Insight into the Mechanism of Phenylacetate Decarboxylase (PhdB), a Toluene‐Producing Glycyl Radical Enzyme

We recently reported the discovery of phenylacetate decarboxylase (PhdB), representing one of only ten glycyl‐radical‐enzyme reaction types known, and a promising biotechnological tool for first‐time biochemical synthesis of toluene from renewable resources. Here, we used experimental and computational data to evaluate the plausibility of three candidate PhdB mechanisms, involving either attack at the phenylacetate methylene carbon or carboxyl group [via H‐atom ion from COOH or single‐electron oxidation of COO− (Kolbe‐type decarboxylation)]. In vitro experimental data included assays with F‐labeled phenylacetate, kinetic studies, and tests with site‐directed PhdB mutants; computational data involved estimation of reaction energetics using density functional theory (DFT). The DFT results indicated that all three mechanisms are thermodynamically challenging (beyond the range of many known enzymes in terms of endergonicity or activation energy barrier), reflecting the formidable demands on PhdB for catalysis of this reaction. Evidence that PhdB was able to bind α,α‐difluorophenylacetate but was unable to catalyze its decarboxylation supported the enzyme's ion of a methylene H atom. Diminished activity of H327A and Y691F mutants was consistent with proposed proton donor roles for His327 and Tyr691. Collectively, these and other data most strongly support PhdB attack at the methylene carbon. Three candidate mechanisms for the novel glycyl radical enzyme (GRE) phenylacetate decarboxylase (PhdB) were evaluated using a combination of in vitro experimental data and computational data involving estimation of reaction energetics. The data best support a methylene carbon attack (green arrows) that differs substantially from the reported mechanism for p‐hydroxyphenylacetate decarboxylase.