The Experts below are selected from a list of 93 Experts worldwide ranked by ideXlab platform
John D Lipscomb - One of the best experts on this subject based on the ideXlab platform.
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a family of diiron Monooxygenases catalyzing amino acid beta hydroxylation in antibiotic biosynthesis
Proceedings of the National Academy of Sciences of the United States of America, 2010Co-Authors: Thomas M Makris, Mrinmoy Chakrabarti, Eckard Munck, John D LipscombAbstract:The biosynthesis of chloramphenicol requires a β-hydroxylation tailoring Reaction of the precursor L-p-aminophenylalanine (L-PAPA). Here, it is shown that this Reaction is catalyzed by the enzyme CmlA from an operon containing the genes for biosynthesis of L-PAPA and the nonribosomal peptide synthetase CmlP. EPR, Mossbauer, and optical spectroscopies reveal that CmlA contains an oxo-bridged dinuclear iron cluster, a metal center not previously associated with nonribosomal peptide synthetase chemistry. Single-turnover kinetic studies indicate that CmlA is functional in the diferrous state and that its substrate is L-PAPA covalently bound to CmlP. Analytical studies show that the product is hydroxylated L-PAPA and that O2 is the oxygen source, demonstrating a Monooxygenase Reaction. The gene sequence of CmlA shows that it utilizes a lactamase fold, suggesting that the diiron cluster is in a protein environment not previously known to effect Monooxygenase Reactions. Notably, CmlA homologs are widely distributed in natural product biosynthetic pathways, including a variety of pharmaceutically important beta-hydroxylated antibiotics and cytostatics.
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a family of diiron Monooxygenases catalyzing amino acid beta hydroxylation in antibiotic biosynthesis
Proceedings of the National Academy of Sciences of the United States of America, 2010Co-Authors: Thomas M Makris, Mrinmoy Chakrabarti, Eckard Munck, John D LipscombAbstract:The biosynthesis of chloramphenicol requires a beta-hydroxylation tailoring Reaction of the precursor L-p-aminophenylalanine (L-PAPA). Here, it is shown that this Reaction is catalyzed by the enzyme CmlA from an operon containing the genes for biosynthesis of L-PAPA and the nonribosomal peptide synthetase CmlP. EPR, Mossbauer, and optical spectroscopies reveal that CmlA contains an oxo-bridged dinuclear iron cluster, a metal center not previously associated with nonribosomal peptide synthetase chemistry. Single-turnover kinetic studies indicate that CmlA is functional in the diferrous state and that its substrate is L-PAPA covalently bound to CmlP. Analytical studies show that the product is hydroxylated L-PAPA and that O(2) is the oxygen source, demonstrating a Monooxygenase Reaction. The gene sequence of CmlA shows that it utilizes a lactamase fold, suggesting that the diiron cluster is in a protein environment not previously known to effect Monooxygenase Reactions. Notably, CmlA homologs are widely distributed in natural product biosynthetic pathways, including a variety of pharmaceutically important beta-hydroxylated antibiotics and cytostatics.
Ingela Jansson - One of the best experts on this subject based on the ideXlab platform.
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the many roles of cytochrome b5
Pharmacology & Therapeutics, 2003Co-Authors: John B Schenkman, Ingela JanssonAbstract:Four distinct suggestions have been made to explain the mechanism of the cytochrome b(5)-imposed positive modifier action of the cytochrome P450 Monooxygenase Reaction. The first mechanism involves a direct input of an electron into the Monooxygenase cycle. This is the second of the two electrons necessary for activation of molecular oxygen, and appears to be a rate-limiting step in the Monooxygenase Reaction. P450 Monooxygenases all appear to be uncoupled to varying extents, releasing superoxide and hydrogen peroxide instead of oxidized substrate. A second mechanism suggests that cytochrome b(5) acts as a positive modifier of the Monooxygenase by decreasing the extent of uncoupling of the Monooxygenase Reaction. The implication is that a slow input of the second electron allows uncoupling of a superoxide anion instead of formation of two-electron reduced oxygen. Faster input of the second electron via cytochrome b(5) would result in formation of more of the activated oxygen that reacts with substrate to form product. A third suggestion involves formation of a two-hemoprotein complex between cytochrome b(5) and cytochrome P450 that allows acceptance of two electrons from NADPH-cytochrome P450 reductase. Uncomplexed cytochrome P450 accepts an electron from the reductase, dissociates from it, binds oxygen, and re-associates with the reductase to accept another electron. Complexation with cytochrome b(5) enhances the rate of formation of the active oxygen by obviating the need for two interactions with reductase. The fourth mechanism has cytochrome b(5) serving as an effector without a reduction-oxidation role in the monooxygenation Reaction. This effector function may be to enhance the breakdown of the oxygenated hemoprotein to products or to facilitate flow of electrons through the system.
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influence of ionic strength on the p450 Monooxygenase Reaction and role of cytochrome b5 in the process
Archives of Biochemistry and Biophysics, 1994Co-Authors: John B Schenkman, A I Voznesensky, Ingela JanssonAbstract:A stimulatory effect of increased salt content on the metabolism of benzphetamine, 7-ethoxycoumarin, and coumarin by rabbit liver microsomes, CYP2B4 and rabbit CYP1A2, was seen, indicating that the effect was not specific for either substrate or form of cytochrome P450. The stimulation was not due to an action on the cytochrome P450 itself as increased salt concentration minimally affected the substrate turnover when cumene hydroperoxide was used as the source of active oxygen. The elevation of ionic strength increased the coupling efficiency of the Monooxygenase Reaction with benzphetamine as substrate. Cytochrome b5 also can increase the Monooxygenase coupling efficiency. At low ionic strength cytochrome b5 did not much influence the reduction of P450, but the rate constant of the cytochrome b5 reduction was increased about 15-fold by its binding to cytochrome P450. A stimulatory effect of cytochrome b5 on benzphetamine oxidation was seen at low ionic strength, but it was lost at elevated ionic strength as the binding of cytochrome b5 to cytochrome P450 was weakened. At the higher ionic strength cytochrome b5 competes with cytochrome P450 for the reductase, an action that slows cytochrome P450 reduction. Based upon these observations, plus those in the literature, a scheme is suggested that proposes the stimulatory effect of cytochrome b5 on the cytochrome P450-mediated monooxygenation Reaction is due to an increase in the efficiency of the electron transfer Reaction: With cytochrome b5 bound to cytochrome P450, two electrons can be provided from the reductase to the P450-b5 complex in a single interaction, obviating the need for a second interaction with the reductase.
Thomas M Makris - One of the best experts on this subject based on the ideXlab platform.
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a family of diiron Monooxygenases catalyzing amino acid beta hydroxylation in antibiotic biosynthesis
Proceedings of the National Academy of Sciences of the United States of America, 2010Co-Authors: Thomas M Makris, Mrinmoy Chakrabarti, Eckard Munck, John D LipscombAbstract:The biosynthesis of chloramphenicol requires a β-hydroxylation tailoring Reaction of the precursor L-p-aminophenylalanine (L-PAPA). Here, it is shown that this Reaction is catalyzed by the enzyme CmlA from an operon containing the genes for biosynthesis of L-PAPA and the nonribosomal peptide synthetase CmlP. EPR, Mossbauer, and optical spectroscopies reveal that CmlA contains an oxo-bridged dinuclear iron cluster, a metal center not previously associated with nonribosomal peptide synthetase chemistry. Single-turnover kinetic studies indicate that CmlA is functional in the diferrous state and that its substrate is L-PAPA covalently bound to CmlP. Analytical studies show that the product is hydroxylated L-PAPA and that O2 is the oxygen source, demonstrating a Monooxygenase Reaction. The gene sequence of CmlA shows that it utilizes a lactamase fold, suggesting that the diiron cluster is in a protein environment not previously known to effect Monooxygenase Reactions. Notably, CmlA homologs are widely distributed in natural product biosynthetic pathways, including a variety of pharmaceutically important beta-hydroxylated antibiotics and cytostatics.
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a family of diiron Monooxygenases catalyzing amino acid beta hydroxylation in antibiotic biosynthesis
Proceedings of the National Academy of Sciences of the United States of America, 2010Co-Authors: Thomas M Makris, Mrinmoy Chakrabarti, Eckard Munck, John D LipscombAbstract:The biosynthesis of chloramphenicol requires a beta-hydroxylation tailoring Reaction of the precursor L-p-aminophenylalanine (L-PAPA). Here, it is shown that this Reaction is catalyzed by the enzyme CmlA from an operon containing the genes for biosynthesis of L-PAPA and the nonribosomal peptide synthetase CmlP. EPR, Mossbauer, and optical spectroscopies reveal that CmlA contains an oxo-bridged dinuclear iron cluster, a metal center not previously associated with nonribosomal peptide synthetase chemistry. Single-turnover kinetic studies indicate that CmlA is functional in the diferrous state and that its substrate is L-PAPA covalently bound to CmlP. Analytical studies show that the product is hydroxylated L-PAPA and that O(2) is the oxygen source, demonstrating a Monooxygenase Reaction. The gene sequence of CmlA shows that it utilizes a lactamase fold, suggesting that the diiron cluster is in a protein environment not previously known to effect Monooxygenase Reactions. Notably, CmlA homologs are widely distributed in natural product biosynthetic pathways, including a variety of pharmaceutically important beta-hydroxylated antibiotics and cytostatics.
John B Schenkman - One of the best experts on this subject based on the ideXlab platform.
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the many roles of cytochrome b5
Pharmacology & Therapeutics, 2003Co-Authors: John B Schenkman, Ingela JanssonAbstract:Four distinct suggestions have been made to explain the mechanism of the cytochrome b(5)-imposed positive modifier action of the cytochrome P450 Monooxygenase Reaction. The first mechanism involves a direct input of an electron into the Monooxygenase cycle. This is the second of the two electrons necessary for activation of molecular oxygen, and appears to be a rate-limiting step in the Monooxygenase Reaction. P450 Monooxygenases all appear to be uncoupled to varying extents, releasing superoxide and hydrogen peroxide instead of oxidized substrate. A second mechanism suggests that cytochrome b(5) acts as a positive modifier of the Monooxygenase by decreasing the extent of uncoupling of the Monooxygenase Reaction. The implication is that a slow input of the second electron allows uncoupling of a superoxide anion instead of formation of two-electron reduced oxygen. Faster input of the second electron via cytochrome b(5) would result in formation of more of the activated oxygen that reacts with substrate to form product. A third suggestion involves formation of a two-hemoprotein complex between cytochrome b(5) and cytochrome P450 that allows acceptance of two electrons from NADPH-cytochrome P450 reductase. Uncomplexed cytochrome P450 accepts an electron from the reductase, dissociates from it, binds oxygen, and re-associates with the reductase to accept another electron. Complexation with cytochrome b(5) enhances the rate of formation of the active oxygen by obviating the need for two interactions with reductase. The fourth mechanism has cytochrome b(5) serving as an effector without a reduction-oxidation role in the monooxygenation Reaction. This effector function may be to enhance the breakdown of the oxygenated hemoprotein to products or to facilitate flow of electrons through the system.
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influence of ionic strength on the p450 Monooxygenase Reaction and role of cytochrome b5 in the process
Archives of Biochemistry and Biophysics, 1994Co-Authors: John B Schenkman, A I Voznesensky, Ingela JanssonAbstract:A stimulatory effect of increased salt content on the metabolism of benzphetamine, 7-ethoxycoumarin, and coumarin by rabbit liver microsomes, CYP2B4 and rabbit CYP1A2, was seen, indicating that the effect was not specific for either substrate or form of cytochrome P450. The stimulation was not due to an action on the cytochrome P450 itself as increased salt concentration minimally affected the substrate turnover when cumene hydroperoxide was used as the source of active oxygen. The elevation of ionic strength increased the coupling efficiency of the Monooxygenase Reaction with benzphetamine as substrate. Cytochrome b5 also can increase the Monooxygenase coupling efficiency. At low ionic strength cytochrome b5 did not much influence the reduction of P450, but the rate constant of the cytochrome b5 reduction was increased about 15-fold by its binding to cytochrome P450. A stimulatory effect of cytochrome b5 on benzphetamine oxidation was seen at low ionic strength, but it was lost at elevated ionic strength as the binding of cytochrome b5 to cytochrome P450 was weakened. At the higher ionic strength cytochrome b5 competes with cytochrome P450 for the reductase, an action that slows cytochrome P450 reduction. Based upon these observations, plus those in the literature, a scheme is suggested that proposes the stimulatory effect of cytochrome b5 on the cytochrome P450-mediated monooxygenation Reaction is due to an increase in the efficiency of the electron transfer Reaction: With cytochrome b5 bound to cytochrome P450, two electrons can be provided from the reductase to the P450-b5 complex in a single interaction, obviating the need for a second interaction with the reductase.
Edward I. Solomon - One of the best experts on this subject based on the ideXlab platform.
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a cu2o 2 core in cu zsm 5 the active site in the oxidation of methane to methanol
Proceedings of the National Academy of Sciences of the United States of America, 2009Co-Authors: Julia S Woertink, Pieter J Smeets, Marijke H Groothaert, Michael A Vance, Bert F Sels, Robert A Schoonheydt, Edward I. SolomonAbstract:Driven by the depletion of crude oil, the direct oxidation of methane to methanol has been of considerable interest. Promising low-temperature activity of an oxygen-activated zeolite, Cu-ZSM-5, has recently been reported in this selective oxidation and the active site in this Reaction correlates with an absorption feature at 22,700 cm−1. In the present study, this absorption band is used to selectively resonance enhance Raman vibrations of this active site. 18O2 labeling experiments allow definitive assignment of the observed vibrations and exclude all previously characterized copper-oxygen species for the active site. In combination with DFT and normal coordinate analysis calculations, the oxygen activated Cu core is uniquely defined as a bent mono-(μ-oxo)dicupric site. Spectroscopically validated electronic structure calculations show polarization of the low-lying singly-occupied molecular orbital of the [Cu2O]2+ core, which is directed into the zeolite channel, upon approach of CH4. This induces significant oxyl character into the bridging O atom leading to a low transition state energy consistent with experiment and explains why the bent mono-(μ-oxo)dicupric core is highly activated for H atom abstraction from CH4. The oxygen intermediate of Cu-ZSM-5 is now the most well defined species active in the methane Monooxygenase Reaction.
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Oxygen Activation by the Noncoupled Binuclear Copper Site in Peptidylglycine α-Hydroxylating Monooxygenase. Reaction Mechanism and Role of the Noncoupled Nature of the Active Site
Journal of the American Chemical Society, 2004Co-Authors: Peng Chen, Edward I. SolomonAbstract:Reaction thermodynamics and potential energy surfaces are calculated using density functional methods to investigate possible reactive Cu/O(2) species for H-atom abstraction in peptidylglycine alpha-hydroxylating Monooxygenase (PHM), which has a noncoupled binuclear Cu active site. Two possible mononuclear Cu/O(2) species have been evaluated, the 2-electron reduced Cu(II)(M)-OOH intermediate and the 1-electron reduced side-on Cu(II)(M)-superoxo intermediate, which could form with comparable thermodynamics at the catalytic Cu(M) site. The substrate H-atom abstraction Reaction by the Cu(II)(M)-OOH intermediate is found to be thermodynamically accessible due to the contribution of the methionine ligand, but with a high activation barrier ( approximately 37 kcal/mol, at a 3.0-A active site/substrate distance), arguing against the Cu(II)(M)-OOH species as the reactive Cu/O(2) intermediate in PHM. In contrast, H-atom abstraction from substrate by the side-on Cu(II)(M)-superoxo intermediate is a nearly isoenergetic process with a low Reaction barrier at a comparable active site/substrate distance ( approximately 14 kcal/mol), suggesting that side-on Cu(II)(M)-superoxo is the reactive species in PHM. The differential reactivities of the Cu(II)(M)-OOH and Cu(II)(M)-superoxo species correlate to their different frontier molecular orbitals involved in the H-atom abstraction Reaction. After the H-atom abstraction, a reasonable pathway for substrate hydroxylation involves a "water-assisted" direct OH transfer to the substrate radical, which generates a high-energy Cu(II)(M)-oxyl species. This provides the necessary driving force for intramolecular electron transfer from the Cu(H) site to complete the Reaction in PHM. The differential reactivity pattern between the Cu(II)(M)-OOH and Cu(II)(M)-superoxo intermediates provides insight into the role of the noncoupled nature of PHM and dopamine beta-Monooxygenase active sites, as compared to the coupled binuclear Cu active sites in hemocyanin, tyrosinase, and catechol oxidase, in O(2) activation.