An efficient and stable ascorbate/O ₂ ‑driven route for L‑DOPA synthesis by heme‑dependent tyrosine hydroxylase

L-DOPA is a key therapeutic agent for Parkinson’s disease, with growing demand due to global population aging. Here we report that heme-dependent tyrosine hydroxylase (TyrH) can utilize an ascorbate/O ₂ system—as an alternative to H ₂ O ₂ —to synthesize L-DOPA with markedly enhanced operational stability. While exogenous H ₂ O ₂ rapidly inactivates TyrH within minutes, sodium ascorbate (NaAsc) enables sustained catalysis for up to 24 h, surpassing the H ₂ O ₂ -driven yield after only 30 min. UV-vis spectroscopy confirms that H ₂ O ₂ readily degrades the heme center, whereas the heme remains intact in the presence of NaAsc. QM/MM simulations reveal that in situ generated H ₂ O ₂ leads to the active species of Compound I for tyrosine hydroxylation. Through systematic optimization, we establish efficient reaction conditions (40 µM TyrH, 1 mM L-Tyr, 100 mM NaAsc, pH 8.5, 40 °C), achieving >95% conversion of L-Tyr to L-DOPA within 2 h. This work not only provides a robust and sustainable biocatalytic route for L-DOPA production but also highlights the broader applicability of the ascorbate/O ₂ pathway in heme-enzyme catalysis.