Insight into the Mechanism of Phenylacetate Decarboxylase (PhdB), a Toluene-Producing Glycyl Radical Enzyme
Abstract
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.
- Authors:
-
[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
- Publication Date:
- Research Org.:
- Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
- Sponsoring Org.:
- USDOE Office of Science (SC), Biological and Environmental Research (BER); National Science Foundation (NSF)
- OSTI Identifier:
- 1573417
- Alternate Identifier(s):
- OSTI ID: 1573418; OSTI ID: 1609115
- Grant/Contract Number:
- AC02-05CH11231
- Resource Type:
- Published Article
- Journal Name:
- ChemBioChem: a European journal of chemical biology
- Additional Journal Information:
- Journal Name: ChemBioChem: a European journal of chemical biology Journal Volume: 21 Journal Issue: 5; Journal ID: ISSN 1439-4227
- Publisher:
- ChemPubSoc Europe
- Country of Publication:
- France
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; glycyl radical enzyme; Kolbe decarboxylation; phenylacetate decarboxylase; radical reactions; reaction mechanisms
Citation Formats
Rodrigues, Andria V., Tantillo, Dean J., Mukhopadhyay, Aindrila, Keasling, Jay D., and Beller, Harry R. Insight into the Mechanism of Phenylacetate Decarboxylase (PhdB), a Toluene-Producing Glycyl Radical Enzyme. France: N. p., 2019.
Web. doi:10.1002/cbic.201900560.
Rodrigues, Andria V., Tantillo, Dean J., Mukhopadhyay, Aindrila, Keasling, Jay D., & Beller, Harry R. Insight into the Mechanism of Phenylacetate Decarboxylase (PhdB), a Toluene-Producing Glycyl Radical Enzyme. France. https://doi.org/10.1002/cbic.201900560
Rodrigues, Andria V., Tantillo, Dean J., Mukhopadhyay, Aindrila, Keasling, Jay D., and Beller, Harry R. Wed .
"Insight into the Mechanism of Phenylacetate Decarboxylase (PhdB), a Toluene-Producing Glycyl Radical Enzyme". France. https://doi.org/10.1002/cbic.201900560.
@article{osti_1573417,
title = {Insight into the Mechanism of Phenylacetate Decarboxylase (PhdB), a Toluene-Producing Glycyl Radical Enzyme},
author = {Rodrigues, Andria V. and Tantillo, Dean J. and Mukhopadhyay, Aindrila and Keasling, Jay D. and Beller, Harry R.},
abstractNote = {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.},
doi = {10.1002/cbic.201900560},
journal = {ChemBioChem: a European journal of chemical biology},
number = 5,
volume = 21,
place = {France},
year = {2019},
month = {9}
}
Free Publicly Available Full Text
Publisher's Version of Record
https://doi.org/10.1002/cbic.201900560
https://doi.org/10.1002/cbic.201900560
Other availability
Search WorldCat to find libraries that may hold this journal
Cited by: 10 works
Citation information provided by
Web of Science
Web of Science
Figures / Tables:
Figure 1: In vitro production of substituted toluenes in an anaerobic assay containing purified, recombinant PhdB and PhdA, and SAM. The substituted toluenes are [methyl-13C]toluene from phenylacetate-2–13C (positive control, n=2) and [methyl-F2]toluene from $\alpha$,$\alpha$-difluorophenylacetate (n=3).
All figures and tables
(4 total)
Save to My Library
You must Sign In or Create an Account in order to save documents to your library.
Works referenced in this record:
A prominent glycyl radical enzyme in human gut microbiomes metabolizes trans -4-hydroxy-l-proline
journal, February 2017
- Levin, B. J.; Huang, Y. Y.; Peck, S. C.
- Science, Vol. 355, Issue 6325
Discovery of enzymes for toluene synthesis from anoxic microbial communities
journal, March 2018
- Beller, Harry R.; Rodrigues, Andria V.; Zargar, Kamrun
- Nature Chemical Biology, Vol. 14, Issue 5
Indoleacetate decarboxylase is a glycyl radical enzyme catalysing the formation of malodorant skatole
journal, October 2018
- Liu, Dazhi; Wei, Yifeng; Liu, Xuyang
- Nature Communications, Vol. 9, Issue 1
Dissociation constants of phenylacetic acid and three substituted phenylacetic acids
journal, January 1971
- Duer, Wayne C.; Robinson, R. A.
- Journal of the Chemical Society B: Physical Organic
p -Hydroxyphenylacetate decarboxylase from Clostridium difficile : A novel glycyl radical enzyme catalysing the formation of
journal, March 2001
- Selmer, Thorsten; Andrei, Paula I.
- European Journal of Biochemistry, Vol. 268, Issue 5
In vitro Characterization of Phenylacetate Decarboxylase, a Novel Enzyme Catalyzing Toluene Biosynthesis in an Anaerobic Microbial Community
journal, August 2016
- Zargar, K.; Saville, R.; Phelan, R. M.
- Scientific Reports, Vol. 6, Issue 1
The role of fluorine in medicinal chemistry: Review Article
journal, January 2007
- Shah, Poonam; Westwell, Andrew D.
- Journal of Enzyme Inhibition and Medicinal Chemistry, Vol. 22, Issue 5
An evaluation review of the prediction of protonation states in proteins versus crystallographic experiment†
journal, October 2009
- Fisher, Stuart J.; Wilkinson, James; Henchman, Richard H.
- Crystallography Reviews, Vol. 15, Issue 4
Structure and Function of CutC Choline Lyase from Human Microbiota Bacterium Klebsiella pneumoniae
journal, July 2015
- Kalnins, Gints; Kuka, Janis; Grinberga, Solveiga
- Journal of Biological Chemistry, Vol. 290, Issue 35
Anaerobic bacterial metabolism of hydrocarbons
journal, December 1998
- Heider, Johann; Spormann, Alfred M.; Beller, Harry R.
- FEMS Microbiology Reviews, Vol. 22, Issue 5
Theoretical Studies on Synthetic and Biosynthetic Oxidopyrylium−Alkene Cycloadditions: Pericyclic Pathways to Intricarene
journal, February 2008
- Wang, Selina C.; Tantillo, Dean J.
- The Journal of Organic Chemistry, Vol. 73, Issue 4
A glycyl radical enzyme enables hydrogen sulfide production by the human intestinal bacterium Bilophila wadsworthia
journal, February 2019
- Peck, Spencer C.; Denger, Karin; Burrichter, Anna
- Proceedings of the National Academy of Sciences, Vol. 116, Issue 8
New tricks for the glycyl radical enzyme family
journal, September 2017
- Backman, Lindsey R. F.; Funk, Michael A.; Dawson, Christopher D.
- Critical Reviews in Biochemistry and Molecular Biology, Vol. 52, Issue 6
Biochemical and genetic characterization of benzylsuccinate synthase from Thauera aromatica : a new glycyl radical enzyme catalysing the first step in anaerobic toluene metabolism
journal, April 1998
- Leuthner, Birgitta; Leutwein, Christina; Schulz, Henk
- Molecular Microbiology, Vol. 28, Issue 3
Perspective on Fluorocarbon Chemistry
journal, January 2004
- Lemal, David M.
- The Journal of Organic Chemistry, Vol. 69, Issue 1
Structural Basis for a Kolbe-Type Decarboxylation Catalyzed by a Glycyl Radical Enzyme
journal, September 2011
- Martins, Berta M.; Blaser, Martin; Feliks, Mikolaj
- Journal of the American Chemical Society, Vol. 133, Issue 37
Analysis of the Novel Benzylsuccinate Synthase Reaction for Anaerobic Toluene Activation Based on Structural Studies of the Product
journal, January 1998
- Beller, Harry R.; Spormann, Alfred M.
- Journal of Bacteriology, Vol. 180, Issue 20
New glycyl radical enzymes catalysing key metabolic steps in anaerobic bacteria
journal, January 2005
- Selmer, Thorsten; Pierik, Antonio J.; Heider, Johann
- Biological Chemistry, Vol. 386, Issue 10
Catalytic Mechanism of the Glycyl Radical Enzyme 4-Hydroxyphenylacetate Decarboxylase from Continuum Electrostatic and QC/MM Calculations
journal, September 2013
- Feliks, Mikolaj; Martins, Berta M.; Ullmann, G. Matthias
- Journal of the American Chemical Society, Vol. 135, Issue 39
Structural Basis for Allosteric Substrate Specificity Regulation in Anaerobic Ribonucleotide Reductases
journal, August 2001
- Larsson, Karl-Magnus; Andersson, Jessica; Sjöberg, Britt-Marie
- Structure, Vol. 9, Issue 8
Radical-mediated C-S bond cleavage in C2 sulfonate degradation by anaerobic bacteria
journal, April 2019
- Xing, Meining; Wei, Yifeng; Zhou, Yan
- Nature Communications, Vol. 10, Issue 1
Anaerobic activation of toluene and o-xylene by addition to fumarate in denitrifying strain T.
journal, February 1997
- Beller, H. R.; Spormann, A. M.
- Journal of Bacteriology, Vol. 179, Issue 3
Charge Screening and the Dielectric Constant of Proteins: Insights from Molecular Dynamics
journal, January 1996
- Simonson, Thomas; Brooks, Charles L.
- Journal of the American Chemical Society, Vol. 118, Issue 35
Solving the Density Functional Conundrum: Elimination of Systematic Errors To Derive Accurate Reaction Enthalpies of Complex Organic Reactions
journal, May 2017
- Sengupta, Arkajyoti; Raghavachari, Krishnan
- Organic Letters, Vol. 19, Issue 10
Insight into the Mechanism of the B 12 -Independent Glycerol Dehydratase from Clostridium butyricum : Preliminary Biochemical and Structural Characterization ‡
journal, April 2004
- O'Brien, Jessica Rae; Raynaud, Celine; Croux, Christian
- Biochemistry, Vol. 43, Issue 16
Molecular Basis of C–N Bond Cleavage by the Glycyl Radical Enzyme Choline Trimethylamine-Lyase
journal, October 2016
- Bodea, Smaranda; Funk, Michael A.; Balskus, Emily P.
- Cell Chemical Biology, Vol. 23, Issue 10
4-Hydroxyphenylacetate Decarboxylases: Properties of a Novel Subclass of Glycyl Radical Enzyme Systems †
journal, August 2006
- Yu, Lihua; Blaser, Martin; Andrei, Paula I.
- Biochemistry, Vol. 45, Issue 31
Microbial conversion of choline to trimethylamine requires a glycyl radical enzyme
journal, November 2012
- Craciun, S.; Balskus, E. P.
- Proceedings of the National Academy of Sciences, Vol. 109, Issue 52
Substrate range of benzylsuccinate synthase from Azoarcus sp. strain T
journal, September 1999
- Beller, Harry R.; Spormann, Alfred M.
- FEMS Microbiology Letters, Vol. 178, Issue 1
Figures / Tables found in this record:
Figures/Tables have been extracted from DOE-funded journal article accepted manuscripts.