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

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.