Protostadienol synthase from Aspergillus fumigatus: functional conversion into lanosterol synthase

Area of Science:

• Biochemistry and Molecular Biology
• Enzymology
• Fungal Secondary Metabolism

Background:

• Oxidosqualene cyclases (OSCs) are crucial enzymes in sterol biosynthesis.
• Fungal Oxidosqualene:protostadienol cyclase (OSPC) produces protostadienol, a precursor to helvolic acid.
• Oxidosqualene:lanosterol cyclase (OSLC) produces lanosterol and parkeol, common sterol intermediates.

Purpose of the Study:

• To investigate the structure-function relationship of OSPC by altering its C-terminal region.
• To functionally convert OSPC into an oxidosqualene:lanosterol cyclase (OSLC) by introducing OSLC-like residues.
• To elucidate the role of active-site residues in determining product specificity.

Main Methods:

• Site-directed mutagenesis was used to replace the C-terminal residues of Aspergillus fumigatus OSPC with those from human OSLC.
• Enzyme activity assays were performed to analyze the cyclization products of the OSPC mutant.
• Kinetic parameters (Vmax/KM) and substrate binding affinity were determined.
• Homology modeling was employed to visualize potential active-site interactions.

Main Results:

• The engineered OSPC mutant, with C-terminal residues swapped to mimic OSLC, no longer produced protostadienol.
• The mutant efficiently produced a 1:1 mixture of lanosterol and parkeol, characteristic of OSLC activity.
• The catalytic efficiency (Vmax/KM) decreased 14-fold, while substrate binding affinity remained largely unchanged.
• Homology modeling suggested Phe701 stabilizes the C-20 protosteryl cation via cation-pi interactions, influencing product outcome.

Conclusions:

• The C-terminal region of OSPC plays a critical role in determining its product specificity.
• Functional conversion of OSPC to OSLC activity is achievable through targeted mutagenesis of specific residues.
• Active-site residue interactions, particularly cation-pi interactions involving Phe701, are key determinants in the divergence of sterol biosynthesis pathways.