QM/MM Study of the Reaction Mechanism of L‑Tyrosine Hydroxylation Catalyzed by the Enzyme CYP76AD1

We have studied the hydroxylation mechanism of l-Tyr by
the heme-dependent enzyme CYP76AD1 from the sugar beet (Beta vulgaris). This enzyme has a promising biotechnological
application in modified yeast strains to produce medicinal alkaloids,
an alternative to the traditional opium poppy harvest. A generative
machine learning software based on AlphaFold was used to build the
structure of CYP76AD1 since there are no structural data for this
specific enzyme. After model validation, l-Tyr was docked
in the active site of CYP76AD1 to assemble the reactive complex, whose
catalytic distances remained stable throughout the 100 ns of MD simulation.
Subsequent QM/MM calculations elucidated that l-Tyr hydroxylation
occurs in two steps: hydrogen abstraction from l-Tyr by CpdI,
forming an l-Tyr radical, and subsequent radical rebound,
corresponding to a rate-limiting step of 16.0 kcal·mol–1. Our calculations suggest that the hydrogen abstraction step should
occur in the doublet state, while the radical rebound should happen
in the quartet state. The clarification of the reaction mechanism
of CYP76AD1 provides insights into the rational optimization of the
biosynthesis of alkaloids to eliminate the use of opium poppy.