A variant of high biotechnological interest (called 2-1B) was obtained by directed evolution of the Pleurotus eryngii VP (versatile peroxidase) expressed in Saccharomyces cerevisiae [García-Ruiz, González-Pérez, Ruiz-Dueñas, Martínez and Alcalde (2012) Biochem. J. 441, 487–498]. 2-1B shows seven mutations in the mature protein that resulted in improved functional expression, activity and thermostability, along with a remarkable stronger alkaline stability (it retains 60% of the initial activity after 120 h of incubation at pH 9 compared with complete inactivation of the native enzyme after only 1 h). The latter is highly demanded for biorefinery applications. In the present study we investigate the structural basis behind the enhanced alkaline stabilization of this evolved enzyme. In order to do this, several VP variants containing one or several of the mutations present in 2-1B were expressed in Escherichia coli, and their alkaline stability and biochemical properties were determined. In addition, the crystal structures of 2-1B and one of the intermediate variants were solved and carefully analysed, and molecular dynamics simulations were carried out. We concluded that the introduction of three basic residues in VP (Lys-37, Arg-39 and Arg-330) led to new connections between haem and helix B (where the distal histidine residue is located), and formation of new electrostatic interactions, that avoided the hexa-co-ordination of the haem iron. These new structural determinants stabilized the haem and its environment, helping to maintain the structural enzyme integrity (with penta-co-ordinated haem iron) under alkaline conditions. Moreover, the reinforcement of the solvent-exposed area around Gln-305 in the proximal side, prompted by the Q202L mutation, further enhanced the stability.
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The reactions catalysed by enzymes associated with the TCA cycle (Krebs cycle) are commonly thought to operate in a full series of eight steps. However, under appropriate conditions in skeletal muscle and cells of immune system, the ‘cycle’ may be considered to operate in two separate parts. Firstly, in muscle, reactions of the right hand side of the cycle terminate at 2-oxoglutarate dehydrogenase, and the flux of carbon is shifted to the synthesis of glutamate and glutamine (for release into the bloodstream) via transaminases and glutamine synthetase respectively. Secondly, in immune cells such as lymphocytes, neutrophils and macrophages, glutamine may be converted to 2-oxoglutarate where it is metabolised by reactions of the left hand side of the TCA cycle where carbon may leave the cycle as malate or oxaloacetate, being subsequently converted to pyruvate (and lactate), or aspartate respectively. Image adapted from: Newsholme, E.A., Newsholme, P. and Curi, R. (1987) The role of the citric acid cycle in cells of the immune system and its importance in sepsis, trauma and burns. Biochem. Soc. Symp. 54, 145–162. For further details please see pp. 1845–1857. Image kindly provided by Philip Newsholme. - PDF Icon PDF LinkFront Matter
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Research Article|
June 28 2016
Unveiling the basis of alkaline stability of an evolved versatile peroxidase
Verónica Sáez-Jiménez;
Verónica Sáez-Jiménez
*Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain
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Sandra Acebes;
Sandra Acebes
†Joint BSC-CRG-IRB Research Program in Computational Biology, Barcelona Supercomputing Center, Jordi Girona 29, E-08034 Barcelona, Spain
‡Anaxomics Biotech, Balmes 89, E-08008 Barcelona, Spain
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Eva Garcia-Ruiz;
Eva Garcia-Ruiz
§Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL 61801, U.S.A.
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Antonio Romero;
Antonio Romero
*Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain
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Victor Guallar;
Victor Guallar
†Joint BSC-CRG-IRB Research Program in Computational Biology, Barcelona Supercomputing Center, Jordi Girona 29, E-08034 Barcelona, Spain
║ICREA, Passeig Lluis Companys 23, E-08010 Barcelona, Spain
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Miguel Alcalde;
Miguel Alcalde
¶Institute of Catalysis and Petroleochemistry, CSIC, Marie Curie 2, Cantoblanco, E-28049 Madrid, Spain
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Francisco J. Medrano;
Francisco J. Medrano
*Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain
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Angel T. Martínez;
Angel T. Martínez
*Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain
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Francisco J. Ruiz-Dueñas
Francisco J. Ruiz-Dueñas
1
*Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain
1To whom correspondence should be addressed (email fjruiz@cib.csic.es).
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Publisher: Portland Press Ltd
Received:
January 04 2016
Revision Received:
April 22 2016
Accepted:
April 26 2016
Accepted Manuscript online:
April 26 2016
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society
2016
Biochem J (2016) 473 (13): 1917–1928.
Article history
Received:
January 04 2016
Revision Received:
April 22 2016
Accepted:
April 26 2016
Accepted Manuscript online:
April 26 2016
Citation
Verónica Sáez-Jiménez, Sandra Acebes, Eva Garcia-Ruiz, Antonio Romero, Victor Guallar, Miguel Alcalde, Francisco J. Medrano, Angel T. Martínez, Francisco J. Ruiz-Dueñas; Unveiling the basis of alkaline stability of an evolved versatile peroxidase. Biochem J 1 July 2016; 473 (13): 1917–1928. doi: https://doi.org/10.1042/BCJ20160248
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