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Download fileInsights into the Glycyl Radical Enzyme Active Site of Benzylsuccinate Synthase: A Computational Study
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posted on 2013-08-21, 00:00 authored by Vivek
S. Bharadwaj, Anthony M. Dean, C. Mark MaupinThe
fumarate addition reaction, catalyzed by the enzyme benzylsuccinate
synthase (BSS), is considered to be one of the most intriguing and
energetically challenging reactions in biology. BSS belongs to the
glycyl radical enzyme family and catalyzes the fumarate addition reaction,
which enables microorganisms to utilize hydrocarbons as an energy
source under anaerobic conditions. Unfortunately, the extreme sensitivity
of the glycyl radical to oxygen has hampered the structural and kinetic
characterization of BSS, thereby limiting our knowledge on this enzyme.
To enhance our molecular-level understanding of BSS, a computational
approach involving homology modeling, docking studies, and molecular
dynamics (MD) simulations has been used to deduce the structure of
BSS’s catalytic subunit (BSSα) and illuminate the molecular
basis for the fumarate addition reaction. We have identified two conserved
and distinct binding pockets at the BSSα active site: a hydrophobic
pocket for toluene binding and a polar pocket for fumaric acid binding.
Subsequent dynamical and energetic evaluations have identified Glu509,
Ser827, Leu390, and Phe384 as active site residues critical for substrate
binding. The orientation of substrates at the active site observed
in MD simulations is consistent with experimental observations of
the syn addition of toluene to fumaric acid. It is
also found that substrate binding tightens the active site and restricts
the conformational flexibility of the thiyl radical, leading to hydrogen
transfer distances conducive to the proposed reaction mechanism. The
stability of substrates at the active site and the occurrence of feasible
radical transfer distances between the thiyl radical, substrates,
and the active site glycine indicate a substrate-assisted radical
transfer pathway governing fumarate addition.