The Experts below are selected from a list of 177 Experts worldwide ranked by ideXlab platform
Eric M. Brustad - One of the best experts on this subject based on the ideXlab platform.
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A serine-substituted P450 catalyzes highly efficient carbene transfer to olefins in vivo
Nature Chemical Biology, 2013Co-Authors: Pedro S. Coelho, Z. Jane Wang, Maraia E. Ener, S.a. Baril, Arvind Kannan, Frances H. Arnold, Eric M. BrustadAbstract:Whole-cell catalysts for non-natural chemical reactions will open new routes to sustainable production of chemicals. We designed a cytochrome 'P411' with unique serine-heme ligation that catalyzes efficient and selective olefin cyclopropanation in intact Escherichia coli cells. The mutation C400S in cytochrome P450_BM3 gives a signature ferrous CO Soret Peak at 411 nm, abolishes monooxygenation activity, raises the resting-state Fe^III-to-Fe^II reduction potential and substantially improves NAD(P)H-driven activity. The exchange of a heme-ligating cysteine for a serine residue in an engineered P450 alters the redox potential of the enzyme, deactivating wild-type function and enabling efficient, NADPH-mediated carbene transfer in cells.
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A Serine-Substituted P450 Catalyzes Highly Efficient Carbene Transfer to Olefins In Vivo
Nature Chemical Biology, 2013Co-Authors: Pedro S. Coelho, Z. Jane Wang, Maraia E. Ener, S.a. Baril, Arvind Kannan, Frances H. Arnold, Eric M. BrustadAbstract:Whole-cell catalysts for non-natural chemical reactions will open new routes to sustainable production of chemicals. We designed a cytochrome 'P411' with unique serine-heme ligation that catalyzes efficient and selective olefin cyclopropanation in intact Escherichia coli cells. The mutation C400S in cytochrome P450_(BM3) gives a signature ferrous CO Soret Peak at 411 nm, abolishes monooxygenation activity, raises the resting-state FeIII-to-FeII reduction potential and substantially improves NAD(P)H-driven activity.
Miguel Teixeira - One of the best experts on this subject based on the ideXlab platform.
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An archaeal b-type cytochrome containing a nonfunctional carbonic anhydrase-like domain
JBIC Journal of Biological Inorganic Chemistry, 2002Co-Authors: Cláudio M. Gomes, Arnulf Kletzin, Miguel TeixeiraAbstract:A new type of cytochrome b was isolated from the cytoplasmatic fraction of the archaeon Acidianus ambivalens , which is the first soluble cytochrome found in this member of the thermoacidophilic order of the Sulfolobales . The protein is a monomeric and monohemic cytochrome b with a molecular mass of 22 kDa. Visible spectroscopy of the as-purified protein shows a Soret Peak at 405 nm and a broad band at 625 nm, indicating the presence of a high-spin ferric heme. Upon reduction, the Soret band shifts to 422 nm and a broad band at 560 nm develops, again characteristic of high-spin ferrous heme. The reduced form can bind carbon monoxide, with visible absorption bands arising at 411 and 566 nm. EPR spectroscopy of the oxidized protein shows a spectrum typical of a high-spin heme, with major g values at 6.56 and 5.85. The reduction potential of the heme cofactor was determined to be –16±10 mV, at pH 6.5. Analysis of the protein amino acid sequence shows that it consists of a novel arrangement of domains. The first domain, at the N-terminus, has a remarkable similarity towards β class carbonic anhydrases, whereas the second region comprises a putative cytochrome domain. The latter presumably consists of a novel fold, as it bears no sequence similarities towards other known cytochromes, or towards known domains. Strikingly, the first module contains the C-X_ n -H-X_2-C motif that accounts for the binding of the catalytic zinc in carbonic anhydrases, but lacks several other critical residues required for substrate binding and proper active site geometry. In agreement with this finding, the isolated cytochrome contains one bound zinc atom, but has no carbonic anhydrase activity. Inspection of the sequences available from the genomic sequencing project of the close relative archaeon Sulfolobus solfataricus shows the presence of an identical protein, suggesting its dissemination among the Sulfolobales . The role of zinc as a key element for the intrinsic thermal stability of these proteins is discussed.
Suman Kundu - One of the best experts on this subject based on the ideXlab platform.
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penta and hexa coordinate ferric hemoglobins display distinct ph titration profiles measured by Soret Peak shifts
Analytical Biochemistry, 2016Co-Authors: Sheetal Uppal, Amit Kumar, Manish Shandilya, Nitika Mukhi, Amit Singh, Suneel Kateriya, Jagreet Kaur, Suman KunduAbstract:Hemoglobins with diverse characteristics have been identified in all kingdoms of life. Their ubiquitous presence indicates that these proteins play important roles in physiology, though function for all hemoglobins are not yet established with certainty. Their physiological role may depend on their ability to bind ligands, which in turn is dictated by their heme chemistry. However, we have an incomplete understanding of the mechanism of ligand binding for these newly discovered hemoglobins and the measurement of their kinetic parameters depend on their coordination at the heme iron. To gain insights into their functional role, it is important to categorize the new hemoglobins into either penta- or hexa-coordinated varieties. We demonstrate that simple pH titration and absorbance measurements can determine the coordination state of heme iron atom in ferric hemoglobins, thus providing unambiguous information about the classification of new globins. This method is rapid, sensitive and requires low concentration of protein. Penta- and hexa-coordinate hemoglobins displayed distinct pH titration profiles as observed in a variety of hemoglobins. The pentacoordinate distal histidine mutant proteins of hexacoordinate hemoglobins and ligand-bound hexacoordinate forms of pentacoordinate hemoglobins reverse the pH titration profiles, thus validating the sensitivity of this spectroscopic technique.
Angela Wilks - One of the best experts on this subject based on the ideXlab platform.
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characterization of the outer membrane receptor shua from the heme uptake system of shigella dysenteriae substrate specificity and identification of the heme protein ligands
Journal of Biological Chemistry, 2007Co-Authors: Kimberly Burkhard, Angela WilksAbstract:Abstract Shigella dysenteriae, like many bacterial pathogens, has evolved outer membrane receptor-mediated pathways for the uptake and utilization of heme as an iron source. As a first step toward understanding the mechanism of heme uptake we have undertaken a site-directed mutagenesis, spectroscopic, and kinetic analysis of the outer membrane receptor ShuA of S. dysenteriae. Purification of the outer membrane receptor gave a single band of molecular mass 73 kDa on SDS-PAGE. Initial spectroscopic analysis of the protein in either detergent micelles or lipid bicelles revealed residual heme bound to the receptor, with a Soret maximum at 413 nm. Titration of the protein with exogenous heme gave a Soret Peak at 437 nm in detergent micelles, and 402 nm in lipid bicelles. However, transfer of heme from hemoglobin yields a Soret maximum at 413 nm identical to that of the isolated protein. Further spectroscopic and kinetic analysis revealed that hemoglobin in the oxidized state is the most likely physiological substrate for ShuA. In addition, mutation of the conserved histidines, H86A or H420A, resulted in a loss of the ability of the receptor to efficiently extract heme from hemoglobin. In contrast the double mutant H86A/H420A was unable to extract heme from hemoglobin. These findings taken together confirm that both His-86 and His-420 are essential for substrate recognition, heme coordination, and transfer. Furthermore, the full-length TonB was shown to form a 1:1 complex with either apo-ShuA H86A/H420A or the wild-type ShuA. These observations provide a basis for future studies on the coordination and transport of heme by the TonB-dependent outer membrane receptors.
Pedro S. Coelho - One of the best experts on this subject based on the ideXlab platform.
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A serine-substituted P450 catalyzes highly efficient carbene transfer to olefins in vivo
Nature Chemical Biology, 2013Co-Authors: Pedro S. Coelho, Z. Jane Wang, Maraia E. Ener, S.a. Baril, Arvind Kannan, Frances H. Arnold, Eric M. BrustadAbstract:Whole-cell catalysts for non-natural chemical reactions will open new routes to sustainable production of chemicals. We designed a cytochrome 'P411' with unique serine-heme ligation that catalyzes efficient and selective olefin cyclopropanation in intact Escherichia coli cells. The mutation C400S in cytochrome P450_BM3 gives a signature ferrous CO Soret Peak at 411 nm, abolishes monooxygenation activity, raises the resting-state Fe^III-to-Fe^II reduction potential and substantially improves NAD(P)H-driven activity. The exchange of a heme-ligating cysteine for a serine residue in an engineered P450 alters the redox potential of the enzyme, deactivating wild-type function and enabling efficient, NADPH-mediated carbene transfer in cells.
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A Serine-Substituted P450 Catalyzes Highly Efficient Carbene Transfer to Olefins In Vivo
Nature Chemical Biology, 2013Co-Authors: Pedro S. Coelho, Z. Jane Wang, Maraia E. Ener, S.a. Baril, Arvind Kannan, Frances H. Arnold, Eric M. BrustadAbstract:Whole-cell catalysts for non-natural chemical reactions will open new routes to sustainable production of chemicals. We designed a cytochrome 'P411' with unique serine-heme ligation that catalyzes efficient and selective olefin cyclopropanation in intact Escherichia coli cells. The mutation C400S in cytochrome P450_(BM3) gives a signature ferrous CO Soret Peak at 411 nm, abolishes monooxygenation activity, raises the resting-state FeIII-to-FeII reduction potential and substantially improves NAD(P)H-driven activity.