title: Calculation of substrate binding affinities for a bacterial GH78 rhamnosidase through molecular dynamics simulations.
abstract: Ram2 fromPediococcus acidilacticiis a rhamnosidase from the glycoside hydrolase family 78. It shows remarkable selectivity for rutinose rather than para-nitrophenyl-alpha-l-rhamnopyranoside (p-NPR). Molecular dynamics simulations were performed using a homology model of this enzyme, in complex with both substrates. Free energy calculations lead to predicted binding affinities of -34.4 and -30.6 kJ mol-1respectively, agreeing well with an experimentally estimated relative free energy of 5.4 kJ mol-1. Further, the most relevant binding poses could be determined. While p-NPR preferably orients its rhamnose moiety toward the active site, rutinose interacts most strongly with its glucose moiety. A detailed hydrogen bond analysis confirms previously implicated residues in the active site (Asp217, Asp222, Trp226, Asp229 and Glu488) and quantifies the importance of individual residues for the binding. The most important amino acids are Asp229 and Phe339 which are involved in many interactions during the simulations. While Phe339 was observed in more simulations, Asp229 was involved in more persistent interactions (forming an average of at least 2 hydrogen bonds during the simulation). These analyses directly suggest mutations that could be used in a further experimental characterization of the enzyme. This study shows once more the strength of computer simulations to rationalize and guide experiments at an atomic level.
authors.affiliation: Department of Material Sciences and Process Engineering, Institute of Molecular Modeling and Simulation, University of Natural Resources and Life Sciences Vienna, Muthgasse 18, A-1190 Vienna, Austria.
authors.forename: Melanie
authors.initials: M
authors.lastname: Grandits
authors.forename: Herbert
authors.initials: H
authors.lastname: Michlmayr
authors.forename: Christoph
authors.initials: C
authors.lastname: Sygmund
authors.forename: Chris
authors.initials: C
authors.lastname: Oostenbrink
keywords: Binding thermodynamics; Free energy calculations; GH78, glycoside hydrolase family 78; GROMOS; Glycoside hydrolase; Rutinose; p-NPR, para-nitrophenyl-alpha-l-rhamnopyranoside
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