≥96.5% (HPLC), ≥96.5% (spectrophotometric assay), ≥96.5% (enzymatic), from yeast53-84-9Relevant articles and documents
Oxygen Activation and Electron Transfer in Flavocytochrome P450 BM3
Ost, Tobias W. B.,Clark, Jonathan,Mowat, Christopher G.,Miles, Caroline S.,Walkinshaw, Malcolm D.,Reid, Graeme A.,Chapman, Stephen K.,Daff, Simon
, p. 15010 - 15020 (2003)
In flavocytochrome P450 BM3, there is a conserved phenylalanine residue at position 393 (Phe393), close to Cys400, the thiolate ligand to the heme. Substitution of Phe393 by Ala, His, Tyr, and Trp has allowed us to modulate the reduction potential of the heme, while retaining the structural integrity of the enzyme's active site. Substrate binding triggers electron transfer in P450 BM3 by inducing a shift from a low- to high-spin ferric heme and a 140 mV increase in the heme reduction potential. Kinetic analysis of the mutants indicated that the spin-state shift alone accelerates the rate of heme reduction (the rate determining step for overall catalysis) by 200-fold and that the concomitant shift in reduction potential is only responsible for a modest 2-fold rate enhancement. The second step in the P450 catalytic cycle involves binding of dioxygen to the ferrous heme. The stabilities of the oxy-ferrous complexes in the mutant enzymes were also analyzed using stopped-flow kinetics. These were found to be surprisingly stable, decaying to superoxide and ferric heme at rates of 0.01-0.5 s-1. The stability of the oxy-ferrous complexes was greater for mutants with higher reduction potentials, which had lower catalytic turnover rates but faster heme reduction rates. The catalytic rate-determining step of these enzymes can no longer be the initial heme reduction event but is likely to be either reduction of the stabilized oxy-ferrous complex, i.e., the second flavin to heme electron transfer or a subsequent protonation event. Modulating the reduction potential of P450 BM3 appears to tune the two steps in opposite directions; the potential of the wild-type enzyme appears to be optimized to maximize the overall rate of turnover. The dependence of the visible absorption spectrum of the oxy-ferrous complex on the heme reduction potential is also discussed.
The Peroxidase-NADH Biochemical Oscillator. 1. Examination of Oxygen Mass Transport, the Effect of Light, and the Role of Methylene Blue
Olson, Dean L.,Scheeline, Alexander
, p. 1204 - 1211 (1995)
The peroxidase-NADH oscillator examined here initially consists of four chemical components.The well-mixed aqueous solution includes native horseradish peroxidase, reduced β-nicotinamide adenine dinucleotide (NADH), methylene blue (MB+), and dissolved oxygen combined in a semi-batch reactor under a set of standard conditions.In this system, the macroscopic appearance of the process of oxygen dissolution from the gas phase is dependent on k-m, the mass transport constant of oxygen out of solution.Additional details of oxygen mass transport are derived.The amplitude of oxygen oscillations is decreased by continuous illumination by the deuterium source of a diode array spectrophotometer.This attenuation effect of light is dependent on wavelengths =+ allows several damped oscillations of small amplitude.Subsequent addition of MB+ to the oscillator results in oscillations of much larger amplitude.MB+ is seen to either directly or indirectly enhance the conversion of peroxidase compound III to the native enzyme and then inhibit oxygen consumption, allowing the initiation of relatively large, prolonged oscillations.MB+ is seen to function either as a system catalyst, or as a peroxidase inhibitor in the oxidation of NADH by oxygen.
NADH oxidase activity of Bacillus subtilis nitroreductase NfrA1: Insight into its biological role
Cortial, Sylvie,Chaignon, Philippe,Iorga, Bogdan I.,Aymerich, Stephane,Truan, Gilles,Gueguen-Chaignon, Virginie,Meyer, Philippe,Morera, Solange,Ouazzani, Jamal
, p. 3916 - 3922 (2010)
NfrA1 nitroreductase from the Gram-positive bacterium Bacillus subtilis is a member of the NAD(P)H/FMN oxidoreductase family. Here, we investigated the reactivity, the structure and kinetics of NfrA1, which could provide insight into the unclear biological role of this enzyme. We could show that NfrA1 possesses an NADH oxidase activity that leads to high concentrations of oxygen peroxide and an NAD+ degrading activity leading to free nicotinamide. Finally, we showed that NfrA1 is able to rapidly scavenge H2O2 produced during the oxidative process or added exogenously. Structured summary: MINT- 7990140: nfrA1 (uniprotkb:. P39605) and nfrA1 (uniprotkb:. P39605) bind (MI:. 0407) by X-ray crystallography (MI:. 0114).
The Peroxidase-NADH Biochemical Oscillator. 2. Examination of the Roles of Hydrogen Peroxide and Superoxide
Olson, Dean L.,Scheeline, Alexander
, p. 1212 - 1217 (1995)
The peroxidase-NADH oscillator examined here initially consists of a well-mixed aqueous solution of native horseradish peroxidase, reduced β-nicotinamide adenine dinucleotide (NADH), methylene blue (MB+), and dissolved oxygen combined in a semi-batch reactor under a set of standard conditions.Hydrogen peroxide and superoxide have been implicated as important chemical intermediates.A comprehensive model which includes such intermediates and all initial chemical species has appeared elsewhere.To experimentally explore the role of hydrogen peroxide in the oscillator, H2O2 was substituted for MB+ as an initial ingredient.This substitution allows relatively small, quasi-sinusoidal oscillations sensitive to the oxygen mass transport constant, and predicted earlier in a theoretical model.The oscillations become much larger when MB+ is added, suggesting that MB+ might serve as a chemical mediator between the small oscillations seen when H2O2 is substituted for MB+, and the relatively large oscillations observed when MB+ is present.Catalase and superoxide dismutase are used as enzymatic scavengers for H2O2 and O2.-, respectively.The enzymes are added individually to a working oscillator at oxygen minima and maxima to examine the roles and approximate the concentrations of H2O2 and O2.-.For the enzyme addition experiments, a perturbation model for oxygen behavior is proposed and applied to the interpretation of experimental data.Two methods of analysis for the addition of the enzyme probes indicate a higher concentration of H2O2 and O2.- at oxygen maxima than at minima.Comparison of experimental and simulated data indicate that the relatively simple model presented here is a resonable, yet apparently incomplete, representation of oxygen dynamics for the addition of scavenger enzymes to this oscillator.
Magneto-stimulated hydrodynamic control of electrocatalytic and bioelectrocatalytic processes.
Katz, Eugenii,Willner, Itamar
, p. 10290 - 10291 (2002)
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Altering the substrate specificity of glutamate dehydrogenase from Bacillus subtilis by site-directed mutagenesis
Khan, Md. Iqbal Hassan,Kim, Hyeung,Ashida, Hiroyuki,Ishikawa, Takahiro,Shibata, Hitoshi,Sawa, Yoshihiro
, p. 1802 - 1805 (2005)
The Lys80, Gly82 and Met101 residues of glutamate dehydrogenase from Bacillus subtilis were mutated into a series of single mutants. The wild-type enzyme was highly specific for 2-oxoglutarate, whereas G82K and M101S dramatically switched to increased specificity for oxaloacetate with k cat values 3.45 and 5.68s-1, which were 265-fold and 473-fold higher respectively than those for 2-oxoglutarate.
Fluorescent half-sandwich phosphine-sulfonate iridium(III) and ruthenium(II) complexes as potential lysosome-targeted anticancer agents
Du, Qing,Yang, Yuliang,Guo, Lihua,Tian, Meng,Ge, Xingxing,Tian, Zhenzhen,Zhao, Liping,Xu, Zhishan,Li, Juanjuan,Liu, Zhe
, p. 821 - 830 (2018/11/23)
The synthesis, characterization and biological activity of neutral fluorescent Ir(III) and Ru(II) half-sandwich organometallic complexes containing phosphine-sulfonate ligands are reported. X-ray crystal structure of complexes 1–3, 10 and 11 exhibits the expected half-sandwich “three-legged piano-stool” pseudo-octahedral geometry. Spectroscopic properties study displays that these complexes show rich fluorescence properties. With the exception of 9, 10 and 11 toward A549 human lung cancer cells and 10 towards HeLa human cervical cancer cells, each complex shows promising cytotoxicity toward HeLa and A549 cells line with IC50 values in the range of 3.6–53.1 μM, and 6.5–34.5 μM, respectively. Hydrolysis, DNA cleavage and depolarization of the mitochondrial membrane potential (MMP) appear not to be the main mechanism of action. However, these complexes are able to covert NADH to NAD+ via the transfer hydrogenation. Mechanism studies by flow cytometry display that the complexes exert their anticancer efficacy by inducing apoptosis, perturbing the cell cycle and increasing the intracellular ROS level. Furthermore, fluorescence property of these complexes provides a tool to investigate the microscopic mechanism by confocal microscopy. Notably, the typical Ir(III) complex 3 can specially localize to lysosome and damage it. In addition, complex 3 enters into HeLa cells mainly through energy-dependent pathway.
Toward Automated Enzymatic Glycan Synthesis in a Compartmented Flow Microreactor System
Heinzler, Raphael,Fisch?der, Thomas,Elling, Lothar,Franzreb, Matthias
supporting information, p. 4506 - 4516 (2019/08/20)
Immobilized microfluidic enzyme reactors (IMER) are of particular interest for automation of enzyme cascade reactions. Within an IMER, substrates are converted by paralleled immobilized enzyme modules and intermediate products are transported for further conversion by subsequent enzyme modules. By optimizing substrate conversion in the spatially separated enzyme modules purification of intermediate products is not necessary, thus shortening process time and increasing space-time yields. The IMER enables the development of efficient enzyme cascades by combining compatible enzymatic reactions in different arrangements under optimal conditions and the possibility of a cost-benefit analysis prior to scale-up. These features are of special interest for automation of enzymatic glycan synthesis. We here demonstrate a compartmented flow microreactor system using six magnetic enzyme beads (MEBs) for the synthesis of the non-sulfated human natural killer cell-1 (HNK-1) glycan epitope. MEBs are assembled to build compartmented enzyme modules, consisting of enzyme cascades for the synthesis of uridine 5′- diphospho-α- d-galactose (UDP-Gal) and uridine 5′-diphospho-α-d-glucuronic acid (UDP-GlcA), the donor substrates for the Leloir glycosyltransferases β4-galactosyltransferase and β3-glucuronosyltransferase, respectively. Glycan synthesis was realized in an automated microreactor system by a cascade of individual enzyme module compartments each performing under optimal conditions. The products were analyzed inline by an MS-system connected to the microreactor. The high synthesis yield of 96% for the non-sulfated HNK-1 glycan epitope indicates the excellent performance of the automated enzyme module cascade. Furthermore, combinations of other MEBs for nucleotide sugars synthesis with MEBs of glycosyltransferases have the potential for a fully automated and programmed glycan synthesis in a compartmented flow microreactor system. (Figure presented.).
