly understood. Conceivably, cofactors for Rev-erb may play an important role in the regulation of lipid metabolism. In this study, a zinc finger HIT domain-containing protein, ZNHIT-1, interacted with Rev-erb,. ZNHIT-1 was found to be a conserved protein in eukaryotes and was highly abundant in human liver. Furthermore, ZNHIT-1 was identified as a nuclear protein. Serial truncated fragments and substitution mutations established a putative nuclear localization signal at amino acids 38,47 of ZNHIT-1. A putative ligand-binding domain of Rev-erb, and the FxxLL motif of ZNHIT-1 were required for their interaction. Finally, ZNHIT-1 was recruited by Rev-erb, to the apoCIII promoter and removed the Rev-erb,-induced inhibition of apoCIII transcription. These findings demonstrate that ZNHIT-1 functions as a cofactor to regulate the activity of Rev-erb,, and may play a role in lipid metabolism. [source] Analysis of the NADH-dependent retinaldehyde reductase activity of amphioxus retinol dehydrogenase enzymes enhances our understanding of the evolution of the retinol dehydrogenase familyFEBS JOURNAL, Issue 14 2007Diana Dalfó In vertebrates, multiple microsomal retinol dehydrogenases are involved in reversible retinol/retinal interconversion, thereby controlling retinoid metabolism and retinoic acid availability. The physiologic functions of these enzymes are not, however, fully understood, as each vertebrate form has several, usually overlapping, biochemical roles. Within this context, amphioxus, a group of chordates that are simpler, at both the functional and genomic levels, than vertebrates, provides a suitable evolutionary model for comparative studies of retinol dehydrogenase enzymes. In a previous study, we identified two amphioxus enzymes, Branchiostoma floridae retinol dehydrogenase 1 and retinol dehydrogenase 2, both candidates to be the cephalochordate orthologs of the vertebrate retinol dehydrogenase enzymes. We have now proceeded to characterize these amphioxus enzymes. Kinetic studies have revealed that retinol dehydrogenase 1 and retinol dehydrogenase 2 are microsomal proteins that catalyze the reduction of all- trans -retinaldehyde using NADH as cofactor, a remarkable combination of substrate and cofactor preferences. Moreover, evolutionary analysis, including the amphioxus sequences, indicates that Rdh genes were extensively duplicated after cephalochordate divergence, leading to the gene cluster organization found in several mammalian species. Overall, our data provide an evolutionary reference with which to better understand the origin, activity and evolution of retinol dehydrogenase enzymes. [source] Site-directed mutagenesis of selected residues at the active site of aryl-alcohol oxidase, an H2O2 -producing ligninolytic enzymeFEBS JOURNAL, Issue 21 2006Patricia Ferreira Aryl-alcohol oxidase provides H2O2 for lignin biodegradation, a key process for carbon recycling in land ecosystems that is also of great biotechnological interest. However, little is known of the structural determinants of the catalytic activity of this fungal flavoenzyme, which oxidizes a variety of polyunsaturated alcohols. Different alcohol substrates were docked on the aryl-alcohol oxidase molecular structure, and six amino acid residues surrounding the putative substrate-binding site were chosen for site-directed mutagenesis modification. Several Pleurotus eryngii aryl-alcohol oxidase variants were purified to homogeneity after heterologous expression in Emericella nidulans, and characterized in terms of their steady-state kinetic properties. Two histidine residues (His502 and His546) are strictly required for aryl-alcohol oxidase catalysis, as shown by the lack of activity of different variants. This fact, together with their location near the isoalloxazine ring of FAD, suggested a contribution to catalysis by alcohol activation, enabling its oxidation by flavin-adenine dinucleotide (FAD). The presence of two aromatic residues (at positions 92 and 501) is also required, as shown by the conserved activity of the Y92F and F501Y enzyme variants and the strongly impaired activity of Y92A and F501A. By contrast, a third aromatic residue (Tyr78) does not seem to be involved in catalysis. The kinetic and spectral properties of the Phe501 variants suggested that this residue could affect the FAD environment, modulating the catalytic rate of the enzyme. Finaly, L315 affects the enzyme kcat, although it is not located in the near vicinity of the cofactor. The present study provides the first evidence for the role of aryl-alcohol oxidase active site residues. [source] Prediction of coenzyme specificity in dehydrogenases/ reductasesFEBS JOURNAL, Issue 6 2006A hidden Markov model-based method, its application on complete genomes Dehydrogenases and reductases are enzymes of fundamental metabolic importance that often adopt a specific structure known as the Rossmann fold. This fold, consisting of a six-stranded ,-sheet surrounded by ,-helices, is responsible for coenzyme binding. We have developed a method to identify Rossmann folds and predict their coenzyme specificity (NAD, NADP or FAD) using only the amino acid sequence as input. The method is based upon hidden Markov models and sequence pattern analysis. The prediction sensitivity is 79% and the selectivity close to 100%. The method was applied on a set of 68 genomes, representing the three kingdoms archaea, bacteria and eukaryota. In prokaryotes, 3% of the genes were found to code for Rossmann-fold proteins, while the corresponding ratio in eukaryotes is only around 1%. In all genomes, NAD is the most preferred cofactor (41,49%), followed by NADP with 30,38%, while FAD is the least preferred cofactor (21%). However, the NAD preponderance over NADP is most pronounced in archaea, and least in eukaryotes. In all three kingdoms, only 3,8% of the Rossmann proteins are predicted to have more than one membrane-spanning segment, which is much lower than the frequency of membrane proteins in general. Analysis of the major protein types in eukaryotes reveals that the most common type (26%) of the Rossmann proteins are short-chain dehydrogenases/reductases. In addition, the identified Rossmann proteins were analyzed with respect to further protein types, enzyme classes and redundancy. The described method is available at http://www.ifm.liu.se/bioinfo, where the preferred coenzyme and its binding region are predicted given an amino acid sequence as input. [source] Stage-specific expression of Caenorhabditis elegans ribonuclease H1 enzymes with different substrate specificities and bivalent cation requirementsFEBS JOURNAL, Issue 2 2006Hiromi Kochiwa Ribonuclease H1 (RNase H1) is a widespread enzyme found in a range of organisms from viruses to humans. It is capable of degrading the RNA moiety of DNA,RNA hybrids and requires a bivalent ion for activity. In contrast with most eukaryotes, which have one gene encoding RNase H1, the activity of which depends on Mg2+ ions, Caenorhabditis elegans has four RNase H1-related genes, and one of them has an isoform produced by alternative splicing. However, little is known about the enzymatic features of the proteins encoded by these genes. To determine the differences between these enzymes, we compared the expression patterns of each RNase H1-related gene throughout the development of the nematode and the RNase H activities of their recombinant proteins. We found gene-specific expression patterns and different enzymatic features. In particular, besides the enzyme that displays the highest activity in the presence of Mg2+ ions, C. elegans has another enzyme that shows preference for Mn2+ ion as a cofactor. We characterized this Mn2+ -dependent RNase H1 for the first time in eukaryotes. These results suggest that there are at least two types of RNase H1 in C. elegans depending on the developmental stage of the organism. [source] Human ATP-dependent RNA/DNA helicase hSuv3p interacts with the cofactor of survivin HBXIPFEBS JOURNAL, Issue 19 2005Michal Minczuk The human SUV3gene encodes an NTP-dependent DNA/RNA DExH box helicase predominantly localized in mitochondria. Its orthologue in yeast is a component of the mitochondrial degradosome complex involved in the mtRNA decay pathway. In contrast to this, the physiological function of human SUV3 remains to be elucidated. In this report we demonstrate that the hSuv3 protein interacts with HBXIP, previously identified as a cofactor of survivin in suppression of apoptosis and as a protein that binds the HBx protein encoded by the hepatitis B virus. Using deletion analysis we identified the region within the hSuv3 protein, which is responsible for binding to HBXIP. The HBXIP binding domain was found to be important for mitochondrial import and stability of the Suv3 protein in vivo. We discuss the possible involvement of the hSuv3p,HBXIP interaction in the survivin-dependent antiapoptotic pathway. [source] Aly/,REF, a factor for mRNA transport, activates RH gene promoter functionFEBS JOURNAL, Issue 11 2005Hiroshi Suganuma The rhesus (Rh) blood group antigens are of considerable importance in transfusion medicine as well as in newborn or autoimmune hemolytic diseases due to their high antigenicity. We identified a major DNaseI hypersensitive site at the 5, flanking regions of both RHD and RHCE exon 1. A 34 bp fragment located at ,191 to ,158 from a translation start position, and containing the TCCCCTCCC sequence, was involved in enhancing promoter activity, which was assessed by luciferase reporter gene assay. A biotin-labelled 34 bp probe isolated an mRNA transporter protein, Aly/REF. The specific binding of Aly/REF to RH promoter in erythroid was confirmed by chromatin immunoprecipitation assay. The silencing of Aly/REF by siRNA reduced not only the RH promoter activity of the reporter gene but also transcription from the native genome. These facts provide second proof of Aly/REF as a transcription coactivator, initially identified as a coactivator for the TCR, enhancer function. Aly/REF might be a novel transcription cofactor for erythroid-specific genes. [source] Characterization of cinnamyl alcohol dehydrogenase of Helicobacter pyloriFEBS JOURNAL, Issue 5 2005An aldehyde dismutating enzyme Cinnamyl alcohol dehydrogenases (CAD; 1.1.1.195) catalyse the reversible conversion of p -hydroxycinnamaldehydes to their corresponding alcohols, leading to the biosynthesis of lignin in plants. Outside of plants their role is less defined. The gene for cinnamyl alcohol dehydrogenase from Helicobacter pylori (HpCAD) was cloned in Escherichia coli and the recombinant enzyme characterized for substrate specificity. The enzyme is a monomer of 42.5 kDa found predominantly in the cytosol of the bacterium. It is specific for NADP(H) as cofactor and has a broad substrate specificity for alcohol and aldehyde substrates. Its substrate specificity is similar to the well-characterized plant enzymes. High substrate inhibition was observed and a mechanism of competitive inhibition proposed. The enzyme was found to be capable of catalysing the dismutation of benzaldehyde to benzyl alcohol and benzoic acid. This dismutation reaction has not been shown previously for this class of alcohol dehydrogenase and provides the bacterium with a means of reducing aldehyde concentration within the cell. [source] Determination of the metal ion dependence and substrate specificity of a hydratase involved in the degradation pathway of biphenyl/chlorobiphenylFEBS JOURNAL, Issue 4 2005Pan Wang BphH is a divalent metal ion-dependent hydratase that catalyzes the formation of 2-keto-4-hydroxypentanoate from 2-hydroxypent-2,4-dienoate (HPDA). This reaction lies on the catabolic pathway of numerous aromatics, including the significant environmental pollutant, polychlorinated biphenyls (PCBs). BphH from the PCB degrading bacterium, Burkholderia xenoverans LB400, was overexpressed and purified to homogeneity. Atomic absorption spectroscopy and Scatchard analysis reveal that only one divalent metal ion is bound to each enzyme subunit. The enzyme exhibits the highest activity when Mg2+ was used as cofactor. Other divalent cations activate the enzyme in the following order of effectiveness: Mg2+ > Mn2+ > Co2+ > Zn2+ > Ca2+. This differs from the metal activation profile of the homologous hydratase, MhpD. UV-visible spectroscopy of the Co2+,BphH complex indicates that the divalent metal ion is hexa-coordinated in the enzyme. The nature of the metal ion affected only the kcat and not the Km values in the BphH hydration of HPDA, suggesting that cation has a catalytic rather than just a substrate binding role. BphH is able to transform alternative substrates substituted with methyl- and chlorine groups at the 5-position of HPDA. The specificity constants (kcat/Km) for 5-methyl and 5-chloro substrates are, however, lowered by eight- and 67-fold compared with the unsubstituted substrate. Significantly, kcat for the chloro-substituted substrate is eightfold lower compared with the methyl-substituted substrate, showing that electron withdrawing substituent at the 5-position of the substrate has a negative influence on enzyme catalysis. [source] Protein farnesyltransferase inhibitors interfere with farnesyl diphosphate binding by rubber transferaseFEBS JOURNAL, Issue 19 2003Christopher J. D. Mau Rubber transferase, a cis -prenyltransferase, catalyzes the addition of thousands of isopentenyl diphosphate (IPP) molecules to an allylic diphosphate initiator, such as farnesyl diphosphate (FPP, 1), in the presence of a divalent metal cofactor. In an effort to characterize the catalytic site of rubber transferase, the effects of two types of protein farnesyltransferase inhibitors, several chaetomellic acid A analogs (2, 4,7) and ,-hydroxyfarnesylphosphonic acid (3), on the ability of rubber transferase to add IPP to the allylic diphosphate initiator were determined. Both types of compounds inhibited the activity of rubber transferases from Hevea brasiliensis and Parthenium argentatum, but there were species,specific differences in the inhibition of rubber transferases by these compounds. Several shorter analogs of chaetomellic acid A did not inhibit rubber transferase activity, even though the analogs contained chemical features that are present in an elongating rubber molecule. These results indicate that the initiator-binding site in rubber transferase shares similar features to FPP binding sites in other enzymes. [source] Isocitrate dehydrogenase of Plasmodium falciparumFEBS JOURNAL, Issue 8 2003Energy metabolism or redox control? Erythrocytic stages of the malaria parasite Plasmodium falciparum rely on glycolysis for their energy supply and it is unclear whether they obtain energy via mitochondrial respiration albeit enzymes of the tricarboxylic acid (TCA) cycle appear to be expressed in these parasite stages. Isocitrate dehydrogenase (ICDH) is either an integral part of the mitochondrial TCA cycle or is involved in providing NADPH for reductive reactions in the cell. The gene encoding P. falciparum ICDH was cloned and analysis of the deduced amino-acid sequence revealed that it possesses a putative mitochondrial targeting sequence. The protein is very similar to NADP+ -dependent mitochondrial counterparts of higher eukaryotes but not Escherichia coli. Expression of full-length ICDH generated recombinant protein exclusively expressed in inclusion bodies but the removal of 27 N-terminal amino acids yielded appreciable amounts of soluble ICDH consistent with the prediction that these residues confer targeting of the native protein to the parasites' mitochondrion. Recombinant ICDH forms homodimers of 90 kDa and its activity is dependent on the bivalent metal ions Mg2+ or Mn2+ with apparent Km values of 13 µm and 22 µm, respectively. Plasmodium ICDH requires NADP+ as cofactor and no activity with NAD+ was detectable; the for NADP+ was found to be 90 µm and that of d -isocitrate was determined to be 40 µm. Incubation of P. falciparum under exogenous oxidative stress resulted in an up-regulation of ICDH mRNA and protein levels indicating that the enzyme is involved in mitochondrial redox control rather than energy metabolism of the parasites. [source] Purification, characterization and subunits identification of the diol dehydratase of Lactobacillus collinoidesFEBS JOURNAL, Issue 22 2002Nicolas Sauvageot The three genes pduCDE encoding the diol dehydratase of Lactobacillus collinoides, have been cloned for overexpression in the pQE30 vector. Although the three subunits of the protein were highly induced, no activity was detected in cell extracts. The enzyme was therefore purified to near homogeneity by ammonium sulfate precipitation and gel filtration chromatography. In fractions showing diol dehydratase activity, three main bands were present after SDS/PAGE with molecular masses of 63, 28 and 22 kDa, respectively. They were identified by mass spectrometry to correspond to the large, medium and small subunits of the dehydratase encoded by the pduC, pduD and pduE genes, respectively. The molecular mass of the native complex was estimated to 207 kDa in accordance with the calculated molecular masses deduced from the pduC, D, E genes (61, 24.7 and 19,1 kDa, respectively) and a ,2,2,2 composition. The Km for the three main substrates were 1.6 mm for 1,2-propanediol, 5.5 mm for 1,2-ethanediol and 8.3 mm for glycerol. The enzyme required the adenosylcobalamin coenzyme for catalytic activity and the Km for the cofactor was 8 µm. Inactivation of the enzyme was observed by both glycerol and cyanocobalamin. The optimal reaction conditions of the enzyme were pH 8.75 and 37 °C. Activity was inhibited by sodium and calcium ions and to a lesser extent by magnesium. A fourth band at 59 kDa copurified with the diol dehydratase and was identified as the propionaldehyde dehydrogenase enzyme, another protein involved in the 1,2-propanediol metabolism pathway. [source] Monoterpene biosynthesis in lemon (Citrus limon)FEBS JOURNAL, Issue 13 2002cDNA isolation, functional analysis of four monoterpene synthases Citrus limon possesses a high content and large variety of monoterpenoids, especially in the glands of the fruit flavedo. The genes responsible for the production of these monoterpenes have never been isolated. By applying a random sequencing approach to a cDNA library from mRNA isolated from the peel of young developing fruit, four monoterpene synthase cDNAs were isolated that appear to be new members of the previously reported tpsb family. Based on sequence homology and phylogenetic analysis, these sequences cluster in two separate groups. All four cDNAs could be functionally expressed in Escherichia coli after removal of their plastid targeting signals. The main products of the enzymes in assays with geranyl diphosphate as substrate were (+)-limonene (two cDNAs) (,)-,-pinene and ,-terpinene. All enzymes exhibited a pH optimum around 7; addition of Mn2+ as bivalent metal ion cofactor resulted in higher activity than Mg2+, with an optimum concentration of 0.6 mm. Km values ranged from 0.7 to 3.1 µm. The four enzymes account for the production of 10 out of the 17 monoterpene skeletons commonly observed in lemon peel oil, corresponding to more than 90% of the main components present. [source] | ||||||||||||||||||||||||||||||||||||||||