TY - JOUR
T1 - Impact of scaffold rigidity on the design and evolution of an artificial Diels-Alderase
AU - Preiswerk, Nathalie
AU - Beck, Tobias
AU - Schulz, Jessica D.
AU - Milovník, Peter
AU - Mayer, Clemens
AU - Siegel, Justin
AU - Baker, David
AU - Hilvert, Donald
PY - 2014/6/3
Y1 - 2014/6/3
N2 - By combining targeted mutagenesis, computational refinement, and directed evolution, amodestly active, computationally designed Diels-Alderase was converted into the most proficient biocatalyst for [4+2] cycloadditions known. The high stereoselectivity and minimal product inhibition of the evolved enzyme enabled preparative scale synthesis of a single product diastereomer. X-ray crystallography of the enzyme-product complex shows that the molecular changes introduced over the course of optimization, including addition of a lid structure, gradually reshaped the pocket for more effective substrate preorganization and transition state stabilization. The good overall agreement between the experimental structure and the original design model with respect to the orientations of both the bound product and the catalytic side chains contrasts with other computationally designed enzymes. Because design accuracy appears to correlate with scaffold rigidity, improved control over backbone conformation will likely be the key to future efforts to design more efficient enzymes for diverse chemical reactions.
AB - By combining targeted mutagenesis, computational refinement, and directed evolution, amodestly active, computationally designed Diels-Alderase was converted into the most proficient biocatalyst for [4+2] cycloadditions known. The high stereoselectivity and minimal product inhibition of the evolved enzyme enabled preparative scale synthesis of a single product diastereomer. X-ray crystallography of the enzyme-product complex shows that the molecular changes introduced over the course of optimization, including addition of a lid structure, gradually reshaped the pocket for more effective substrate preorganization and transition state stabilization. The good overall agreement between the experimental structure and the original design model with respect to the orientations of both the bound product and the catalytic side chains contrasts with other computationally designed enzymes. Because design accuracy appears to correlate with scaffold rigidity, improved control over backbone conformation will likely be the key to future efforts to design more efficient enzymes for diverse chemical reactions.
KW - Biocatalysis
KW - Computational enzyme design
KW - Diels-Alder reaction
KW - Enzyme mechanism
KW - Laboratory evolution
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U2 - 10.1073/pnas.1401073111
DO - 10.1073/pnas.1401073111
M3 - Article
C2 - 24847076
AN - SCOPUS:84901855811
VL - 111
SP - 8013
EP - 8018
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
SN - 0027-8424
IS - 22
ER -