Modeling of the Reaction Mechanism of Enzymatic Radical C–C Coupling by Benzylsuccinate Synthase
Molecular modeling techniques and density functional theory calculations were performed to study the mechanism of enzymatic radical C–C coupling catalyzed by benzylsuccinate synthase (BSS). BSS has been identified as a glycyl radical enzyme that catalyzes the enantiospecific fumarate addition to tol...
Main Authors: | , |
---|---|
Format: | Article |
Language: | English |
Published: |
MDPI AG
2016-04-01
|
Series: | International Journal of Molecular Sciences |
Subjects: | |
Online Access: | http://www.mdpi.com/1422-0067/17/4/514 |
id |
doaj-d3a2c7e255c4455bab1645218d2951ba |
---|---|
record_format |
Article |
spelling |
doaj-d3a2c7e255c4455bab1645218d2951ba2020-11-24T21:11:33ZengMDPI AGInternational Journal of Molecular Sciences1422-00672016-04-0117451410.3390/ijms17040514ijms17040514Modeling of the Reaction Mechanism of Enzymatic Radical C–C Coupling by Benzylsuccinate SynthaseMaciej Szaleniec0Johann Heider1Jerzy Haber Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences, Kraków 30-239, PolandLaboratory of Microbial Biochemistry, and LOEWE-Center for Synthetic Microbiology, Philipps-University of Marburg, Marburg 35043, GermanyMolecular modeling techniques and density functional theory calculations were performed to study the mechanism of enzymatic radical C–C coupling catalyzed by benzylsuccinate synthase (BSS). BSS has been identified as a glycyl radical enzyme that catalyzes the enantiospecific fumarate addition to toluene initiating its anaerobic metabolism in the denitrifying bacterium Thauera aromatica, and this reaction represents the general mechanism of toluene degradation in all known anaerobic degraders. In this work docking calculations, classical molecular dynamics (MD) simulations, and DFT+D2 cluster modeling was employed to address the following questions: (i) What mechanistic details of the BSS reaction yield the most probable molecular model? (ii) What is the molecular basis of enantiospecificity of BSS? (iii) Is the proposed mechanism consistent with experimental observations, such as an inversion of the stereochemistry of the benzylic protons, syn addition of toluene to fumarate, exclusive production of (R)-benzylsuccinate as a product and a kinetic isotope effect (KIE) ranging between 2 and 4? The quantum mechanics (QM) modeling confirms that the previously proposed hypothetical mechanism is the most probable among several variants considered, although C–H activation and not C–C coupling turns out to be the rate limiting step. The enantiospecificity of the enzyme seems to be enforced by a thermodynamic preference for binding of fumarate in the pro(R) orientation and reverse preference of benzyl radical attack on fumarate in pro(S) pathway which results with prohibitively high energy barrier of the radical quenching. Finally, the proposed mechanism agrees with most of the experimental observations, although the calculated intrinsic KIE from the model (6.5) is still higher than the experimentally observed values (4.0) which suggests that both C–H activation and radical quenching may jointly be involved in the kinetic control of the reaction.http://www.mdpi.com/1422-0067/17/4/514benzylsuccinate synthaseanaerobic metabolismDFTkinetic isotope effecttoluene metabolism |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Maciej Szaleniec Johann Heider |
spellingShingle |
Maciej Szaleniec Johann Heider Modeling of the Reaction Mechanism of Enzymatic Radical C–C Coupling by Benzylsuccinate Synthase International Journal of Molecular Sciences benzylsuccinate synthase anaerobic metabolism DFT kinetic isotope effect toluene metabolism |
author_facet |
Maciej Szaleniec Johann Heider |
author_sort |
Maciej Szaleniec |
title |
Modeling of the Reaction Mechanism of Enzymatic Radical C–C Coupling by Benzylsuccinate Synthase |
title_short |
Modeling of the Reaction Mechanism of Enzymatic Radical C–C Coupling by Benzylsuccinate Synthase |
title_full |
Modeling of the Reaction Mechanism of Enzymatic Radical C–C Coupling by Benzylsuccinate Synthase |
title_fullStr |
Modeling of the Reaction Mechanism of Enzymatic Radical C–C Coupling by Benzylsuccinate Synthase |
title_full_unstemmed |
Modeling of the Reaction Mechanism of Enzymatic Radical C–C Coupling by Benzylsuccinate Synthase |
title_sort |
modeling of the reaction mechanism of enzymatic radical c–c coupling by benzylsuccinate synthase |
publisher |
MDPI AG |
series |
International Journal of Molecular Sciences |
issn |
1422-0067 |
publishDate |
2016-04-01 |
description |
Molecular modeling techniques and density functional theory calculations were performed to study the mechanism of enzymatic radical C–C coupling catalyzed by benzylsuccinate synthase (BSS). BSS has been identified as a glycyl radical enzyme that catalyzes the enantiospecific fumarate addition to toluene initiating its anaerobic metabolism in the denitrifying bacterium Thauera aromatica, and this reaction represents the general mechanism of toluene degradation in all known anaerobic degraders. In this work docking calculations, classical molecular dynamics (MD) simulations, and DFT+D2 cluster modeling was employed to address the following questions: (i) What mechanistic details of the BSS reaction yield the most probable molecular model? (ii) What is the molecular basis of enantiospecificity of BSS? (iii) Is the proposed mechanism consistent with experimental observations, such as an inversion of the stereochemistry of the benzylic protons, syn addition of toluene to fumarate, exclusive production of (R)-benzylsuccinate as a product and a kinetic isotope effect (KIE) ranging between 2 and 4? The quantum mechanics (QM) modeling confirms that the previously proposed hypothetical mechanism is the most probable among several variants considered, although C–H activation and not C–C coupling turns out to be the rate limiting step. The enantiospecificity of the enzyme seems to be enforced by a thermodynamic preference for binding of fumarate in the pro(R) orientation and reverse preference of benzyl radical attack on fumarate in pro(S) pathway which results with prohibitively high energy barrier of the radical quenching. Finally, the proposed mechanism agrees with most of the experimental observations, although the calculated intrinsic KIE from the model (6.5) is still higher than the experimentally observed values (4.0) which suggests that both C–H activation and radical quenching may jointly be involved in the kinetic control of the reaction. |
topic |
benzylsuccinate synthase anaerobic metabolism DFT kinetic isotope effect toluene metabolism |
url |
http://www.mdpi.com/1422-0067/17/4/514 |
work_keys_str_mv |
AT maciejszaleniec modelingofthereactionmechanismofenzymaticradicalcccouplingbybenzylsuccinatesynthase AT johannheider modelingofthereactionmechanismofenzymaticradicalcccouplingbybenzylsuccinatesynthase |
_version_ |
1716753078133194752 |