Expression improvement and mechanistic study of the retro-Diels-Alderase catalytic antibody 10F11 by site-directed mutagenesis |
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Authors: | Zheng Lei Goddard Jean-Philippe Baumann Ulrich Reymond Jean-Louis |
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Institution: | Department of Chemistry and Biochemistry, University of Berne, Freiestrasse 3, CH-3012 Berne, Switzerland. |
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Abstract: | Antibody 10F11 catalyzes the retro-Diels-Alder reaction of the bicyclic prodrug 1 releasing HNO and anthracene 4 (kcat/kuncat=2500). Earlier X-ray crystal structures of Fab 10F11 showed that tryptophan H104 at the bottom of the binding pocket interacts by pi-stacking with the aromatic ring of the substrate. Antibody 10F11 was expressed as a chimeric Fab and subjected to site-directed mutagenesis. Expression was improved by substituting a serine for a phenylalanine residue on the Fv-domain surface. Nine active-site mutants were then prepared including replacements at TrpH104, PheH101 and SerH100. Catalysis depends mainly on TrpH104 and PheH101. Catalysis is most likely caused by a combination of shape complementarity and specific electronic interactions between transition state and the aromatic residue H104. Medium and de-solvation effects have no effect on the reaction rate. Catalysis was improved to (kcat/kuncat=6300) by substituting phenylalanine for LeuL101 to indirectly enhance pi-stacking between transition state and TrpH104. |
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Keywords: | catalytic antibody site-directed mutagenesis protein engineering stacking enhancement antibody expression |
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