The synthesis of protein–protein and protein–peptide conjugates is an important capability for producing vaccines, immunotherapeutics, and targeted delivery agents. Herein we show that the enzyme tyrosinase is capable of oxidizing exposed tyrosine residues into o -quinones that react rapidly with cysteine residues on target proteins. This coupling reaction occurs under mild aerobic conditions and has the rare ability to join full-size proteins in under 2 h. The utility of the approach is demonstrated for the attachment of cationic peptides to enhance the cellular delivery of CRISPR-Cas9 20-fold and for the coupling of reporter proteins to a cancer-targeting antibody fragment without loss of its cell-specific binding ability. The broad applicability of this technique provides a new building block approach for the synthesis of protein chimeras.

No abstract

Biocatalytic C–H activation has the potential to merge enzymatic and synthetic strategies for bond formation. Fe II /αKG-dependent halogenases are particularly distinguished for their ability both to control selective C–H activation as well as to direct group transfer of a bound anion along a reaction axis separate from oxygen rebound, enabling the development of new transformations. In this context, we elucidate the basis for the selectivity of enzymes that perform selective halogenation to yield 4-Cl-lysine (BesD), 5-Cl-lysine (HalB), and 4-Cl-ornithine (HalD), allowing us to probe how site-selectivity and chain length selectivity are achieved. We now report the crystal structure of the HalB and HalD, revealing the key role of the substrate-binding lid in positioning the substrate for C 4 vs C 5 chlorination and recognition of lysine vs ornithine. Targeted engineering of the substrate-binding lid further demonstrates that these selectivities can be altered or switched, showcasing the potential to develop halogenases for biocatalytic applications.

<p>Chimeric protein-protein conjugates provide platforms for immunotherapy, targeted drug delivery, and vaccine development. However, many desirable constructs cannot be produced through direct expression, and the targeted coupling of two proteins is chemically challenging. Here we present a new approach for the rapid and site-specific coupling of proteins using native amino acids. Tyrosinase oxidizes exposed tyrosine residues on polypeptides, generating <i>ortho</i>-quinones that react rapidly with strategically placed cysteine residues in other proteins. This approach was used to modify CRISPR-Cas9 and other substrates with small molecules, peptides and even intact proteins. The conjugation of cell penetrating peptides to CRISPR-Cas9 was shown to increase cellular genome editing efficiency by 20-fold relative to unmodified Cas9. This technology represents a new paradigm for biomolecular coupling, and paves the way to an unprecedented range of multifunctional bioconjugates.</p>

<p>Chimeric protein-protein conjugates provide platforms for immunotherapy, targeted drug delivery, and vaccine development. However, many desirable constructs cannot be produced through direct expression, and the targeted coupling of two proteins is chemically challenging. Here we present a new approach for the rapid and site-specific coupling of proteins using native amino acids. Tyrosinase oxidizes exposed tyrosine residues on polypeptides, generating <i>ortho</i>-quinones that react rapidly with strategically placed cysteine residues in other proteins. This approach was used to modify CRISPR-Cas9 and other substrates with small molecules, peptides and even intact proteins. The conjugation of cell penetrating peptides to CRISPR-Cas9 was shown to increase cellular genome editing efficiency by 20-fold relative to unmodified Cas9. This technology represents a new paradigm for biomolecular coupling, and paves the way to an unprecedented range of multifunctional bioconjugates.</p>