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Coupled Reaction (coupled + reaction)
Selected AbstractsPrediction of missing enzyme genes in a bacterial metabolic networkFEBS JOURNAL, Issue 9 2007Reconstruction of the lysine-degradation pathway of Pseudomonas aeruginosa The metabolic network is an important biological network which consists of enzymes and chemical compounds. However, a large number of metabolic pathways remains unknown, and most organism-specific metabolic pathways contain many missing enzymes. We present a novel method to identify the genes coding for missing enzymes using available genomic and chemical information from bacterial genomes. The proposed method consists of two steps: (a) estimation of the functional association between the genes with respect to chromosomal proximity and evolutionary association, using supervised network inference; and (b) selection of gene candidates for missing enzymes based on the original candidate score and the chemical reaction information encoded in the EC number. We applied the proposed methods to infer the metabolic network for the bacteria Pseudomonas aeruginosa from two genomic datasets: gene position and phylogenetic profiles. Next, we predicted several missing enzyme genes to reconstruct the lysine-degradation pathway in P. aeruginosa using EC number information. As a result, we identified PA0266 as a putative 5-aminovalerate aminotransferase (EC 2.6.1.48) and PA0265 as a putative glutarate semialdehyde dehydrogenase (EC 1.2.1.20). To verify our prediction, we conducted biochemical assays and examined the activity of the products of the predicted genes, PA0265 and PA0266, in a coupled reaction. We observed that the predicted gene products catalyzed the expected reactions; no activity was seen when both gene products were omitted from the reaction. [source] Fusion of farnesyldiphosphate synthase and epi -aristolochene synthase, a sesquiterpene cyclase involved in capsidiol biosynthesis in Nicotiana tabacumFEBS JOURNAL, Issue 14 2002Maria Brodelius A clone encoding farnesyl diphosphate synthase (FPPS) was obtained by PCR from a cDNA library made from young leaves of Artemisia annua. A cDNA clone encoding the tobacco epi -aristolochene synthase (eAS) was kindly supplied by J. Chappell (University of Kentucky, Lexington, KY, USA). Two fusions were constructed, i.e. FPPS/eAS and eAS/FPPS. The stop codon of the N-terminal enzyme was removed and replaced by a short peptide (Gly-Ser-Gly) to introduce a linker between the two ORFs. These two fusions and the two single cDNA clones were separately introduced into a bacterial expression vector (pET32). Escherichia coli was transformed with the expression vectors and enzymatically active soluble proteins were obtained after induction with isopropyl thio-,- d -thiogalactoside. The recombinant enzymes were purified using immobilized metal affinity chromatography on Co2+ columns. The fusion enzymes produced epi- aristolochene from isopentenyl diphosphate through a coupled reaction. The Km values of FPPS and eAS for isopentenyl diphosphate and farnesyl diphosphate, respectively, were essentially the same for the single and fused enzymes. The bifunctional enzymes showed a more efficient conversion of isopentenyl diphosphate to epi -aristolochene than the corresponding amount of single enzymes. [source] EVALUATION OF GLOBAL YIELD, COMPOSITION, ANTIOXIDANT ACTIVITY AND COST OF MANUFACTURING OF EXTRACTS FROM LEMON VERBENA (ALOYSIA TRIPHYLLA[L'HÉRIT.] BRITTON) AND MANGO (MANGIFERA INDICA L.) LEAVESJOURNAL OF FOOD PROCESS ENGINEERING, Issue 2 2007CAMILA G. PEREIRA ABSTRACT In this work, the global yields, composition and antioxidant activity (AA) of extracts from lemon verbena (Aloysia triphylla) and mango (Mangifera indica) leaves obtained by different separation processes were determined. Lemon verbena extracts were obtained by supercritical fluid extraction (SFE), while mango leaf extracts were obtained by SFE, low-pressure solvent extraction (LPSE) and hydrodistillation. The extract's constituents were analyzed by thin-layer chromatography, gas chromatography/mass spectrometry and gas chromatography/flame ionization detector. The AA of the extracts was evaluated by the coupled reaction of , -carotene/linolenic acid. The cost of manufacturing (COM) was estimated for the SFE extracts. Higher global yields were obtained using SFE at 350 bar/45C (1.49%) for lemon verbena and LPSE (3.04%) for mango. The AAs of the extracts were larger than that of the , -carotene for both plants. The minimum values of COM were U.S.$26.96 and 52.45/kg of extract for lemon verbena and mango, respectively. [source] Activities of the Bimodal Fluorescent Protein Produced by Photobacterium phosphoreum Strain bmFP in the Luciferase Reaction In VitroPHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 2 2000Hajime Karatani ABSTRACT The activity of the bimodal fluorescent protein (bmFP) (,max, 488 and 517 nm) in the in vitro luciferase reaction has been studied. The bmFP that is produced by Photobacterium phosphoreum strain bmFP is a dimer of two homologous subunits binding four riboflavin 5,-phosphate (FMN)-myristate chromophores. The addition of bmFP to the luciferase reaction in the presence of the lumazine protein prevented the lumazine protein-induced blue shift in the emission band. The bmFP reduced electrochemically serves as a substrate in the luciferase reaction in the absence of added FMN, resulting in light emission with a single maximum at about 487 nm. The bmFP was also active in lieu of FMN in the NADH/FMN oxidoreductase (flavin reductase),luciferase coupled bioluminescence reaction in the absence of added FMN. In the coupled reaction, bioluminescence with the isolated bmFP chromophore was weaker than that with the holo-bmFP. After bmFP was used in luciferase reactions initiated either chemically or electrochemically, it was still capable of emitting bimodal fluorescence. [source] Pyrroloquinoline quinone-dependent carbohydrate dehydrogenase: Activity enhancement and the role of artificial electron acceptorsBIOTECHNOLOGY JOURNAL, Issue 8 2010Juozas Kulys Professor Abstract Pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase (PQQ-GDH) offers a variety of opportunities for applications, e.g. in highly sensitive biosensors and electrosynthetic reactions. Here we report on the acceleration (up to 4.9 x 104 -fold) of enzymatic ferricyanide reduction by artificial redox mediators (enhancers). The reaction mechanism includes reduction of the PQQ-GDH by glucose followed by oxidation of the reduced PQQ cofactor with either ferricyanide or a redox mediator. A synergistic effect occurs through the oxidation of a reduced mediator by ferricyanide. Using kinetic description of the coupled reaction, the second order rate constant for the reaction of an oxidized mediator with the reduced enzyme cofactor (kox) can be calculated. For different mediators this value is 2.2 x 106,1.6 x 108 M -1s -1 at pH 7.2 and 25°C. However, no correlation of the rate constant with the midpoint redox potential of the mediator could be established. For low-potential mediators the synergistic effect is proportional to the ratio of kox(med)/kox(ferricyanide), whereas for the high-potential mediators the effect depends on both this ratio and the concentration of the oxidized mediator, which can be calculated from the Nernst equation. The described effect can be applied in various ways, e.g. for substrate reactivity determination, electrosynthetic PQQ cofactor regeneration or building of new highly sensitive biosensors. [source] A comparative proteomic approach to understand the adaptations of an H+ -ATPase-defective mutant of Corynebacterium glutamicum ATCC14067 to energy deficienciesPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 18 2007Liyuan Li Abstract F172-8, an H+ -ATPase-defective mutant of the glutamic acid-producing bacterium Corynebacterium glutamicum ATCC 14067, exhibits enhanced rates of glucose consumption and respiration compared to the parental strain when cultured in a biotin-rich medium with glucose as the carbon source. We conducted a comparative proteomic analysis to clarify the mechanism by which the enhanced glucose metabolism in this mutant is established using a proteome reference map for strain ATCC 14067. A comparison of the proteomes of the two strains revealed the up-regulated expression of the several important enzymes such as pyruvate kinase (Pyk), malate:quinone oxidoreductase (Mqo), and malate dehydrogenase (Mdh) in the mutant. Because Pyk activates glycolysis in response to cellular energy shortages in this bacterium, its increased expression may contribute to the enhanced glucose metabolism of the mutant. A unique reoxidation system has been suggested for NADH in C. glutamicum consisting of coupled reactions between Mqo and Mdh, together with the respiratory chain; therefore, the enhanced expression of both enzymes might contribute to the reoxidation of NADH during increased respiration. The proteomic analysis allowed the identification of unique physiological changes associated with the H+ -ATPase defect in F172-8 and contributed to the understanding of the adaptations of C. glutamicum to energy deficiencies. [source] | |||||||||||||