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Nigel S. Scrutton - One of the best experts on this subject based on the ideXlab platform.

  • ultrafast vibrational energy transfer between protein and cofactor in a Flavoenzyme
    Journal of Physical Chemistry B, 2020
    Co-Authors: Samantha J O Hardman, Nigel S. Scrutton, Andreea Iulia Iorgu, Derren J Heyes, Igor V Sazanovich, Sam Hay
    Abstract:

    Protein motions and enzyme catalysis are often linked. It is hypothesized that ultrafast vibrations (femtosecond-picosecond) enhance the rate of hydride transfer catalyzed by members of the old yellow enzyme (OYE) family of ene-reductases. Here, we use time-resolved infrared (TRIR) spectroscopy in combination with stable "heavy" isotopic labeling (2H, 13C, 15N) of protein and/or cofactor to probe the vibrational energy transfer (VET) between pentaerythritol tetranitrate reductase (a member of the OYE family) and its noncovalently bound flavin mononucleotide (FMN) cofactor. We show that when the FMN cofactor is photoexcited with visible light, vibrational energy is transferred from the flavin to the surrounding protein environment on the picosecond timescale. This finding expands the scope of VET investigation in proteins, which are limited by suitable intrinsic probes, and may have implications in the understanding of the mechanism of recently discovered photoactive Flavoenzymes.

  • 1h 15n and 13c backbone resonance assignments of pentaerythritol tetranitrate reductase from enterobacter cloacae pb2
    Biomolecular Nmr Assignments, 2018
    Co-Authors: Andreea Iulia Iorgu, Sam Hay, Nicola J Baxter, Matthew J Cliff, Jonathan P Waltho, Nigel S. Scrutton
    Abstract:

    Pentaerythritol tetranitrate reductase (PETNR) is a Flavoenzyme possessing a broad substrate specificity and is a member of the Old Yellow Enzyme family of oxidoreductases. As well as having high potential as an industrial biocatalyst, PETNR is an excellent model system for studying hydrogen transfer reactions. Mechanistic studies performed with PETNR using stopped-flow methods have shown that tunneling contributes towards hydride transfer from the NAD(P)H coenzyme to the flavin mononucleotide (FMN) cofactor and fast protein dynamics have been inferred to facilitate this catalytic step. Herein, we report the near-complete 1H, 15N and 13C backbone resonance assignments of PETNR in a stoichiometric complex with the FMN cofactor in its native oxidized form, which were obtained using heteronuclear multidimensional NMR spectroscopy. A total of 97% of all backbone resonances were assigned, with 333 out of a possible 344 residues assigned in the 1H–15N TROSY spectrum. This is the first report of an NMR structural study of a Flavoenzyme from the Old Yellow Enzyme family and it lays the foundation for future investigations of functional dynamics in hydride transfer catalytic mechanism.

  • proton coupled electron transfer and adduct configuration are important for c4a hydroperoxyflavin formation and stabilization in a Flavoenzyme
    Journal of the American Chemical Society, 2014
    Co-Authors: Thanyaporn Wongnate, Nigel S. Scrutton, Panida Surawatanawong, Surawit Visitsatthawong, Jeerus Sucharitakul, Pimchai Chaiyen
    Abstract:

    Determination of the mechanism of dioxygen activation by Flavoenzymes remains one of the most challenging problems in flavoenzymology for which the underlying theoretical basis is not well understood. Here, the reaction of reduced flavin and dioxygen catalyzed by pyranose 2-oxidase (P2O), a Flavoenzyme oxidase that is unique in its formation of C4a-hydroperoxyflavin, was investigated by density functional calculations, transient kinetics, and site-directed mutagenesis. Based on work from the 1970s–1980s, the current understanding of the dioxygen activation process in Flavoenzymes is believed to involve electron transfer from flavin to dioxygen and subsequent proton transfer to form C4a-hydroperoxyflavin. Our findings suggest that the first step of the P2O reaction is a single electron transfer coupled with a proton transfer from the conserved residue, His548. In fact, proton transfer enhances the electron acceptor ability of dioxygen. The resulting ·OOH of the open-shell diradical pair is placed in an opt...

  • covalent attachment of flavin adenine dinucleotide fad and flavin mononucleotide fmn to enzymes the current state of affairs
    Protein Science, 1998
    Co-Authors: Martin Mewies, William S Mcintire, Nigel S. Scrutton
    Abstract:

    The first identified covalent flavoprotein, a component of mammalian succinate dehydrogenase, was reported 42 years ago. Since that time, more than 20 covalent Flavoenzymes have been described, each possessing one of five modes of FAD or FMN linkage to protein. Despite the early identification of covalent flavoproteins, the mechanisms of covalent bond formation and the roles of the covalent links are only recently being appreciated. The main focus of this review is, therefore, one of mechanism and function, in addition to surveying the types of linkage observed and the methods employed for their identification. Case studies are presented for a variety of covalent Flavoenzymes, from which general findings are beginning to emerge.

Shuguang Zhang - One of the best experts on this subject based on the ideXlab platform.

  • peptergents peptide detergents that improve stability and functionality of a membrane protein glycerol 3 phosphate dehydrogenase
    Biochemistry, 2005
    Co-Authors: Joanne I Yeh, Antoni Tortajada, Joao Paulo, Shuguang Zhang
    Abstract:

    Toward enhancing in vitro membrane protein studies, we have utilized small self-assembling peptides with detergent properties ("peptergents") to extract and stabilize the integral membrane Flavoenzyme, glycerol-3-phosphate dehydrogenase (GlpD), and the soluble redox Flavoenzyme, NADH peroxidase (Npx). GlpD is a six transmembrane spanning redox enzyme that catalyzes the oxidation of glycerol-3-phosphate to dihydroxyacetone phosphate. Although detergents such as n-octyl-beta-D-glucpyranoside can efficiently solubilize the enzyme, GlpD is inactivated within days once reconstituted into detergent micelles. In contrast, peptergents can efficiently extract and solubilize GlpD from native Escherichia coli membrane and maintain its enzymatic activity up to 10 times longer than in traditional detergents. Intriguingly, peptergents also extended the activity of a soluble Flavoenzyme, Npx, when used as an additive. Npx is a Flavoenzyme that catalyzes the two-electron reduction of hydrogen peroxide to water using a cysteine-sulfenic acid as a secondary redox center. The lability of the peroxidase results from oxidation of the sulfenic acid to the sulfinic or sulfonic acid forms. Oxidation of the sulfenic acid, the secondary redox center, results in inactivation, and this reaction proceeds in vitro even in the presence of reducing agents. Although the exact mechanism by which peptergents influence solution stability of Npx remains to be determined, the positive effects may be due to antioxidant properties of the peptides. Peptide-based detergents can be beneficial for many applications and may be particularly useful for structural and functional studies of membrane proteins due to their propensity to enhance the formation of ordered supramolecular assemblies.

  • peptergents peptide detergents that improve stability and functionality of a membrane protein glycerol 3 phosphate dehydrogenase
    Biochemistry, 2005
    Co-Authors: Shoucheng Du, Antoni Tortajada, Joao Paulo, Shuguang Zhang
    Abstract:

    Toward enhancing in vitro membrane protein studies, we have utilized small self-assembling peptides with detergent properties ("peptergents") to extract and stabilize the integral membrane Flavoenzyme, glycerol-3-phosphate dehydrogenase (GlpD), and the soluble redox Flavoenzyme, NADH peroxidase (Npx). GlpD is a six transmembrane spanning redox enzyme that catalyzes the oxidation of glycerol-3-phosphate to dihydroxyacetone phosphate. Although detergents such as n-octyl-‚-D-glucpyra- noside can efficiently solubilize the enzyme, GlpD is inactivated within days once reconstituted into detergent micelles. In contrast, peptergents can efficiently extract and solubilize GlpD from native Escherichia coli membrane and maintain its enzymatic activity up to 10 times longer than in traditional detergents. Intriguingly, peptergents also extended the activity of a soluble Flavoenzyme, Npx, when used as an additive. Npx is a Flavoenzyme that catalyzes the two-electron reduction of hydrogen peroxide to water using a cysteine-sulfenic acid as a secondary redox center. The lability of the peroxidase results from oxidation of the sulfenic acid to the sulfinic or sulfonic acid forms. Oxidation of the sulfenic acid, the secondary redox center, results in inactivation, and this reaction proceeds in vitro even in the presence of reducing agents. Although the exact mechanism by which peptergents influence solution stability of Npx remains to be determined, the positive effects may be due to antioxidant properties of the peptides. Peptide-based detergents can be beneficial for many applications and may be particularly useful for structural and functional studies of membrane proteins due to their propensity to enhance the formation of ordered supramolecular assemblies.

Dale C Poulter - One of the best experts on this subject based on the ideXlab platform.

  • site selective synthesis of 15n and 13c enriched flavin mononucleotide coenzyme isotopologues
    Journal of Organic Chemistry, 2016
    Co-Authors: Syam Sundar Neti, Dale C Poulter
    Abstract:

    Flavin mononucleotide (FMN) is a coenzyme for numerous proteins involved in key cellular and physiological processes. Isotopically labeled flavin is a powerful tool for studying the structure and mechanism of Flavoenzyme-catalyzed reactions by a variety of techniques, including NMR, IR, Raman, and mass spectrometry. In this report, we describe the preparation of labeled FMN isotopologues enriched with 15N and 13C isotopes at various sites in the pyrazine and pyrimidine rings of the isoalloxazine core of the cofactor from readily available precursors by a five-step chemo-enzymatic synthesis.

  • Site-Selective Synthesis of 15N- and 13C‑Enriched Flavin Mononucleotide Coenzyme Isotopologues
    2016
    Co-Authors: Syam Sundar Neti, Dale C Poulter
    Abstract:

    Flavin mononucleotide (FMN) is a coenzyme for numerous proteins involved in key cellular and physiological processes. Isotopically labeled flavin is a powerful tool for studying the structure and mechanism of Flavoenzyme-catalyzed reactions by a variety of techniques, including NMR, IR, Raman, and mass spectrometry. In this report, we describe the preparation of labeled FMN isotopologues enriched with 15N and 13C isotopes at various sites in the pyrazine and pyrimidine rings of the isoalloxazine core of the cofactor from readily available precursors by a five-step chemo-enzymatic synthesis

Tadhg P. Begley - One of the best experts on this subject based on the ideXlab platform.

Syam Sundar Neti - One of the best experts on this subject based on the ideXlab platform.

  • site selective synthesis of 15n and 13c enriched flavin mononucleotide coenzyme isotopologues
    Journal of Organic Chemistry, 2016
    Co-Authors: Syam Sundar Neti, Dale C Poulter
    Abstract:

    Flavin mononucleotide (FMN) is a coenzyme for numerous proteins involved in key cellular and physiological processes. Isotopically labeled flavin is a powerful tool for studying the structure and mechanism of Flavoenzyme-catalyzed reactions by a variety of techniques, including NMR, IR, Raman, and mass spectrometry. In this report, we describe the preparation of labeled FMN isotopologues enriched with 15N and 13C isotopes at various sites in the pyrazine and pyrimidine rings of the isoalloxazine core of the cofactor from readily available precursors by a five-step chemo-enzymatic synthesis.

  • Site-Selective Synthesis of 15N- and 13C‑Enriched Flavin Mononucleotide Coenzyme Isotopologues
    2016
    Co-Authors: Syam Sundar Neti, Dale C Poulter
    Abstract:

    Flavin mononucleotide (FMN) is a coenzyme for numerous proteins involved in key cellular and physiological processes. Isotopically labeled flavin is a powerful tool for studying the structure and mechanism of Flavoenzyme-catalyzed reactions by a variety of techniques, including NMR, IR, Raman, and mass spectrometry. In this report, we describe the preparation of labeled FMN isotopologues enriched with 15N and 13C isotopes at various sites in the pyrazine and pyrimidine rings of the isoalloxazine core of the cofactor from readily available precursors by a five-step chemo-enzymatic synthesis