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A Stable Artificial Multienzymatic Complex Using a Heterotrimeric Protein From Metallosphaera sedula.

Fumiya Iwata1, Hidehiko Hirakawa1,2, Teruyuki Nagamune1,3

  • 1Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Tokyo 113-8656, Japan.

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|April 18, 2018
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Summary

Metallosphaera sedula PCNA enhances bacterial cytochrome P450 monooxygenase activity by 2.1-fold. This stable assembly overcomes previous limitations, enabling efficient biocatalysis for chemical synthesis.

Keywords:
PCNAcytochrome P450enzyme engineeringmultienzyme complexprotein assembly

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Area of Science:

  • Biocatalysis
  • Enzyme Engineering
  • Biochemistry

Background:

  • Bacterial cytochrome P450 monooxygenases (P450s) are valuable biocatalysts for hydrocarbon oxidation.
  • Their in vitro application is limited by the need for auxiliary electron transfer proteins and reductases.
  • Previous attempts to create self-sufficient P450s using Sulfolobus solfataricus PCNA showed reduced activity due to partial complex dissociation.

Purpose of the Study:

  • To improve the efficiency of self-sufficient bacterial P450 systems.
  • To investigate the use of a more stable heterotrimeric PCNA from Metallosphaera sedula for P450 assembly.
  • To enhance the apparent specific monooxygenase activity of bacterial P450s.

Main Methods:

  • Assembly of bacterial P450 with auxiliary proteins using Metallosphaera sedula PCNA.
  • Characterization of the P450-PCNA complex at submicromolar protein concentrations.
  • Comparison of enzymatic activity with P450 assembled using Sulfolobus solfataricus PCNA.

Main Results:

  • The M. sedula PCNA-assembled P450 system achieved saturated activity at 40 nM protein concentration.
  • The apparent specific monooxygenase activity was 2.1-fold higher compared to the S. solfataricus PCNA-assembled system.
  • M. sedula PCNA demonstrated superior stability and efficiency in facilitating the P450 monooxygenase system.

Conclusions:

  • Metallosphaera sedula PCNA is a highly effective tool for creating stable and active self-sufficient bacterial P450 systems.
  • This approach significantly enhances biocatalytic efficiency for P450-mediated oxidations.
  • M. sedula PCNA offers a versatile platform for various enzymatic reactions, including P450 monooxygenases.