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

Rodrigues, Andria V.; Tantillo, Dean J.; Mukhopadhyay, Aindrila; Keasling, Jay D.; Beller, Harry R.

doi:10.1002/cbic.201900560

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

Journal Article · Wed Sep 11 00:00:00 EDT 2019 · ChemBioChem: a European journal of chemical biology

DOI:https://doi.org/10.1002/cbic.201900560· OSTI ID:1573417

Rodrigues, Andria V. ^[1]; ^[2]; ^[1]; ^[3]; ^[1]

Joint BioEnergy Institute (JBEI) 5885 Hollis Street Emeryville CA 94608 USA, Biological Systems and EngineeringLawrence Berkeley National Laboratory 1 Cyclotron Road Berkeley CA 94720 USA
Department of ChemistryUniversity of California 1 Shields Avenue Davis CA 95616 USA
Joint BioEnergy Institute (JBEI) 5885 Hollis Street Emeryville CA 94608 USA; Biological Systems and EngineeringLawrence Berkeley National Laboratory 1 Cyclotron Road Berkeley CA 94720 USA; Department of BioengineeringUniversity of California 306 Stanley Hall Berkeley CA 94720 USA; Department of Chemical and Biomolecular EngineeringUniversity of California 201 Gilman Hall Berkeley CA 94720 USA; Novo Nordisk Foundation Center for BiosustainabilityTechnical University of Denmark Building 220; Kemitorvet 2800 Kgs. Lyngby Denmark

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 abstraction 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 abstraction 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.

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Research Organization:: Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

Sponsoring Organization:: National Science Foundation (NSF); USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)

Grant/Contract Number:: AC02-05CH11231

OSTI ID:: 1573417

Alternate ID(s):: OSTI ID: 1609115
OSTI ID: 1573418

Journal Information:: ChemBioChem: a European journal of chemical biology, Journal Name: ChemBioChem: a European journal of chemical biology Journal Issue: 5 Vol. 21; ISSN 1439-4227

Publisher:: ChemPubSoc EuropeCopyright Statement

Country of Publication:: France

Language:: English

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Kolbe decarboxylation
glycyl radical enzyme
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radical reactions
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Insight into the Mechanism of Phenylacetate Decarboxylase (PhdB), a Toluene-Producing Glycyl Radical Enzyme

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