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Application of directed evolution to the study of structural enzyme dynamics

Application of directed evolution to the study of structural enzyme dynamics

Objective

We propose to study the importance of structural dynamics to enzymatic catalysis. This will utilise simplified model systems in which a variety of catalytically distinct mutants have been generated though directed evolution. This research will focus on whether changes to the conformational landscape of enzymes affect catalysis and how the structural scaffold can determine the optimum temperature for enzymatic activity. Two model systems will be used: (i) the lactate dehydrogenase family, chosen because it is the most heavily studied family with regards to thermophilicity; (ii) a designed Kemp elimination catalyst, chosen because it is the first example of an enzyme in which the evolution of the active site and structural scaffold has been decoupled. A synergistic experimental approach will be used to study the mutants generated through directed evolution: - Biophysics (temperature-controlled cryo-crystallography, incoherent neutron scattering, in-cristallo spectroscopy) and detailed data analysis (anisotropic and multiple crystallographic refinement) - Enzyme kinetics (temperature dependent kinetics and Arrhenius plots) - Computational simulation (normal mode analysis and molecular dynamics) This project will constitute, to our knowledge, one of the first attempts to apply directed evolution to the systematic analysis of the importance and evolution of pathways for dynamic conformational change in enzymatic catalysis.
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Coordinator

CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE CNRS

Address

Rue Michel Ange 3
75794 Paris

France

Activity type

Higher or Secondary Education Establishments

EU Contribution

€ 167 145,57

Administrative Contact

Jean-Xavier Boucherle (Dr.)

Project information

Grant agreement ID: 237065

Status

Closed project

  • Start date

    1 June 2009

  • End date

    31 May 2011

Funded under:

FP7-PEOPLE

Coordinated by:

CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE CNRS

France