Oxygen activation by P450(cin): Protein and substrate mutagenesis

Area of Science:

• Biochemistry
• Enzymology
• Chemical catalysis

Background:

• Most cytochromes P450 (P450s) utilize a conserved threonine residue for dioxygen activation during catalysis.
• P450(cin) (CYP176A) is an exception, featuring an asparagine at this position.
• Previous work showed P450(cin)'s Asn-242 is crucial for regioselectivity and stereocontrol in cineole oxidation, not for functional replacement of threonine.

Purpose of the Study:

• To investigate how P450(cin) controls dioxygen activation despite lacking the conserved threonine.
• To explore the roles of specific residues and substrate modifications in P450(cin)'s catalytic mechanism.

Main Methods:

• Site-directed mutagenesis of P450(cin) to create Thr-243 and Asn-242 mutants.
• Enzymatic assays using wild-type and mutant P450(cin) with native substrate (cineole) and modified substrates (camphane, cinane).
• Analysis of NADPH cofactor coupling and product formation to assess catalytic turnover and oxygen activation.

Main Results:

• P450(cin) Thr-243 was not involved in protonating the hydroperoxy intermediate, and the N242T mutant did not improve catalytic efficiency.
• Modifying the substrate by removing the ethereal oxygen (creating camphane or cinane) did not abolish NADPH coupling in P450(cin).
• Unlike P450(EryF), P450(cin) showed significant NADPH coupling even with modified substrates lacking the ethereal oxygen, indicating the substrate is not key for oxygen activation control.

Conclusions:

• The substrate structure is essential for regioselectivity and stereoselectivity in P450(cin) catalysis, but not for controlling oxygen activation.
• The mechanism of dioxygen activation in P450(cin) remains unclear, as it deviates from the typical threonine-dependent pathway and substrate requirements seen in other P450s.