K. Anton Feenstra, Rolien Bosch, Karin Hofstetter*, Jennifer Venhorst, Jan N.M. Commandeur, Andreas Schmid* and Nico P.E. Vermeulen
* Dept. of Biochemical and Chemical Engineering, University of Dortmund, Germany
Molecular Dynamics optimization in Homology Building of Styrene Mono-Oxygenase Catalytic Component, Analysis by Essential Dynamics and Validation by Automated Docking in preparation (2005).

The styrene mono-oxygenase (SMO) two component flavo-enyzme produces >99% enantiopure S-styrene oxide. Of scientific and industrial interest is the availability of mutants that would be highly selective and highly active in the production of pure R-styrene oxide [1], for which purpose approaches using directed evolution are being applied. Of further interest is the development of methods to rationalize and design such mutants from crystallographic structural data and/or from homology model(s).

In the present study, a homology model of StyA (SMO large & catalytic domain) was developed based on the X-ray crystallographic structure of para-hydroxybenzoate hydroxylase (1PBE[2], 15% sequence identity). An optimization scheme employing energy minimization (EM) and molecular dynamics (MD) simulations with and without positional restraints on specific sub-sets of atoms was performed to provide verification of model stability and as a means of refinement of the model. The substrate styrene was docked into the models generated to provide final validation of model quality and appropriate substrate binding location and orientation.

The initial homology model was found to be reasonably stable in EM and MD, with RMSD values for Ca atoms rising over 1 ns of free MD to 3-4 Å. Optimized models (i.e. before free MD) were significantly more stable with corresponding values of no more than 1-2 Å. One optimized model was found to bind styrene in a single tight cluster (maximum deviation 0.5 Å) in a catalytically active position with respect to the FAD co-factor and an orientation pro-chiral to the formation of S-styrene oxide. In the other models, styrene was bound in a non-catalytically relevant position, in widely varying positions, or in a mixed pro-S/pro-R orientation.

Ongoing work focuses on the fine details of the model, and on determination of key interactions in the enantiospecific binding. Mutants of the enzyme are being produced by the group of Prof. A. Schmid in Zürich. These mutants will be modelled also and investigated further in a similar fashion. Attempts at producing high-resolution grade protein crystals of StyA (by the group of Prof. A. Mattevi in Padua) have not been successful to date.

  1.  A. Schmid, K. Hofstetter, H-J. Feiten, F. Hollmann, B. Witholt. “Integrated Biocatalytic Synthesis on Gram Scale: The Highly Enantioselective Preparation of Chiral Oxiranes with Styrene Monooxygenase”. Adv. Synth. Catal. 343, 732-737, 2001.
  2. H.A. Schreuder, P.A.J. Prick, R.K. Wierenga, G. Vriend, K.S. Wilson, W.G.J. Hol, J. Drenth. “Crystal Structure Of The P-Hydroxybenzoate Hydroxylase-Substrate Complex Refined At 1.9 Angstroms Resolution. Analysis Of The Enzyme-Substrate And Enzyme-Product Complexes”. J.Mol.Biol. 208, 679, 1989.