Enzymatic Modification of N-Terminal Proline Residues Using Phenol Derivatives

Abstract

A convenient enzymatic strategyis reported for the modification of proline residues in the N-terminal positions of proteins. Using a tyrosinase enzyme isolated from Agaricus bisporus(abTYR), phenols and catechols are oxidized to highly reactive o-quinone intermediates that then couple to N-terminal proline residues in high yield. Key advantages of this bioconjugation method include (1) the use of air-stable precursors that can be prepared on large scale if needed, (2) mild reaction conditions, including low temperatures, (3) the targeting of native functional groups that can be introduced readily on most proteins, and (4) the use of molecular oxygen as the sole oxidant. This coupling strategy was successfully demonstrated for the attachment of a variety of phenol-derivatized cargo molecules to a series of protein substrates, including self-assembled viral capsids, enzymes, and a chitin binding domain (CBD). The ability of the CBD to bind to the surfaces of yeast cells was found to be unperturbed by this modification reaction. File list (2) download file view on ChemRxiv Maza 2018. ChemRxiv Final.pdf (0.97 MiB) download file view on ChemRxiv SI. Maza. ChemRxiv.pdf (1.50 MiB)

Tyrosinase-Mediated Oxidative Coupling of Tyrosine Tags on Peptides and Proteins

“…It readily oxidizes phenols that are appended with NHS-activated cargo molecules, allowing simple tyramine derivatives to be used as readily available and storable building blocks. Most importantly, in previous work, 14 we and others have found that it leaves native tyrosine residues on proteins untouched if they are within the secondary and tertiary structural elements of folded structure.The ability of tyrosinases to generate o-quinones on proteins is well documented, at least at high substrate concentrations. Tyrosinases and phenol oxidases have been known for decades to mediate crosslinking between proteins in meat 35 , whey 36 , and flour 37 via non-selective tyrosine-tyrosine, tyrosinecysteine, and tyrosine-lysine linkages.…”

“…It readily oxidizes phenols that are appended with NHS-activated cargo molecules, allowing simple tyramine derivatives to be used as readily available and storable building blocks. Most importantly, in previous work, 14 we and others have found that it leaves native tyrosine residues on proteins untouched if they are within the secondary and tertiary structural elements of folded structure.…”

“…Recently, we reported that the tyrosinase enzyme from the common button mushroom Agaricus bisporus (abTYR) is capable of oxidizing simple phenols attached to cargo molecules to yield o-quinones in situ (Figure 1b). 14 The enzyme relies on two copper ions chelated in a deep active site pocket to transfer an atom from molecular O2 to the ortho-position of a bound phenol (Figure 1c-e). 33,34 The enzyme operates rapidly at 4 °C and room temperature at a mild pH of 6.5, is commercially available, and can be stored at -80 °C in solution for months.…”

Enabling protein-hosted organocatalytic transformations

“…57,84,85 Chemical methodologies for protein labelling have vastly diversied and improved in recent years, showing ne-tuned reactivity and biocompatibility with labelling achieved within live cells. [86][87][88][89][90][91][92][93][94] One can anticipate that efficient articial enzymes can be made by adapting these novel technologies.…”

Chemical Methods for Selective Labeling of Proteins

“…Here, the ligation of an acyl‐donor peptide ester and N‐terminal α‐amine of the acceptor protein is catalyzed by mutated subtiligase. Recently, Francis and co‐workers reported a tyrosinase mediate labeling of N‐terminal proline with an oxidized phenol or catechol . The enzymatic methods provide excellent tools for the labeling of proteins.…”