Interplay between two spin states determines the hydroxylation catalyzed by P₄₅₀ monooxygenase from Trichoderma...

Area of Science:

• Biochemistry
• Mycology
• Computational Chemistry

Background:

• Trichoderma brevicompactum produces trichodermin/harzianum A, valuable secondary metabolites.
• Cytochrome P450 monooxygenases are key enzymes in fungal secondary metabolism.
• The enzyme tri22 (formerly Tri11) is involved in the biosynthesis of trichothecenes.

Purpose of the Study:

• To elucidate the mechanism of C-4 C-H hydroxylation of 12,13-epoxytrichothec-9-ene (EPT) by tri22.
• To investigate the role of spin states in the catalytic activity of P450.
• To understand the formation of trichodermol in the trichodermin/harzianum A biosynthetic pathway.

Main Methods:

• Density Functional Theory (DFT) and Quantum Mechanics (QM) calculations.
• Modeling of the P450 active site (Cpd I) and substrate (EPT).
• Analysis of transition states, intermediates, and product complexes across different spin states (doublet and quartet).

Main Results:

• The C-4 C-H hydroxylation of EPT to trichodermol was computationally elucidated.
• A two-state reactivity (TSR) mechanism was identified, involving interplay between doublet and quartet spin states.
• Rebound-free product formation was observed, driven by the spin state dynamics.

Conclusions:

• The study provides a detailed mechanistic insight into P450-catalyzed trichothecene biosynthesis.
• The interplay of spin states is crucial for the efficiency and selectivity of the hydroxylation reaction.
• This work contributes to understanding fungal P450 enzyme mechanisms and potential applications.