Structural Characterization of Two Short Unspecific Peroxygenases: Two Different Dimeric Arrangements.

Area of Science:

• Biochemistry
• Structural Biology
• Enzymology

Background:

• Unspecific peroxygenases (UPOs) are fungal enzymes with biotechnological potential, serving as stable alternatives to cytochrome P450 monooxygenases.
• Limited structural information exists for UPOs, hindering the exploitation of their vast genetic diversity for industrial applications.
• Only 19 Protein Data Bank entries represent UPOs, with published structures from just two species prior to this study.

Purpose of the Study:

• To determine the crystal structures of two novel UPOs, rMroUPO and rCviUPO, from *Marasmius rotula* and *Collariella virescens*, respectively.
• To elucidate the structural basis for the dimeric nature of these UPOs.
• To engineer a UPO variant with enhanced capabilities for producing valuable compounds.

Main Methods:

• Sequence optimization and expression of rMroUPO and rCviUPO in *Escherichia coli*.
• Crystallization of the enzymes and collection of diffraction data at high resolution.
• Structure determination by molecular replacement and analysis of dimer interfaces using biophysical and molecular biology techniques.

Main Results:

• High-resolution crystal structures (1.45 Å for rMroUPO, 1.95 Å for rCviUPO) of the two UPOs were solved, revealing dimeric arrangements.
• Diverse structural interactions, including disulfide bridges and alpha-helix associations, were identified at the dimer interfaces.
• A modified rCviUPO variant lacking the C-terminal tail demonstrated an enhanced ability to produce fatty acid diepoxides.

Conclusions:

• The study provides novel structural insights into two distinct UPO enzymes, highlighting varied dimeric arrangements.
• The findings contribute to understanding UPO structure-function relationships and open avenues for protein engineering.
• The engineered rCviUPO variant shows potential for industrial applications in producing cross-linking agents.