Catalytic Mechanism of the Glycyl Radical Enzyme 4‑Hydroxyphenylacetate Decarboxylase from Continuum Electrostatic and QC/MM Calculations

Using continuum electrostatics and
QC/MM calculations, we investigate
the catalytic cycle of the glycyl radical enzyme 4-hydroxyphenylacetate
decarboxylase, an enzyme involved in the fermentative production of p-cresol from tyrosine in clostridia. On the basis of our
calculations, we propose a five-step mechanism for the reaction. In
the first step, the substrate 4-hydroxyphenylacetate is activated
by an unusual concerted abstraction of an electron and a proton. Namely,
Cys503 radical abstracts an electron from the substrate and Glu637
abstracts a proton. Thus in total, a hydrogen atom is abstracted from
the substrate. In the second step, the carboxylic group readily splits
off from the phenoxy-acetate radical anion to give carbon dioxide.
This decarboxylation step is coupled to a proton transfer from Glu637
back to the phenolic hydroxyl group which leads to a p-hydroxybenzyl radical. The remaining steps of the reaction involve
a rotation of the Cys503 side chain followed by a proton transfer
from Glu505 to Cys503 and a hydrogen atom transfer from Cys503 to
the p-hydroxybenzyl radical to form p-cresol. The calculated mechanism agrees with experimental data suggesting
that both Cys503 and Glu637 are essential for the catalytic function
of 4-hydroxyphenylacetate decarboxylase and that the substrate requires
a hydroxyl group in para-position to the acetate
moiety.