scholarly journals Mapping Potential Determinants of Peroxidative Activity in an Evolved Fungal Peroxygenase from Agrocybe aegerita

2021 ◽  
Vol 9 ◽  
Author(s):  
Patricia Molina-Espeja ◽  
Alejandro Beltran-Nogal ◽  
Maria Alejandra Alfuzzi ◽  
Victor Guallar ◽  
Miguel Alcalde
Keyword(s):  
Computational Analysis ◽  
Saturation Mutagenesis ◽  
Oxidation Reactions ◽  
Protein Surface ◽  
Activity Profile ◽  
Agrocybe Aegerita ◽  
Access Channel ◽  
Heme Domain ◽  
Striking Change ◽  
Point Of Departure

Fungal unspecific peroxygenases (UPOs) are hybrid biocatalysts with peroxygenative activity that insert oxygen into non-activated compounds, while also possessing convergent peroxidative activity for one electron oxidation reactions. In several ligninolytic peroxidases, the site of peroxidative activity is associated with an oxidizable aromatic residue at the protein surface that connects to the buried heme domain through a long-range electron transfer (LRET) pathway. However, the peroxidative activity of these enzymes may also be initiated at the heme access channel. In this study, we examined the origin of the peroxidative activity of UPOs using an evolved secretion variant (PaDa-I mutant) from Agrocybe aegerita as our point of departure. After analyzing potential radical-forming aromatic residues at the PaDa-I surface by QM/MM, independent saturation mutagenesis libraries of Trp24, Tyr47, Tyr79, Tyr151, Tyr265, Tyr281, Tyr293 and Tyr325 were constructed and screened with both peroxidative and peroxygenative substrates. These mutant libraries were mostly inactive, with only a few functional clones detected, none of these showing marked differences in the peroxygenative and peroxidative activities. By contrast, when the flexible Gly314-Gly318 loop that is found at the outer entrance to the heme channel was subjected to combinatorial saturation mutagenesis and computational analysis, mutants with improved kinetics and a shift in the pH activity profile for peroxidative substrates were found, while they retained their kinetic values for peroxygenative substrates. This striking change was accompanied by a 4.5°C enhancement in kinetic thermostability despite the variants carried up to four consecutive mutations. Taken together, our study proves that the origin of the peroxidative activity in UPOs, unlike other ligninolytic peroxidases described to date, is not dependent on a LRET route from oxidizable residues at the protein surface, but rather it seems to be exclusively located at the heme access channel.

scholarly journals Directed Evolution of Unspecific Peroxygenase from Agrocybe aegerita

2014 ◽  
Vol 80 (11) ◽  
pp. 3496-3507 ◽  
Author(s):  
Patricia Molina-Espeja ◽  
Eva Garcia-Ruiz ◽  
David Gonzalez-Perez ◽  
René Ullrich ◽  
Martin Hofrichter ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Signal Peptide ◽  
Oxygen Transfer ◽  
Colorimetric Assay ◽  
Total Activity ◽  
Functional Expression ◽  
Hydrophobic Core ◽  
Content Type ◽  
Agrocybe Aegerita ◽  
Access Channel

ABSTRACTUnspecific peroxygenase (UPO) represents a new type of heme-thiolate enzyme with self-sufficient mono(per)oxygenase activity and many potential applications in organic synthesis. With a view to taking advantage of these properties, we subjected theAgrocybe aegeritaUPO1-encoding gene to directed evolution inSaccharomyces cerevisiae. To promote functional expression, several different signal peptides were fused to the mature protein, and the resulting products were tested. Over 9,000 clones were screened using anad hocdual-colorimetric assay that assessed both peroxidative and oxygen transfer activities. After 5 generations of directed evolution combined with hybrid approaches, 9 mutations were introduced that resulted in a 3,250-fold total activity improvement with no alteration in protein stability. A breakdown between secretion and catalytic activity was performed by replacing the native signal peptide of the original parental type with that of the evolved mutant; the evolved leader increased functional expression 27-fold, whereas an 18-fold improvement in thekcat/Kmvalue for oxygen transfer activity was obtained. The evolved UPO1 was active and highly stable in the presence of organic cosolvents. Mutations in the hydrophobic core of the signal peptide contributed to enhance functional expression up to 8 mg/liter, while catalytic efficiencies for peroxidative and oxygen transfer reactions were increased by several mutations in the vicinity of the heme access channel. Overall, the directed-evolution platform described is a valuable point of departure for the development of customized UPOs with improved features and for the study of structure-function relationships.

scholarly journals Structural insights of mediated electron transfer by P450 BM3 monooxygenase

2014 ◽  
Vol 70 (a1) ◽  
pp. C1169-C1169
Author(s):  
Saravanan Panneerselvam ◽  
Aamir Shehzad ◽  
Jochen Mueller-Dieckmann ◽  
Ulrich Schwaneberg
Keyword(s):  
Electron Transfer ◽  
Active Site ◽  
Water Soluble ◽  
Electron Sources ◽  
P450 Bm3 ◽  
Redox Partner ◽  
Access Channel ◽  
Mediated Electron Transfer ◽  
Heme Domain ◽  
Drug Synthesis

P450 BM3 is a 119-kDa water-soluble heme monooxygenase originating from Bacillus megaterium. P450 BM3 and variants are known to oxidize structurally diverse substrates. However, the requirement for the natural cofactor, NADPH, limits cell-free applications of P450 BM3 in drug synthesis, fuelling efforts to establish alternative cofactor system. Hence, P450 BM3 variants have been generated which circumvent the requirement for NADPH, and enabled P450 BM3 to be driven with alternative electron sources. In this study, crystal structures of the P450 BM3 M7 heme domain variant (F87A, V281G, M354S) with and without cobalt (III) sepulchrate are reported. Cobalt (III) sepulchrate acts as an electron shuttle in an alternative cofactor system employing zinc dust as the electron source. The crystal structure shows a binding site for the mediator cobalt (III) sepulchrate at the entrance of the substrate access channel. The mediator occupies a position which is far from the active site and distinct from the binding of the natural redox partner (FAD/NADPH binding domain). The unusual binding position suggests that the mediator shuttles electrons to the heme-centre through new routes. Electron transfer could occur by a `through-protein' or a `substrate-relayed' pathway. The latter seems more plausible since it would ensure efficient use of electrons only in the presence of a substrate in the active site. The structural evidence also indicates that the use of a positively charged mediator is important to effectively reduce the catalytic heme domain. Understanding the mediator-monooxygenase interface opens new avenues for tailoring P450 BM3 to match application demands. Structural and molecular understanding of mediated electron transfer enables a paradigm shift from a mediator acceptance screening to a rational mediator design which considers only stability and electron transfer performance parameters.

The Contribution of Pavlov's Typology of CNS Properties to Personality Research

1997 ◽  
Vol 2 (2) ◽  
pp. 125-138 ◽  
Author(s):  
Jan Strelau
Keyword(s):  
Soviet Union ◽  
Former Soviet Union ◽  
Animal Species ◽  
Special Significance ◽  
The West ◽  
Personality Dimensions ◽  
Personality Research ◽  
Personality Theories ◽  
Strength Of Excitation ◽  
Point Of Departure

This paper presents Pavlov's contribution to the development of biological-oriented personality theories. Taking a short description of Pavlov's typology of central nervous system (CNS) properties as a point of departure, it shows how, and to what extent, this typology influenced further research in the former Soviet Union as well as in the West. Of special significance for the development of biologically oriented personality dimensions was the conditioned reflex paradigm introduced by Pavlov for studying individual differences in dogs. This paradigm was used by Russian psychologists in research on types of nervous systems conducted in different animal species as well as for assessing temperament in children and adults. Also, personality psychologists in the West, such as Eysenck, Spence, and Gray, incorporated the CR paradigm into their theories. Among the basic properties of excitation and inhibition on which Pavlov's typology was based, strength of excitation and the basic indicator of this property, protective inhibition, gained the highest popularity in arousaloriented personality theories. Many studies have been conducted in which the Pavlovian constructs of CNS properties have been related to different personality dimensions. In current research the behavioral expressions of the Pavlovian constructs of strength of excitation, strength of inhibition, and mobility of nervous processes as measured by the Pavlovian Temperament Survey (PTS) have been related to over a dozen of personality dimensions, mostly referring to temperament.

Performance-based validation of the human activity profile

2007 ◽  
Author(s):  
Luci Fuscaldi Teixeira-Salmela ◽  
Sandra J. Olney ◽  
Revathy Devaraj
Keyword(s):  
Human Activity ◽  
Activity Profile

Migrant Diplomacies: Rethinking Diplomacy Beyond State-Centric Perspectives. A Civic Bi-Nationality Experience from North America

2020 ◽  
Vol 17 (1) ◽  
pp. 37-46 ◽  
Author(s):  
Antonio Alejo
Keyword(s):  
North America ◽  
Mexico City ◽  
Theoretical Framework ◽  
Global Politics ◽  
National Interests ◽  
Interdisciplinary Dialogue ◽  
Contemporary Politics ◽  
Migrant Activism ◽  
Point Of Departure

There is a pressing need to extend our thinking about diplomacy beyond state-centric perspectives, as in the name of sovereignty and national interests, people on move are confronting virtual, symbolic and/or material walls and frames of policies inhibiting their free movement. My point of departure is to explore migrant activism and global politics through the transformation of diplomacy in a globalised world. Developing an interdisciplinary dialogue between new diplomacy and sociology, I evidence the emergence of global sociopolitical formations created through civic bi-nationality organisations. Focusing on the agent in interaction with structures, I present a theoretical framework and strategy for analysing the practices of migrant diplomacies as an expression of contemporary politics. A case study from North America regarding returned families in Mexico City provides evidence of how these alternative diplomacies are operating.

A Comparative Analysis of the Poetics of Anger in John Osborne’s Look Back in Anger and Femi Osofisan’s The Chattering and the Song

2019 ◽  
Keyword(s):  
Comparative Analysis ◽  
Human Emotion ◽  
Fundamental Component ◽  
Look Back ◽  
Point Of Departure ◽  
Desire To Change

Anger is a basic human emotion that has a force for constructive or destructive ends. Its expression in any circumstance can be a trigger for a desire to change a prevailing situation. In all cases, anger is a fundamental component of art. This study examines the use of anger in Osborne’s Look Back in Anger and Osofisan’s The Chattering and the Song. Osborne and Osofisan are two writers who are very anxious to change their societies through their art. In spite of differences in their origin (Osborne was a Briton while Osofisan is a Nigerian), they wrote at a time of certain social and political upheavals in their countries. They also share similar concerns and attitudes towards art. My focus in this paper is on the early plays of Osborne and Osofisan where anger is strongest and where their artistic triumph is most poignant. Working within the formalist approach, the paper reveals that in Osborne and Osofisan, extreme anger is both material and style and is what marks their art out. The reification by the intellect provides a potent instrument for investigating society. Anger becomes the point of departure for their art, it is not mere hysterics but a cerebral one and it is the motivating force for their writings.

Hopscotch Modernism: On Everyday Life and the Blurring of Art and Social Science

2006 ◽  
Vol 2 (1) ◽  
pp. 70-79 ◽  
Author(s):  
Ben Highmore
Keyword(s):  
Twentieth Century ◽  
Social Science ◽  
Everyday Life ◽  
Systematic Study ◽  
Cultural Practices ◽  
Health Centre ◽  
Small Scale ◽  
Modernist Literature ◽  
The Everyday ◽  
Point Of Departure

From a remarkably innovative point of departure, Ben Highmore (University of Sussex) suggests that modernist literature and art were not the only cultural practices concerned with reclaiming the everyday and imbuing it with significance. At the same time, Roger Caillois was studying the spontaneous interactions involved in games such as hopscotch, while other small scale institutions such as the Pioneer Health Centre in Peckham, London attempted to reconcile systematic study and knowledge with the non-systematic exchanges in games and play. Highmore suggests that such experiments comprise a less-often recognised ‘modernist heritage’, and argues powerfully for their importance within early-twentieth century anthropology and the newly-emerged field of cultural studies.

The Transfer of Complaint: A Narcissistic Time-Share

Paragraph ◽  
2017 ◽  
Vol 40 (3) ◽  
pp. 279-293
Author(s):  
Avital Ronell
Keyword(s):  
French Studies ◽  
Time Share ◽  
Point Of Departure ◽  
Insight Into

Reflecting on the debts collected by Shoshana Felman's work, within the theoretical contexts of the time in which the 1977 Yale French Studies issue of ‘Psychoanalysis and Literature’ first appeared, this article takes as its point of departure Lacan's analysis of Hamlet's father as the barred Other, focusing on Hamlet's ‘complaint’. The nature of the complaint (plainte, or Klage, also closely allied to Anklage, or accusation) is then explored in relation to various writers and thinkers — Rilke, Benjamin, Nietzsche, Heidegger, among others — and more specifically via a reading of François Roustang's La Fin de la plainte (The End of the Complaint), and his own interpretations of Freudian narcissism. Scanning the wreckage for which the little narcissists are responsible, the article aims to give more insight into the structuring principles of those who whine incessantly.

Notes on Loving a Mourner (with Roland Barthes and Others)

Paragraph ◽  
2017 ◽  
Vol 40 (2) ◽  
pp. 211-227
Author(s):  
Matt Phillips
Keyword(s):  
Sigmund Freud ◽  
Ethics Of Care ◽  
Roland Barthes ◽  
Melanie Klein ◽  
Personal Observation ◽  
Late Work ◽  
Point Of Departure

This essay examines the place of love in grief, staging a relation between a mourner and her lover. Taking as its point of departure Freud's observation that mourning leads to a ‘loss of the capacity to love’, it considers the effects bereavement might have on the bereaved's relations with those that love them, and the possibilities, pitfalls and ethics of care in such a context. This is explored largely through a reading of Roland Barthes's late work (both as a writer of grief and a theorist of love), as well as ideas drawn from Sigmund Freud, Melanie Klein, Sara Ahmed, Hamlet and personal observation. Love and care are thought through alongside notions of ‘tact’, ‘benevolence’ and ‘parrying against reduction’ in late Barthes.

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