Enhancing enzymatic bioconjugation efficiency via installation of a substrate recruitment domain

Abstract

Enzyme mediated bioconjugation provides a method for easy and rapid formation of protein-protein and protein-small molecule conjugates under mild conditions. Promiscuous enzymes are of particular interest because they can catalyze conjugation reactions on a broad set of substrates. However, this promiscuity carries the risk of undesirable off-target modifications. To mitigate this effect, we used computational design to install a substrate recruitment domain (SRD) onto the promiscuous enzyme, tyrosinase. The redesigned tyrosinase, called D42, preferentially modifies tyrosine residues within the peptide core (core) linked to a 6-amino acid recognition motif/sequence (RS) specific for the SRD. Incorporation of the recognition sequence along with a neighboring tyrosine in peptides or proteins allows for rapid D42-mediated conversion of the tyrosine to an orthoquinone, which can be selectively modified with a variety of nucleophiles. We demonstrate the utility of our design system by rapidly installing cytotoxic molecules on a monoclonal antibody. For Table of Contents Only O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=72 SRC="FIGDIR/small/684804v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1a3b274org.highwire.dtl.DTLVardef@18eeedorg.highwire.dtl.DTLVardef@ee0aaeorg.highwire.dtl.DTLVardef@1ff3fa1_HPS_FORMAT_FIGEXP M_FIG C_FIG