Unexpected Differences between Two Closely Related Bacterial P450 Camphor Monooxygenases

Area of Science:

• Biochemistry
• Enzymology
• Structural Biology

Background:

• Bacterial cytochrome P450cam and CYP101D1 catalyze camphor oxidation for carbon assimilation.
• A newly discovered P450tcu shares high sequence identity and similar enzymatic function with P450cam.
• P450tcu exhibits distinct characteristics not observed in P450cam, suggesting novel catalytic mechanisms.

Purpose of the Study:

• To investigate the unusual features of P450tcu compared to P450cam.
• To elucidate the structural and dynamic differences influencing their enzymatic properties.
• To understand the mechanisms of substrate hydroxylation and activation in both enzymes.

Main Methods:

• X-ray crystallography to determine the structure of P450tcu with bound substrate.
• Molecular dynamics simulations to assess enzyme flexibility and proton access.
• Isothermal titration calorimetry to analyze substrate binding thermodynamics.

Main Results:

• X-ray structures reveal product in multiple orientations for P450tcu, indicating *in crystallo* hydroxylation via X-ray reduction.
• P450tcu displays slower spin transition upon substrate binding compared to P450cam.
• Isothermal titration calorimetry shows entropically driven binding in P450cam (ΔH > 0) versus enthalpically driven binding in P450tcu (ΔH < 0).

Conclusions:

• Despite high sequence and functional similarity, P450tcu and P450cam exhibit significantly different substrate binding and activation properties.
• Conformational dynamics play a crucial role in the distinct catalytic behaviors of these closely related P450 enzymes.
• P450tcu's flexibility facilitates proton access and O2 activation, leading to unique hydroxylation mechanisms.