53-59-8Relevant articles and documents
Oxidative activity of organometallic compounds in relation to the coenzymes NADH and NADPH
Medvedev,Tyurin,Rozhkova,Milaeva
, p. 907 - 911 (1999)
Kinetic investigations of the interaction of Hg and Sn-containing organometallic compounds with NADH and NADPH using spectra-photometric methods have shown that these compounds may act as oxidizing agents in relation to the coenzyme. Their oxidative activity depends on the nature and number of organic groups in the molecule. Comparison of the kinetic data for the activity of Hg and Sn compounds with those for Fe porphyrins imitating the active centers of redox enzymes indicates the competitiveness of the organometallic compounds and models of natural electron acceptors. 1999 KluwerAcademic/Plenum Publishers.
Biochemical and structural basis of triclosan resistance in a novel enoyl-acyl carrier protein reductase
Khan, Raees,Zeb, Amir,Roy, Nazish,Magar, Roniya Thapa,Kim, Hyo Jeong,Lee, Keun Woo,Lee, Seon-Woo
, (2018)
Enoyl-acyl carrier protein reductases (ENR), such as FabI, FabL, FabK, and FabV, catalyze the last reduction step in bacterial type II fatty acid biosynthesis. Previously, we reported metagenome-derived ENR homologs resistant to triclosan (TCL) and highly similar to 7- hydroxysteroid dehydrogenase (7-AHSDH). These homologs are commonly found in Epsilonproteobacteria, a class that contains several human-pathogenic bacteria, including the genera Helicobacter and Campylobacter. Here we report the biochemical and predicted structural basis of TCL resistance in a novel 7-AHSDH-like ENR. The purified protein exhibited NADPH-dependent ENR activity but no 7-AHSDH activity, despite its high homology with 7-AHSDH (69% to 96%). Because this ENR was similar to FabL (41%), we propose that this metagenome-derived ENR be referred to as FabL2. Homology modeling, molecular docking, and molecular dynamic simulation analyses revealed the presence of an extrapolated six-amino-acid loop specific to FabL2 ENR, which prevented the entry of TCL into the active site of FabL2 and was likely responsible for TCL resistance. Elimination of this extrapolated loop via site-directed mutagenesis resulted in the complete loss of TCL resistance but not enzyme activity. Phylogenetic analysis suggested that FabL, FabL2, and 7-AHSDH diverged from a common short-chain dehydrogenase reductase family. This study is the first to report the role of the extrapolated loop of FabL2-type ENRs in conferring TCL resistance. Thus, the FabL2 ENR represents a new drug target specific for pathogenic Epsilonproteobacteria.
Degradation of 3-nitrophenol by Pseudomonas putida B2 occurs via 1,2,4-benzenetriol
Meulenberg, Rogier,Pepi, Milva,De Bont, Jan A.M.
, p. 303 - 311 (1996)
Growth of Pseudomonas putida B2 in chemostat cultures on a mixture of 3-nitrophenol and glucose induced 3-nitrophenol and 1,2,4- benzenetriol-dependent oxygen uptake activities. Anaerobic incubations of cell suspensions with 3-nitrophenol resulted in complete conversion of the substrate to ammonia and 1,2,4-benzenetriol. This indicates that P. putida B2 degrades 3-nitrophenol via 1,2,4-benzenetriol, via a pathway involving a hydroxylaminolyase. Involvement of this pathway in nitroaromatic metabolism has previously only been found for degradation of 4-nitrobenzoate. Reduction of 3 nitrophenol by cell-free extracts was strictly NADPH-dependent. Attempts to purify the enzymes responsible for 3-nitrophenol metabolism were unsuccessful, because their activities were extremely unstable. 3-Nitrophenol reductase was therefore characterized in cell-free extracts. The enzyme had a sharp pH optimum at pH 7 and a temperature optimum at 25°C. At 30°C, reductase activity was completely destroyed within one hour, while at 0°C, the activity in cell-free extracts was over 100-fold more stable. The Km values for NADPH and 3-nitrophenol were estimated at 0.17 mM and below 2 μM, respectively. The substrate specificity of the reductase activity was very broad: all 17 nitroaromatics tested were reduced by cell-free extracts. However, neither intact cells nor cell-free extracts could convert a set of synthesized hydroxylaminoaromatic compounds to the corresponding catechols and ammonia. Apparently, the hydroxylaminolyase of P. putida B2 has a very narrow substrate specificity, indicating that this organism is not a suitable biocatalyst for the industrial production of catechols from nitroaromatics.
Pd (core)-Au (shell) nanoparticles catalyzed conversion of NADH to NAD+ by UV-vis spectroscopy-A kinetic analysis
Gopalan,Ragupathy,Kim,Manesh,Lee
, p. 678 - 684 (2009)
Kinetics of Pd (core)-Au (shell) nanoparticles (NPs) catalyzed transformation of dihydronicotinamide adenine dinucleotide (NADH) to NAD+ was monitored by UV-vis spectroscopy. Pd (core)-Au (shell) NPs were prepared by microwave irradiation method. High resolution transmission electron microscopy image reveals the core-shell morphology. X-ray diffraction pattern shows the presence of distinct crystalline domains for Pd and Au. The changes in absorbances at 340 nm were followed for various time intervals. Rates of conversion of NADH to NAD+ were determined for different conditions. The conversion of NADH to NAD+ was to be first order with respect to NADH at lower concentrations (upto 0.04 mM) and pseudo-first-order beyond 0.04 mM. Rate constants for the Pd (core) Au-(shell) NPs catalyzed transformation of NADH to NAD+ were deduced.
Function of thioredoxin reductase as a peroxynitrite reductase using selenocystine or ebselen
Arteel, Gavin E.,Briviba, Karlis,Sies, Helmut
, p. 264 - 269 (1999)
The activity of mammalian thioredoxin reductase as a peroxynitrite reductase was investigated. Peroxynitrite was infused to maintain a 0.2 μM steady-state concentration in potassium phosphate buffer (pH 7.4). Benzoate hydroxylation and nitrite formation were used as indices of oxidation reactions of peroxynitrite and of peroxynitrite reduction, respectively. In the presence of NADPH (10 μM), thioredoxin reductase at 50 nM alone did not significantly scavenge peroxynitrite, as shown by there being no significant effect on benzoate hydroxylation or nitrite formation. However, when selenocystine (1 μM) or ebselen (2 μM) was present in the reaction mixture, there was significant suppression of benzoate hydroxylation and an increase in nitrite formation until all the NADPH was oxidized. The addition of thioredoxin did not enhance these effects. In contrast, peroxynitrite reduction by ebselen complexed with BSA was enhanced by the presence of thioredoxin. In parallel experiments, thioredoxin reductase efficiently reduced ebselen selenoxide back to ebselen.
Purification and kinetics of bovine kidney cortex glutathione reductase
Tandogan, Berivan,Nuray Ulusu
, p. 667 - 674 (2010)
Glutathione reductase was purified 34806-fold with a final yield of 85% from the bovine kidney cortex. Some molecular and kinetic properties of purified enzyme are investigated. Product inhibition studies showed that the enzyme obeys 'branched' mechanism: KmNADPH 18 ± 3 μM and KmGSSG 65 ± 5 μM were determined.
Photochemical coenzyme regeneration in an enzymatically active optical material
Rickus, Jenna L.,Chang, Pauline L.,Tobin, Allan J.,Zink, Jeffrey I.,Dunn, Bruce
, p. 9325 - 9332 (2004)
The photoinduced electron transfer between immobilized thionine and the dinucleotide enzyme cofactors NADH and NADPH in a SiO2 sol-gel matrix is reported. The electron-transfer quenching of thionine luminescence is used to monitor the rate of NADPH oxidation. Using Stern-Volmer quenching curves, the quenching rates in the silica matrix are 1 to 2 orders of magnitude smaller than those in solution, The rate constants for oxidation of NADPH by thionine were measured to be 9.8(?±2.9) ?? 10-3 s-1 in solution and 8.8(?±1.0) ?? 10-4 s-1 in the gel. Within the silica matrix, the photoinduced oxidation of NADPH is combined with the enzymatic reaction of isocitrate dehydrogenase, which uses the oxidized cofactor, NADP+, as an electron acceptor in the oxidation of isocitrate. The encapsulated isocitrate dehydrogenase is active with a Michaelis-Menten constant, KM, of 3 ??M and a kcat of 0.67 ??M/s per mgenzyme?· Because optical sensors use NADPH fluorescence as an indicator of the presence and relative concentration of enzyme substrate, the successful demonstration of photoinduced regeneration of NADP+ makes possible continuous monitoring by the family of dehydrogenase enzymes.
Purification and Characterization of NfrA1, a Bacillus subtilis Nitro/flavin Reductase Capable of Interacting with the Bacterial Luciferase
Zenno, Shuhei,Kobori, Toshiro,Tanokura, Masaru,Saigo, Kaoru
, p. 1978 - 1987 (1998)
ipa-43d is a hypothetical gene identified by the Bacillus subtilis genome project (Mol. Microbiol. 10, 371-384 1993; Nature 390, 249-256 1997). The ipa-43d protein overexpressed in E. coli was purified to homogeneity and its properties were analyzed biochemically. The ipa-43d protein was found to be tightly associated with FMN and to be capable of reducing both nitrofurazone and FMN effectively. Although the ipa-43d protein catalysis obeys the ping-pong Bi-Bi mechanism, catalysis mode was changed to the sequential mechanism upon coupling with the bioluminescent reaction. Database search showed that B. subtilis possessed four genes (ipa-44d, ytmO, yddN, and yvbT), encoding proteins similar in amino acid sequence to LuxA and LuxB of Photobacterium fischeri, and, in particular, ipa-44d is immediately adjacent to the ipa-43d gene on the chromosome.
Factors influencing the operational stability of NADPH-dependent alcohol dehydrogenase and an NADH-dependent variant thereof in gas/solid reactors
Kulishova, Liliya,Dimoula, Kerasina,Jordan, Max,Wirtz, Astrid,Hofmann, Diana,Santiago-Schuebel, Beatrix,Fitter, Joerg,Pohl, Martina,Spiess, Antje C.
experimental part, p. 271 - 283 (2011/02/22)
The continuous enzymatic gas/solid bio-reactor serves both for the production of volatile fine chemicals and flavors on an industrial scale and for thermodynamically controlled investigation of substrate and water effects on enzyme preparations for research purposes. Here, we comparatively investigated the molecular effects on the operational stability of NADPH-dependent Lactobacillus brevis alcohol dehydrogenase and an NADH-dependent variant thereof, LbADH G37D, in the gas/solid bioreactor. The reference reaction is the reduction of acetophenone to (R)-1-phenylethanol with concomitant oxidation of 2-propanol to acetone for the purpose of regeneration of the redox cofactor. It could be clearly shown that not the thermostability of the cofactor, but the thermostability of the proteins in the solid dry state govern the order of magnitude of the operational stability of both purified enzymes in the gas/solid reactor at low thermodynamic activity of water and substrate. However, at higher thermodynamic activity the operational stability in the gas/solid reactor is overlaid by stabilizing and destabilizing effects of the substrates that require further investigation. We demonstrated first evidence that the substrate affinity of the two variants in the gas/solid reactor is similar to the affinity in aqueous medium. We could also show that partial unfolding of the proteins with subsequent aggregation are the factors governing protein thermo-in-stability both in the dissolved and in the dry state. Thus, stability investigations of enzymes in the dry state are suggested to predict their basal level of operational stability in gas/solid reactions.
Molecular cloning and heterologous expression of progesterone 5β-reductase from Digitalis lanata Ehrh.
Herl, Vanessa,Fischer, Gabriele,Müller-Uri, Frieder,Kreis, Wolfgang
, p. 225 - 231 (2008/02/09)
A full-length cDNA clone that encodes progesterone 5β-reductase (5β-POR) was isolated from Digitalis lanata leaves. The reading frame of the 5β-POR gene is 1170 nucleotides corresponding to 389 amino acids. For expression, a Sph1/Sal1 5β-POR fragment was cloned into the pQE vector and was transformed into Escherichia coli strain M15[pREP4]. The recombinant gene was functionally expressed and the recombinant enzyme was characterized. The Km and vmax values for the putative natural substrate progesterone were calculated to be 0.120 mM and 45 nkat mg-1 protein, respectively. Only 5β-pregnane-3,20-dione but not its α-isomer was formed when progesterone was used as the substrate. Kinetic constants for cortisol, cortexone, 4-androstene-3,17-dione and NADPH were also determined. The molecular organization of the 5β-POR gene in D. lanata was determined by Southern blot analysis. The 5β-POR is highly conserved within the genus Digitalis and the respective genes and proteins share considerable homology to putative progesterone reductases from other plant species.
