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

• Published in: Journal of the American Chemical Society Vol. 135; no. 39; pp. 14574 - 14585
• Main Authors: Feliks, Mikolaj, Martins, Berta M, Ullmann, G. Matthias
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 02-10-2013
• Subjects: Carboxy-Lyases - chemistry; Carboxy-Lyases - metabolism; Catalytic Domain; Clostridium - chemistry; Clostridium - enzymology; Decarboxylation; Models, Molecular; Quantum Theory; Static Electricity
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/ja402379q

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.