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Published March 6, 2012 | Published + Supplemental Material
Journal Article Open

Iterative approach to computational enzyme design


A general approach for the computational design of enzymes to catalyze arbitrary reactions is a goal at the forefront of the field of protein design. Recently, computationally designed enzymes have been produced for three chemical reactions through the synthesis and screening of a large number of variants. Here, we present an iterative approach that has led to the development of the most catalytically efficient computationally designed enzyme for the Kemp elimination to date. Previously established computational techniques were used to generate an initial design, HG-1, which was catalytically inactive. Analysis of HG-1 with molecular dynamics simulations (MD) and X-ray crystallography indicated that the inactivity might be due to bound waters and high flexibility of residues within the active site. This analysis guided changes to our design procedure, moved the design deeper into the interior of the protein, and resulted in an active Kemp eliminase, HG-2. The cocrystal structure of this enzyme with a transition state analog (TSA) revealed that the TSA was bound in the active site, interacted with the intended catalytic base in a catalytically relevant manner, but was flipped relative to the design model. MD analysis of HG-2 led to an additional point mutation, HG-3, that produced a further threefold improvement in activity. This iterative approach to computational enzyme design, including detailed MD and structural analysis of both active and inactive designs, promises a more complete understanding of the underlying principles of enzymatic catalysis and furthers progress toward reliably producing active enzymes.

Additional Information

© 2011 National Academy of Sciences. Contributed by Stephen L. Mayo, November 4, 2011 (sent for review September 6, 2011). We thank Jens Kaiser and Pavle Nikolovski at the Caltech Molecular Observatory for assistance in crystal screening, crystallographic data collection, and structure determination. We are grateful to Daniela Röthlisberger and David Baker for providing genes for the KE positive controls and to Marie Ary and Scott A. Johnson for assistance with the manuscript. Data for the HG-2 and 1A53-2 structures were collected at beamline 12-2 at the Stanford Synchrotron Radiation Lightsource (SSRL, SLAC National Accelerator Laboratory, Menlo Park, CA).We acknowledge the Gordon and Betty Moore Foundation for support of the Molecular Observatory at Caltech and the Department of Energy and National Institutes of Health for supporting the SSRL. This work was supported by the Defense Advanced Research Projects Agency, a Department of Defense National Security Science and Engineering Faculty Fellowship (S.L.M.), and a Lawrence Livermore National Laboratory Lawrence Scholars Fellowship (G.K.). Fellowship support from the Fonds des Verbandes der chemischen Industrie and the Studienstiftung des deutschen Volkes (R.B.) is gratefully acknowledged. Author contributions: H.K.P., G.K., T.M.L., D.H., K.N.H., and S.L.M. designed research; H.K.P., G.K., T.M.L., R.B., R.A.C., and L.M.T. performed research; H.K.P., G.K., T.M.L., R.B., R.A.C., L.M.T., D.H., K.N.H., and S.L.M. analyzed data; and H.K.P., G.K., T.M.L., R.B., R.A.C., L.M.T., D.H., K.N.H., and S.L.M. wrote the paper. Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 3O2L, 3NYD, 3NYZ, and 3NZ1).

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Published - Privett2012p17517P_Natl_Acad_Sci_Usa.pdf

Supplemental Material - Appendix.pdf


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