title: Insights into the glycyl radical enzyme active site of benzylsuccinate synthase: a computational study.
abstract: The 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.
authors.affiliation: Chemical and Biological Engineering Department, Colorado School of Mines, 1500 Illinois Street, Golden, Colorado 80401, USA.
authors.forename: Vivek S
authors.initials: VS
authors.lastname: Bharadwaj
authors.forename: Anthony M
authors.initials: AM
authors.lastname: Dean
authors.forename: C Mark
authors.initials: CM
authors.lastname: Maupin
mesh_terms: D000595:Amino Acid Sequence; D019755:Carbon-Carbon Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D005650:Fumarates / Q000378:metabolism; D005998:Glycine; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D009154:Mutation; D017385:Sequence Homology; D013386:Succinates / Q000378:metabolism; D020628:Thauera / Q000201:enzymology; D014050:Toluene / Q000378:metabolism
chemical_list: D005650:Fumarates; D013386:Succinates; C030196:benzylsuccinate; D014050:Toluene; C032005:fumaric acid; D019755:Carbon-Carbon Lyases; C114354:benzylsuccinate synthase; D005998:Glycine