Unveiling the basis of alkaline stability of an evolved versatile peroxidase.
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.
Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain.
Verónica
V
Sáez-Jiménez
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.
Sandra
S
Acebes
Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL 61801, U.S.A.
Eva
E
Garcia-Ruiz
Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain.
Antonio
A
Romero
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.
Victor
V
Guallar
Institute of Catalysis and Petroleochemistry, CSIC, Marie Curie 2, Cantoblanco, E-28049 Madrid, Spain.
Miguel
M
Alcalde
Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain.
Francisco J
FJ
Medrano
Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain.
Angel T
AT
Martínez
Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain fjruiz@cib.csic.es.
Francisco J
FJ
Ruiz-Dueñas
D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D009195:Peroxidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020076:Pleurotus / Q000201:enzymology; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism
D005656:Fungal Proteins; D009195:Peroxidase
directed evolution; pH stability; structure–function relationships; versatile peroxidase