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Dependent Enzymes (dependent + enzyme)
Selected AbstractsEvaluating Enzyme Cascades for Methanol/Air Biofuel Cells Based on NAD+ -Dependent EnzymesELECTROANALYSIS, Issue 7-8 2010Abstract Previous work by the group has entailed encapsulating enzymes in polymeric micelles at bioelectrode surfaces by utilizing hydrophobically modified Nafion membranes, which are modified in order to eliminate the harsh acidity of Nafion while tailoring the size of the polymer micelles to optimize for the encapsulation of an individual enzyme. This polymer encapsulation has been shown to provide high catalytic activity and enzyme stability. In this study, we employed this encapsulation technique in developing a methanol/air biofuel cell through the combined immobilization of NAD+ -dependent alcohol dehydrogenase (ADH), aldehyde dehydrogenase (AldDH) and formate dehydrogenase (FDH) within a tetrabutylammonium bromide (TBAB) modified Nafion to oxidize methanol to carbon dioxide with poly(methylene green) acting as the NADH electrocatalyst electropolymerized on the surface of the electrode. The methanol biofuel/air cell resulted in a maximum power density of 261±7.6,,W/cm2 and current density of 845±35.5,,A/cm2. This system was characterized for the effects of degree of oxidation, temperature, pH, and concentration of fuel and NAD. [source] Enantioselective oxidation of 2-hydroxy carboxylic acids by glycolate oxidase and catalase coexpressed in methylotrophic Pichia pastorisBIOTECHNOLOGY PROGRESS, Issue 3 2010Shuvendu Das Abstract Glycolate oxidase (GO; (S)-2-hydroxyacid oxidase, EC 1.1.3.15) is a flavin mononucleotide (FMN)-dependent enzyme, which catalyzes the oxidation of 2-hydroxy carboxylic acids to the corresponding 2-keto acids. Catalase has been used as cocatalyst to decompose hydrogen peroxide produced in the reaction, thus limiting peroxide-based side reactions and GO deactivation. GO from spinach and catalase T from Saccharomyces cerevisiae previously coexpressed in Pichia pastoris strain NRRL Y-21001, was permeabilized and used for the oxidation of 3-phenyllactic acid, 3-indolelactic acid, 3-chlorolactic acid, 2-hydroxybutanoic acid, and 2-hydroxydecanoic acid to demonstrate high degree of selectivity to the (S)-enantiomers, leaving (R)-isomers intact. The rates of oxidation ranged from 1.3 to 120.0%, relative to the oxidation of lactic acid to pyruvic acid. The best substrates were 3-chlorolactic acid (110%) and 2-hydroxybutanoic acid (120%). Oxidation was carried out with (R)-, (S)-, and (RS)-3-phenyllactic acid, (RS)-lactic acid, and (RS)-2-hydroxybutanoic acid in 500 mL scale to characterize the products and stoichiometry of the reaction. All (RS)- and (S)-2-hydroxy acids produced 2-keto acids at close to the theoretical yield in 1,9 h. (R)-3-Phenyllactic acid was not oxidized over a period of 9 h. Addition of exogenous FMN and catalase were not required for this oxidation using double recombinant Pichia pastoris whole cells. As GO is absolutely specific to (S)-enantiomers, it can be used for resolution of racemic 2-hydroxy acids to (R)-2-hydroxy acids as well as for production of 2-keto acids. This is the first report on the selectivity of a broad range of 2-hydroxy acids by GO. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source] Structure of the ThDP-dependent enzyme benzaldehyde lyase refined to 1.65,Å resolutionACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2007Andy Maraite Benzaldehyde lyase (BAL; EC 4.1.2.38) is a thiamine diphosphate (ThDP) dependent enzyme that catalyses the enantioselective carboligation of two molecules of benzaldehyde to form (R)-benzoin. BAL has hence aroused interest for its potential in the industrial synthesis of optically active benzoins and derivatives. The structure of BAL was previously solved to a resolution of 2.6,Å using MAD experiments on a selenomethionine derivative [Mosbacher et al. (2005), FEBS J.272, 6067,6076]. In this communication of parallel studies, BAL was crystallized in an alternative space group (P212121) and its structure refined to a resolution of 1.65,Å, allowing detailed observation of the water structure, active-site interactions with ThDP and also the electron density for the co-solvent 2-methyl-2,4-pentanediol (MPD) at hydrophobic patches of the enzyme surface. [source] Crystallization, X-ray diffraction analysis and phasing of 17,-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatusACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 12 2005Alberto Cassetta 17,-Hydroxysteroid dehydrogenase from the filamentous fungus Cochliobolus lunatus (17,-HSDcl) is an NADP(H)-dependent enzyme that preferentially catalyses the oxidoreduction of oestrogens and androgens. The enzyme belongs to the short-chain dehydrogenase/reductase superfamily and is the only fungal hydroxysteroid dehydrogenase known to date. 17,-HSDcl has recently been characterized and cloned and has been the subject of several functional studies. Although several hypotheses on the physiological role of 17,-HSDcl in fungal metabolism have been formulated, its function is still unclear. An X-ray crystallographic study has been undertaken and the optimal conditions for crystallization of 17,-HSDcl (apo form) were established, resulting in well shaped crystals that diffracted to 1.7,Å resolution. The space group was identified as I4122, with unit-cell parameters a = b = 67.14, c = 266.77,Å. Phasing was successfully performed by Patterson search techniques. A catalytic inactive mutant Tyr167Phe was also engineered, expressed, purified and crystallized for functional and structural studies. [source] Structure of Citrobacter freundiil -methionine ,-lyaseACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 6 2005D. V. Mamaeva l -Methionine ,-lyase (MGL) is a pyridoxal 5,-phosphate (PLP) dependent enzyme that catalyzes ,-elimination of l -methionine. The crystal structure of MGL from Citrobacter freundii has been determined at 1.9,Å resolution. The spatial fold of the protein is similar to those of MGLs from Pseudomonas putida and Trichomonas vaginalis. The comparison of these structures revealed that there are differences in PLP-binding residues and positioning of the surrounding flexible loops. [source] Comparative biochemical and functional studies of family I soluble inorganic pyrophosphatases from photosynthetic bacteriaFEBS JOURNAL, Issue 15 2007María R. Gómez-García Soluble inorganic pyrophosphatases (inorganic diphosphatases, EC 3.6.1.1) were isolated and characterized from three phylogenetically diverse cyanobacteria , Synechocystis sp. PCC 6803, Anabaena sp. PCC 7120, and Pseudanabaena sp. PCC 6903 , and one anoxygenic photosynthetic bacterium, Rhodopseudomonas viridis (purple nonsulfur). These enzymes were found to be family I soluble inorganic pyrophosphatases with c. 20 kDa subunits with diverse oligomeric structures. The corresponding ppa genes were cloned and functionally validated by heterologous expression. Cyanobacterial family I soluble inorganic pyrophosphatases were strictly Mg2+ -dependent enzymes. However, diverse cation cofactor dependence was observed for enzymes from other groups of photosynthetic bacteria. Immunochemical studies with antibodies to cyanobacterial soluble inorganic pyrophosphatases showed crossreaction with orthologs of other main groups of phototrophic prokaryotes and suggested a close relationship with the enzyme of heliobacteria, the nearest photosynthetic relatives of cyanobacteria. A slow-growing Escherichia coli JP5 mutant strain, containing a very low level of soluble inorganic pyrophosphatase activity, was functionally complemented up to wild-type growth rates with ppa genes from diverse photosynthetic prokaryotes expressed under their own promoters. Overall, these results suggest that the bacterial family I soluble inorganic pyrophosphatases described here have retained functional similarities despite their genealogies and their adaptations to diverse metabolic scenarios. [source] Identification of substrates for transglutaminase in Physarum polycephalum, an acellular slime mold, upon cellular mechanical damageFEBS JOURNAL, Issue 11 2007Fumitaka Wada Transglutaminases are Ca2+ -dependent enzymes that post-translationally modify proteins by crosslinking or polyamination at specific polypeptide-bound glutamine residues. Physarum polycephalum, an acellular slime mold, is the evolutionarily lowest organism expressing a transglutimase whose primary structure is similar to that of mammalian transglutimases. We observed transglutimase reaction products at injured sites in Physarum macroplasmodia upon mechanical damage. With use of a biotin-labeled primary amine, three major proteins constituting possible transglutimase substrates were affinity-purified from the damaged slime mold. The purified proteins were Physarum actin, a 40 kDa Ca2+ -binding protein with four EF-hand motifs (CBP40), and a novel 33 kDa protein highly homologous to the eukaryotic adenine nucleotide translocator, which is expressed in mitochondria. Immunochemical analysis of extracts from the damaged macroplasmodia indicated that CBP40 is partly dimerized, whereas the other proteins migrated as monomers on SDS/PAGE. Of the three proteins, CBP40 accumulated most significantly around injured areas, as observed by immunofluoresence. These results suggested that transglutimase reactions function in the response to mechanical injury. [source] Performance of DFT in modeling electronic and structural properties of cobalaminsJOURNAL OF COMPUTATIONAL CHEMISTRY, Issue 12 2006Jadwiga Kuta Abstract Computational modeling of the enzymatic activity of B12 -dependent enzymes requires a detailed understanding of the factors that influence the strength of the CoC bond and the limits associated with a particular level of theory. To address this issue, a systematic analysis of the electronic and structural properties of coenzyme B12 models has been performed to establish the performance of three different functionals including B3LYP, BP86, and revPBE. In particular the cobalt,carbon bond dissociation energies, axial bond lengths, and selected stretching frequencies have been analyzed in detail. Current analysis shows that widely used B3LYP functional significantly underestimates the strength of the CoC bond while the nonhybrid BP86 functional produces very consistent results in comparison to experimental data. To explain such different performance of these functionals molecular orbital analysis associated with axial bonds has been performed to show differences in axial bonding provided by hybrid and nonhybrid functionals. © 2006 Wiley Periodicals, Inc. J Comput Chem 27: 1429,1437, 2006 [source] Calcium in the mechanism of ammonia-induced astrocyte swellingJOURNAL OF NEUROCHEMISTRY, Issue 2009Arumugam R. Jayakumar Abstract Brain edema, due largely to astrocyte swelling, is an important clinical problem in patients with acute liver failure. While mechanisms underlying astrocyte swelling in this condition are not fully understood, ammonia and associated oxidative/nitrosative stress appear to be involved. Mechanisms responsible for the increase in reactive oxygen/nitrogen species (RONS) and their role in ammonia-induced astrocyte swelling, however, are poorly understood. Recent studies have demonstrated a transient increase in intracellular Ca2+ in cultured astrocytes exposed to ammonia. As Ca2+ is a known inducer of RONS, we investigated potential mechanisms by which Ca2+ may be responsible for the production of RONS and cell swelling in cultured astrocytes after treatment with ammonia. Exposure of cultured astrocytes to ammonia (5 mM) increased the formation of free radicals, including nitric oxide, and such increase was significantly diminished by treatment with the Ca2+ chelator 1,2-bis-(o -aminophenoxy)-ethane- N,N,- N,,N, -tetraacetic acid tetraacetoxy-methyl ester (BAPTA). We then examined the activity of Ca2+ -dependent enzymes that are known to generate RONS and found that ammonia significantly increased the activities of NADPH oxidase (NOX), constitutive nitric oxide synthase (cNOS), and phospholipase A2 (PLA2) and such increases in activity were significantly diminished by BAPTA. Pre-treatment of cultures with 7-nitroindazole, apocyanin, and quinacrine, respective inhibitors of cNOS, NOX, and PLA2, all significantly diminished RONS production. Additionally, treatment of cultures with BAPTA or with inhibitors of cNOS, NOX, and PLA2 reduced ammonia-induced astrocyte swelling. These studies suggest that the ammonia-induced rise in intracellular Ca2+ activates free radical producing enzymes that ultimately contribute to the mechanism of astrocyte swelling. [source] The roles of NADPH oxidase and phospholipases A2 in oxidative and inflammatory responses in neurodegenerative diseasesJOURNAL OF NEUROCHEMISTRY, Issue 1 2007Grace Y. Sun Abstract Reactive oxygen species (ROS) are produced in mammalian cells through enzymic and non-enzymic mechanisms. Although some ROS production pathways are needed for specific physiological functions, excessive production is detrimental and is regarded as the basis of numerous neurodegenerative diseases. Among enzymes producing superoxide anions, NADPH oxidase is widespread in mammalian cells and is an important source of ROS in mediating physiological and pathological processes in the cardiovascular and the CNS. ROS production is linked to the alteration of intracellular calcium homeostasis, activation of Ca2+ -dependent enzymes, alteration of cytoskeletal proteins, and degradation of membrane glycerophospholipids. There is evolving evidence that ROS produced by NADPH oxidase regulate neuronal functions and degrade membrane phospholipids through activation of phospholipases A2 (PLA2). This review is intended to cover recent studies describing ROS generation from NADPH oxidase in the CNS and its downstream activation of PLA2, namely, the group IV cytosolic cPLA2 and the group II secretory sPLA2. A major focus is to elaborate the dual role of NADPH oxidase and PLA2 in mediating the oxidative and inflammatory responses in neurodegenerative diseases, including cerebral ischemia and Alzheimer's disease. Elucidation of the signaling pathways linking NADPH oxidase with the multiple forms of PLA2 will be important in understanding the oxidative and degradative mechanisms that underline neuronal damage and glial activation and will facilitate development of therapeutic intervention for prevention and treatment of these and other neurodegenerative diseases. [source] From cofactor to enzymes.THE CHEMICAL RECORD, Issue 6 2001-phosphate-dependent enzymes, The molecular evolution of pyridoxal- Abstract The pyridoxal-5,-phosphate (vitamin B6)-dependent enzymes that act on amino acid substrates have multiple evolutionary origins. Thus, the common mechanistic features of B6 enzymes are not accidental historical traits but reflect evolutionary or chemical necessities. The B6 enzymes belong to four independent evolutionary lineages of paralogous proteins, of which the , family (with aspartate aminotransferase as the prototype enzyme) is by far the largest and most diverse. The considerably smaller , family (tryptophan synthase , as the prototype enzyme) is structurally and functionally more homogenous. Both the D -alanine aminotransferase family and the alanine racemase family consist of only a few enzymes. The primordial pyridoxal-5,-phosphate-dependent protein catalysts apparently first diverged into reaction-specific protoenzymes, which then diverged further by specializing for substrate specificity. Aminotransferases as well as amino acid decarboxylases are found in two different evolutionary lineages, providing examples of convergent enzyme evolution. The functional specialization of most B6 enzymes seems to have already occurred in the universal ancestor cell before the divergence of eukaryotes, archebacteria, and eubacteria 1500 million years ago. Pyridoxal-5,-phosphate must have emerged very early in biological evolution; conceivably, metal ions and organic cofactors were the first biological catalysts. To simulate particular steps of molecular evolution, both the substrate and reaction specificity of existent B6 enzymes were changed by substitution of active-site residues, and monoclonal pyridoxal-5,-phosphate-dependent catalytic antibodies were produced with selection criteria that might have been operative in the evolution of protein-assisted pyridoxal catalysis. © 2001 John Wiley & Sons, Inc. and The Japan Chemical Journal Forum Chem Rec 1:436,447, 2001 [source] Stereospecificity for the hydrogen transfer of pyridoxal enzyme reactionsTHE CHEMICAL RECORD, Issue 5 2001Kenji Soda Abstract We have studied the stereospecificities of various pyridoxal 5,-phosphate dependent enzymes for the hydrogen transfer between the C-4, of a bound coenzyme and the C-2 of a substrate in the transamination catalyzed by the enzymes. Prior to our studies, pyridoxal enzymes so far studied were reported to catalyze the hydrogen transfer only on the si -face of the planar imine intermediate formed from substrate and coenzyme. This finding had been considered as the evidence that pyridoxal enzymes have evolved divergently from a common ancestral protein, because identity in the stereospecificity reflects the similarity in the active-site structure, in particular in the geometrical relationship between the coenzyme and the active site base participating in the hydrogen transfer. However, we found that d -amino acid aminotransferase, branched-chain l -amino acid aminotransferase, and 4-amino-4-deoxychorismate lyase catalyze the re -face specific hydrogen transfer, and that amino acid racemases catalyze the nonstereospecific hydrogen transfer. These findings suggest the convergent evolution of pyridoxal enzymes. Crystallographical studies have shown that the stereospecificity reflects the active-site structure of the enzymes, and that the enzymes with the same fold exhibit the same stereospecificity. The active site structure with the catalytic base being situated on the specific face of the cofactor has been conserved during the evolution among the pyridoxal enzymes of the same family. © 2001 John Wiley & Sons, Inc. and The Japan Chemical Journal Forum Chem Rec 1:373,384, 2001 [source] Folate deficiency followed by ionizing radiation perturbs hepatic dihydrofolate reducatse activityBIOFACTORS, Issue 4 2008Vipen Batra Abstract There is lot of interest in the folate metabolism because of the essential role of folate coenzymes in nucleic acid synthesis. Gamma (,) radiation is well known for inducing damage in the DNA. To counteract these damage, a variety of DNA repair pathways have evolved that require regular supply of DNA bases whose biosynthesis in turn depends on sufficient pools of folate dependent enzymes like dihydrofolate reductase (DHFR). In the present study, we examined the ionizing radiation mediated perturbation of DHFR activity in folate deficient and folate sufficient conditions. In folate deficient animals a potent inhibition of liver DHFR activity was observed. Our results showed that combination of folate starvation and ionizing radiation might adversely affect the DHFR activity, compared to their individual treatments. Measurement of apurinic/apyrimidinic sites (AP sites), a major type of DNA damage generated by radiation induced loss of purine and/or pyrimidine base, indicated a dose dependent DNA damage in folate deficient animals. In conclusion our data suggest an interactive role of folate deficiency and radiation injury in inhibiting DHFR activity. [source] | |||||||||||||