11 results on '"Mary Ortmayer"'
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2. Design and evolution of an enzyme with a non-canonical organocatalytic mechanism.
- Author
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Ashleigh J. Burke, Sarah L. Lovelock, Amina Frese, Rebecca Crawshaw, Mary Ortmayer, Mark Dunstan, Colin Levy, and Anthony P. Green
- Published
- 2019
- Full Text
- View/download PDF
3. Engineering enzyme activity using an expanded amino acid alphabet
- Author
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Zachary Birch-Price, Christopher J Taylor, Mary Ortmayer, and Anthony P Green
- Subjects
Bioengineering ,Molecular Biology ,Biochemistry ,Biotechnology - Abstract
Enzyme design and engineering strategies are typically constrained by the limited size of nature’s genetic alphabet, comprised of only 20 canonical amino acids. In recent years, site-selective incorporation of non-canonical amino acids (ncAAs) via an expanded genetic code has emerged as a powerful means of inserting new functional components into proteins, with hundreds of structurally diverse ncAAs now available. Here, we highlight how the emergence of an expanded repertoire of amino acids has opened new avenues in enzyme design and engineering. ncAAs have been used to probe complex biological mechanisms, augment enzyme function and, most ambitiously, embed new catalytic mechanisms into protein active sites that would be challenging to access within the constraints of nature’s genetic code. We predict that the studies reviewed in this article, along with further advances in genetic code expansion technology, will establish ncAA incorporation as an increasingly important tool for biocatalysis in the coming years.
- Published
- 2022
4. A noncanonical tryptophan analogue reveals an active site hydrogen bond controlling ferryl reactivity in a heme peroxidase
- Author
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Matthew G. Quesne, Florence J. Hardy, Stephen E. J. Rigby, Sam Hay, C. Richard A. Catlow, Sam P. de Visser, Karl Fisher, Colin Levy, Anthony P. Green, Derren J. Heyes, and Mary Ortmayer
- Subjects
Letter ,proton-coupled electron transfer ,Stereochemistry ,tryptophan analogue ,metal-oxo reactivity ,010402 general chemistry ,01 natural sciences ,Redox ,03 medical and health sciences ,chemistry.chemical_compound ,Reactivity (chemistry) ,Expanded genetic code ,Heme ,QD1-999 ,030304 developmental biology ,0303 health sciences ,biology ,Hydrogen bond ,Cytochrome c peroxidase ,cytochrome c peroxidase ,Active site ,hydrogen bonding ,Porphyrin ,0104 chemical sciences ,Chemistry ,genetic code expansion ,chemistry ,biology.protein ,heme enzyme - Abstract
Nature employs high-energy metal-oxo intermediates embedded within enzyme active sites to perform challenging oxidative transformations with remarkable selectivity. Understanding how different local metal-oxo coordination environments control intermediate reactivity and catalytic function is a long-standing objective. However, conducting structure–activity relationships directly in active sites has proven challenging due to the limited range of amino acid substitutions achievable within the constraints of the genetic code. Here, we use an expanded genetic code to examine the impact of hydrogen bonding interactions on ferryl heme structure and reactivity, by replacing the N–H group of the active site Trp51 of cytochrome c peroxidase by an S atom. Removal of a single hydrogen bond stabilizes the porphyrin π-cation radical state of CcP W191F compound I. In contrast, this modification leads to more basic and reactive neutral ferryl heme states, as found in CcP W191F compound II and the wild-type ferryl heme-Trp191 radical pair of compound I. This increased reactivity manifests in a >60-fold activity increase toward phenolic substrates but remarkably has negligible effects on oxidation of the biological redox partner cytc. Our data highlight how Trp51 tunes the lifetimes of key ferryl intermediates and works in synergy with the redox active Trp191 and a well-defined substrate binding site to regulate catalytic function. More broadly, this work shows how noncanonical substitutions can advance our understanding of active site features governing metal-oxo structure and reactivity.
- Published
- 2021
5. Recent Advances in Understanding, Enhancing and Creating Heme Peroxidases
- Author
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Mary Ortmayer, Claire E.M. Noble, J. L. Ross Anderson, Anthony P. Green, and Florence J. Hardy
- Subjects
chemistry.chemical_compound ,biology ,Biochemistry ,Chemistry ,biology.protein ,Heme ,Peroxidase - Published
- 2021
6. Heme Peroxidases
- Author
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Mary Ortmayer and Anthony P. Green
- Published
- 2020
7. Rewiring the ‘Push-Pull’ Catalytic Machinery of a Heme Enzyme using an Expanded Genetic Code
- Author
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Stephen E. J. Rigby, Mary Ortmayer, Emmanuel Wolde-Michael, Emma Lloyd Raven, Sarah L. Lovelock, Sam Hay, Jaswir Basran, Anthony P. Green, Derren J. Heyes, Colin Levy, J. L. Ross Anderson, and Karl Fisher
- Subjects
proton-coupled electron transfer ,metal-oxo reactivity ,Computational biology ,010402 general chemistry ,01 natural sciences ,Catalysis ,chemistry.chemical_compound ,Manchester Institute of Biotechnology ,Heme ,Expanded genetic code ,Push pull ,chemistry.chemical_classification ,noncanonical ligand ,010405 organic chemistry ,Chemistry ,General Chemistry ,ResearchInstitutes_Networks_Beacons/manchester_institute_of_biotechnology ,0104 chemical sciences ,Enzyme ,genetic code expansion ,Functional significance ,heme enzyme ,Proton-coupled electron transfer ,human activities ,Research Article - Abstract
Nature employs a limited number of genetically encoded axial ligands to control diverse heme enzyme activities. Deciphering the functional significance of these ligands requires a quantitative understanding of how their electron-donating capabilities modulate the structures and reactivities of the iconic ferryl intermediates compounds I and II. However, probing these relationships experimentally has proven to be challenging as ligand substitutions accessible via conventional mutagenesis do not allow fine tuning of electron donation and typically abolish catalytic function. Here, we exploit engineered translation components to replace the histidine ligand of cytochrome c peroxidase (CcP) by a less electron-donating Nδ-methyl histidine (Me-His) with little effect on the enzyme structure. The rate of formation (k1) and the reactivity (k2) of compound I are unaffected by ligand substitution. In contrast, proton-coupled electron transfer to compound II (k3) is 10-fold slower in CcP Me-His, providing a direct link between electron donation and compound II reactivity, which can be explained by weaker electron donation from the Me-His ligand ("the push") affording an electron-deficient ferryl oxygen with reduced proton affinity ("the pull"). The deleterious effects of the Me-His ligand can be fully compensated by introducing a W51F mutation designed to increase "the pull" by removing a hydrogen bond to the ferryl oxygen. Analogous substitutions in ascorbate peroxidase lead to similar activity trends to those observed in CcP, suggesting that a common mechanistic strategy is employed by enzymes using distinct electron transfer pathways. Our study highlights how noncanonical active site substitutions can be used to directly probe and deconstruct highly evolved bioinorganic mechanisms.
- Published
- 2020
8. Design and evolution of an enzyme with a non-canonical organocatalytic mechanism
- Author
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Anthony P. Green, Mary Ortmayer, Rebecca Crawshaw, Mark S. Dunstan, Sarah L. Lovelock, Colin Levy, Ashleigh J. Burke, and Amina Frese
- Subjects
0301 basic medicine ,Models, Molecular ,Hydrolases ,010402 general chemistry ,Crystallography, X-Ray ,Protein Engineering ,01 natural sciences ,Catalysis ,Substrate Specificity ,03 medical and health sciences ,Nucleophile ,Catalytic Domain ,Hydrolase ,4-Aminopyridine ,Expanded genetic code ,Multidisciplinary ,Chemistry ,Hydrolysis ,Esters ,Protein engineering ,Directed evolution ,Methylhistidines ,Combinatorial chemistry ,0104 chemical sciences ,030104 developmental biology ,Biocatalysis ,Genetic Code ,Mutagenesis ,Mutation ,Directed Molecular Evolution ,Pyrococcus horikoshii - Abstract
The combination of computational design and laboratory evolution is a powerful and potentially versatile strategy for the development of enzymes with new functions1-4. However, the limited functionality presented by the genetic code restricts the range of catalytic mechanisms that are accessible in designed active sites. Inspired by mechanistic strategies from small-molecule organocatalysis5, here we report the generation of a hydrolytic enzyme that uses Nδ-methylhistidine as a non-canonical catalytic nucleophile. Histidine methylation is essential for catalytic function because it prevents the formation of unreactive acyl-enzyme intermediates, which has been a long-standing challenge when using canonical nucleophiles in enzyme design6-10. Enzyme performance was optimized using directed evolution protocols adapted to an expanded genetic code, affording a biocatalyst capable of accelerating ester hydrolysis with greater than 9,000-fold increased efficiency over free Nδ-methylhistidine in solution. Crystallographic snapshots along the evolutionary trajectory highlight the catalytic devices that are responsible for this increase in efficiency. Nδ-methylhistidine can be considered to be a genetically encodable surrogate of the widely employed nucleophilic catalyst dimethylaminopyridine11, and its use will create opportunities to design and engineer enzymes for a wealth of valuable chemical transformations.
- Published
- 2019
- Full Text
- View/download PDF
9. Heme-Dependent Oxidative N-Demethylase
- Author
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Mary Ortmayer and David Leys
- Subjects
chemistry.chemical_classification ,Amine oxidase ,chemistry.chemical_compound ,Biochemistry ,biology ,Chemistry ,Oxidoreductase ,biology.protein ,Demethylase ,Oxidative phosphorylation ,Heme - Published
- 2018
10. An oxidative N-demethylase reveals PAS transition from ubiquitous sensor to enzyme
- Author
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Stephen E. J. Rigby, Mary Ortmayer, Andrew W. Munro, Lukas Denkhaus, Nigel S. Scrutton, Tewes Tralau, David Leys, Pierre Lafite, Binuraj R. K. Menon, Sam Hay, Karl Fisher, Manchester Institute of Biotechnology, University of Manchester [Manchester], Institut de Chimie Organique et Analytique (ICOA), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université d'Orléans (UO)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Université d'Orléans (UO)-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut de Chimie du CNRS (INC)-Institut National de la Santé et de la Recherche Médicale (INSERM), and LAFITE, Pierre
- Subjects
0301 basic medicine ,Iron-Sulfur Proteins ,Models, Molecular ,Stereochemistry ,Flavin Mononucleotide ,Protein domain ,Coenzymes ,Flavin mononucleotide ,Pseudomonas mendocina ,Flavin group ,Heme ,Crystallography, X-Ray ,Cofactor ,03 medical and health sciences ,chemistry.chemical_compound ,Protein Domains ,Oxidoreductase ,PAS domain ,[SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology ,[SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology ,Binding site ,Dimethylamine ,Tetrahydrofolates ,chemistry.chemical_classification ,Multidisciplinary ,Binding Sites ,030102 biochemistry & molecular biology ,biology ,Oxidoreductases, N-Demethylating ,Oxygen ,Protein Subunits ,030104 developmental biology ,chemistry ,biology.protein ,Oxidation-Reduction ,Dimethylamines ,NADP - Abstract
International audience; The universal Per-ARNT-Sim (PAS) domain functions as a signal transduction module involved in sensing diverse stimuli such as small molecules, light, redox state and gases. The highly evolvable PAS scaffold can bind a broad range of ligands, including haem, flavins and metal ions. However, although these ligands can support catalytic activity, to our knowledge no enzymatic PAS domain has been found. Here we report characterization of the first PAS enzyme: a haem-dependent oxidative N-demethylase. Unrelated to other amine oxidases, this enzyme contains haem, flavin mononucleotide, 2Fe-2S and tetrahydrofolic acid cofactors, and specifically catalyses the NADPH-dependent oxidation of dimethylamine. The structure of the α subunit reveals that it is a haem-binding PAS domain, similar in structure to PAS gas sensors. The dimethylamine substrate forms part of a highly polarized oxygen-binding site, and directly assists oxygen activation by acting as both an electron and proton donor. Our data reveal that the ubiquitous PAS domain can make the transition from sensor to enzyme, suggesting that the PAS scaffold can support the development of artificial enzymes.
- Published
- 2016
11. A Noncanonical Tryptophan Analogue Reveals an Active Site Hydrogen Bond Controlling Ferryl Reactivity in a Heme Peroxidase
- Author
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'Mary Ortmayer
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