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Prashant S. Phale - One of the best experts on this subject based on the ideXlab platform.
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Kinetic and Spectroscopic Characterization of 1-Naphthol 2-hydroxylase from Pseudomonas sp. Strain C5
Applied biochemistry and biotechnology, 2014Co-Authors: Vikas D. Trivedi, Prabin D. Majhi, Prashant S. PhaleAbstract:1-Naphthol 2-hydroxylase (1-NH) catalyzes the conversion of 1-Naphthol to 1,2-dihydroxynaphthalene. 1-NH from carbaryl degrading Pseudomonas strain C5 was purified and characterized for its kinetic and spectroscopic properties. The enzyme was found to be NAD(P)H-dependent external flavin monooxygenase. Though the kinetic parameters of 1-NH from strain C5 appear to be similar to 1-NH enzyme from strains C4 and C6, however, they differ in their N-terminal sequences, mole content of flavin adenine dinucleotide (FAD), reconstitution of apoenzyme, and Ki. 1-NH showed narrow substrate specificity with comparable hydroxylation efficiency on 1-Naphthol and 5-amino 1-Naphthol (~30 %) followed by 4-chloro 1-Naphthol (~9 %). Salicylate was found to be the nonsubstrate effector. The flavin fluorescence of 1-NH was found to increase in the presence of 1-Naphthol (Kd = 11.3 μM) and salicylate (Kd = 1027 μM). The circular dichroism (CD) spectra showed significant perturbations in the presence of NAD(P)H, whereas no changes were observed in the presence of 1-Naphthol. Naphthalene, 1-chloronaphthalene, 2-napthol, and 2-naphthoic acid were found to be the mixed inhibitors. Chemical modification studies showed the probable involvement of His, Cys, and Tyr in the binding of 1-Naphthol, whereas Trp was found to be involved in the binding of NAD(P)H.
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1-Naphthol 2-hydroxylase from Pseudomonas sp. strain C6: purification, characterization and chemical modification studies
Biodegradation, 2011Co-Authors: Prashant S. PhaleAbstract:1-Naphthol 2-hydroxylase (1-NH) which catalyzes the conversion of 1-Naphthol to 1,2-dihydroxynaphthalene was purified to homogeneity from carbaryl-degrading Pseudomonas sp. strain C6. The enzyme was found to be a homodimer with subunit molecular weight of 66 kDa. UV, visible and fluorescence spectral properties, identification of flavin moiety by HPLC as FAD, and reconstitution of apoenzyme by FAD suggest that enzyme is FAD-dependent. 1-NH accepts electron from NADH as well as NADPH. Besides 1-Naphthol ( K _m, 9.1 μM), the enzyme also accepts 5-amino 1-Naphthol ( K _m, 6.4 μM) and 4-chloro 1-Naphthol ( K _m, 2.3 μM) as substrates. Enzyme showed substrate inhibition phenomenon at high concentration of 1-Naphthol ( K _i, 283 μM). Stoichiometric consumption of oxygen and NADH, and biochemical properties suggest that 1-NH belongs to FAD containing external flavomonooxygenase group of oxido-reductase class of enzymes. Based on biochemical and kinetic properties, 1-NH from Pseudomonas sp. strain C6 appears to be different than that reported earlier from Pseudomonas sp. strain C4. Chemical modification and protection by 1-Naphthol and NADH suggest that His, Arg, Cys, Tyr and Trp are at or near the active site of 1-NH.
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Purification and Characterization of 1-Naphthol-2-Hydroxylase from Carbaryl-Degrading Pseudomonas Strain C4
Journal of bacteriology, 2007Co-Authors: Vandana P. Swetha, Aditya Basu, Prashant S. PhaleAbstract:Pseudomonas sp. strain C4 metabolizes carbaryl (1-naphthyl-N-methylcarbamate) as the sole source of carbon and energy via 1-Naphthol, 1,2-dihydroxynaphthalene, and gentisate. 1-Naphthol-2-hydroxylase (1-NH) was purified 9.1-fold to homogeneity from Pseudomonas sp. strain C4. Gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the enzyme is a homodimer with a native molecular mass of 130 kDa and a subunit molecular mass of 66 kDa. The enzyme was yellow, with absorption maxima at 274, 375, and 445 nm, indicating a flavoprotein. High-performance liquid chromatography analysis of the flavin moiety extracted from 1-NH suggested the presence of flavin adenine dinucleotide (FAD). Based on the spectral properties and the molar extinction coefficient, it was determined that the enzyme contained 1.07 mol of FAD per mol of enzyme. Although the enzyme accepts electrons from NADH, it showed maximum activity with NADPH and had a pH optimum of 8.0. The kinetic constants Km and Vmax for 1-Naphthol and NADPH were determined to be 9.6 and 34.2 μM and 9.5 and 5.1 μmol min−1 mg−1, respectively. At a higher concentration of 1-Naphthol, the enzyme showed less activity, indicating substrate inhibition. The Ki for 1-Naphthol was determined to be 79.8 μM. The enzyme showed maximum activity with 1-Naphthol compared to 4-chloro-1-Naphthol (62%) and 5-amino-1-Naphthol (54%). However, it failed to act on 2-Naphthol, substituted naphthalenes, and phenol derivatives. The enzyme utilized one mole of oxygen per mole of NADPH. Thin-layer chromatographic analysis showed the conversion of 1-Naphthol to 1,2-dihydroxynaphthalene under aerobic conditions, but under anaerobic conditions, the enzyme failed to hydroxylate 1-Naphthol. These results suggest that 1-NH belongs to the FAD-containing external flavin mono-oxygenase group of the oxidoreductase class of proteins.
Elliot R. Bernstein - One of the best experts on this subject based on the ideXlab platform.
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Excited-state intermolecular proton transfer In molecular jet-cooled 1-Naphthol/H 2 and 1-Naphthol/NH 3 clusters
1991Co-Authors: Seong K. Kim, Elliot R. BernsteinAbstract:Mass-resolved excitation/ionization spectroscopy and excitation/emission spectroscopy are used to investigate the proton transfer reaction in the excited singlet state of the jet-cooled 1-Naphthol (NH3)n and 1-Naphthol (H2O)n. Results indicate no proton transfer for 1-Naphthol(NH3)12 and 1-Naphthol(H2O)n and proton transfer for 1-Naphthol(NH3)n (n≥4) clusters, which is consistent with other reports,1-3 However, 1-Naphthol(NH3)3 exists in multiple conformations, as evidenced by the changes of the peak intensities around the electronic origin with the variations of the second-color energy in the mass-resolved excitation/ionization spectra (Fig.1).
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Excited state intermolecular proton transfer in isolated clusters: 1‐Naphthol/ammonia and water
The Journal of Chemical Physics, 1991Co-Authors: Seong Keun Kim, Elliot R. BernsteinAbstract:The excited singlet state intermolecular proton transfer reaction in jet‐cooled clusters of 1‐Naphthol/ammonia and water is investigated employing mass resolved excitation, threshold photoionization, and emission spectroscopy. The complete data set indicates that no proton transfer occurs for 1‐Naphthol(NH3)1,2 and (H2O)n, n=1,...,20 clusters. Proton transfer occurs for (at least) one configuration of the 1‐Naphthol(NH3)3 cluster, as well as all 1‐Naphthol(NH3)n, n≥4, clusters. The (at least) two configurations of 1‐Naphthol(NH3)3 clusters are distinguished by threshold photoionization studies. The 1‐Naphthol(NH3)3 cluster for which proton transfer is indicated has a threshold photoionization energy ∼2000 cm−1 below the other 1‐Naphthol(NH3)3 cluster configurations. These results are employed to explain the previous discrepancy between static spectroscopic experiments and picosecond time resolved dynamic experiments concerning proton transfer in the 1‐Naphthol(NH3)3 cluster. Calculations of cluster geomet...
Seong Keun Kim - One of the best experts on this subject based on the ideXlab platform.
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Excited state intermolecular proton transfer in isolated clusters: 1‐Naphthol/ammonia and water
The Journal of Chemical Physics, 1991Co-Authors: Seong Keun Kim, Elliot R. BernsteinAbstract:The excited singlet state intermolecular proton transfer reaction in jet‐cooled clusters of 1‐Naphthol/ammonia and water is investigated employing mass resolved excitation, threshold photoionization, and emission spectroscopy. The complete data set indicates that no proton transfer occurs for 1‐Naphthol(NH3)1,2 and (H2O)n, n=1,...,20 clusters. Proton transfer occurs for (at least) one configuration of the 1‐Naphthol(NH3)3 cluster, as well as all 1‐Naphthol(NH3)n, n≥4, clusters. The (at least) two configurations of 1‐Naphthol(NH3)3 clusters are distinguished by threshold photoionization studies. The 1‐Naphthol(NH3)3 cluster for which proton transfer is indicated has a threshold photoionization energy ∼2000 cm−1 below the other 1‐Naphthol(NH3)3 cluster configurations. These results are employed to explain the previous discrepancy between static spectroscopic experiments and picosecond time resolved dynamic experiments concerning proton transfer in the 1‐Naphthol(NH3)3 cluster. Calculations of cluster geomet...
Ehud Pines - One of the best experts on this subject based on the ideXlab platform.
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Ultrafast excited-state proton-transfer reaction of 1-Naphthol-3,6-disulfonate and several 5-substituted 1-Naphthol derivatives.
The journal of physical chemistry. B, 2013Co-Authors: Mirabelle Prémont-schwarz, Tamar Barak, Dina Pines, Erik T. J. Nibbering, Ehud PinesAbstract:The 1-Naphthol molecule has been the subject of intense research activity for the past 60 years due to its complex behavior as a photoacid upon optical excitation. We have utilized femtosecond mid-infrared spectroscopy and time-resolved fluorescence spectroscopy to investigate the excited-state proton-transfer reaction of 1-Naphthol-3,6-disulfonate (1N-3,6diS) and several 5-substituted 1-Naphthol derivatives. The proton dissociation rate constant of 1N-3,6-diS was found to be about 3 times faster and the pKa* about 2 pKa units more acidic than the values previously reported in the literature. A Marcus (free-energy) plot of excited-state proton dissociation rate constants vs the excited-state equilibrium constant of the photoacids, Ka*, was constructed using the C-5 series of 1-Naphthol derivatives. The newly measured values for the ESPT rate constant and pKa* of 1N-3,6diS was found to fit well with the Marcus correlation. We discuss our findings in the context of the photoacidity phenomenon in general, an...
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Competitive geminate quenching and geminate recombination reactions of 1‐Naphthol
Berichte der Bunsengesellschaft für physikalische Chemie, 1998Co-Authors: Ehud Pines, Dimitry Tepper, Ben-zion Magnes, Dina Pines, Tamar BarakAbstract:The back proton recombination following the photo-dissociation of 1-Naphthol was studied by a time-correlated single-photon counting apparatus. The measured picosecond time-resolved fluorescence decay profiles were analyzed using the numerical solution of the Debye-Smoluchowski equation with coupled reversible and irreversible proton back-recombination reactions at the source. A full kinetic analysis shows that both the reversible geminate recombination reaction, which assumes at long times a t -3/2 dependence, and the irreversible geminate quenching reaction, which assumes at long times a t -1/2 dependence, are significant in determining the excited-state kinetics of 1-Naphthol.
R. Knochenmuss - One of the best experts on this subject based on the ideXlab platform.
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Photoionization, fragmentation and proton transfer in 1-Naphthol(NH3)n clusters
Chemical Physics Letters, 1999Co-Authors: R. KnochenmussAbstract:Abstract The fragmentation of photoionized 1-Naphthol·(NH 3 ) n clusters is examined. In one-color resonant two-photon ionization experiments, fragmentation in the ion state is observed for every cluster size above n =1. For small clusters ( n ≤4), a single neutral ammonia is evaporated in the ion state. This ceases at energies 1500–2500 cm −1 above the ionization thresholds. Larger clusters, n ≥5, are readily ionized at much lower energies than n ≤4. They yield (1-Naphthol·(NH 3 ) n ) + , 1-Naphthol·(NH 3 ) n H + , and (NH 3 ) n H + ion fragments. Several neutral ammonia molecules can be lost. These observations support predictions that n ≥5 clusters undergo proton transfer in the ground state.
