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bioRxiv · 10.1101/2025.10.27.684804

Enhancing enzymatic bioconjugation efficiency via installation of a substrate recruitment domain

Shelby, C.·Kuzelka, K. P.·Ellis, J. M.·Yao, Z.·McCue, A. C.·Park, R.·Nair, S. K.·Bowers, A.·Kuhlman, B.
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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

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Shelby, C., Kuzelka, K. P., Ellis, J. M., Yao, Z., McCue, A. C., Park, R., Nair, S. K., Bowers, A., Kuhlman, B.. 2025-10-27. Enhancing enzymatic bioconjugation efficiency via installation of a substrate recruitment domain. https://doi.org/10.1101/2025.10.27.684804

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