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Directed evolution of P450cin for mediated electron transfer.

Ketaki D Belsare1, Thomas Horn1, Anna Joëlle Ruff1

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Protein Engineering, Design & Selection : PEDS
|December 24, 2016
PubMed
Summary

Directed evolution optimized a P450 enzyme fusion protein to utilize a cobalt(III)sepulchrate electron delivery system, enhancing 1,8-cineole hydroxylation activity. This bioelectrocatalytic approach offers a promising alternative to traditional cofactor-dependent methods.

Keywords:
P450cinbioelectrocatalysisdirected Evolutionelectron mediatorprotein engineering

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Area of Science:

  • Biocatalysis and Enzyme Engineering
  • Synthetic Biology
  • Bioelectrochemistry

Background:

  • Cytochrome P450 monooxygenases are crucial for regioselective C-H bond hydroxylation.
  • Cell-free P450 synthesis is limited by low turnover, instability, and reliance on expensive NADPH.
  • Bioelectrocatalysis offers an alternative electron source, but native enzymes often lack compatibility with non-natural systems.

Purpose of the Study:

  • To enhance the activity of a P450 CinA-10aa-CinC fusion protein using directed evolution.
  • To adapt the enzyme for efficient use with a zinc/cobalt(III)sepulchrate electron delivery system.
  • To improve the hydroxylation of 1,8-cineole under bioelectrocatalytic conditions.

Main Methods:

  • Directed evolution using Sequence Saturation Mutagenesis (SeSaM) and multiple site-saturation mutagenesis.
  • Engineering of a P450 CinA-10aa-CinC fusion protein.
  • Assessing enzyme activity with zinc/cobalt(III)sepulchrate and platinum/cobalt(III)sepulchrate electron delivery systems.
  • Comparison with NADPH-dependent reactions.

Main Results:

  • A directed evolution variant (KB8) showed a 3.8-fold increase in catalytic efficiency (28 µM⁻¹ min⁻¹) compared to the parent enzyme (7 µM⁻¹ min⁻¹).
  • KB8 achieved 1.5-fold higher product formation (500 µM µM⁻¹ P450) than the NADPH-dependent system (315 µM µM⁻¹ P450).
  • Electrochemical experiments demonstrated a 4-fold higher product formation rate for KB8 with platinum/cobalt(III)sepulchrate (0.16 nmol (nmol) P450⁻¹ min⁻² cm⁻²) compared to the parent (0.04 nmol (nmol) P450⁻¹ min⁻² cm⁻²).

Conclusions:

  • Directed evolution is effective for creating P450 enzymes compatible with alternative bioelectrocatalytic electron delivery systems.
  • The engineered KB8 variant shows significant improvements in activity and product formation for 1,8-cineole hydroxylation.
  • This work highlights the potential of bioelectrocatalysis for sustainable chemical synthesis using engineered enzymes.