title: Site-specific protein cross-linking by peroxidase-catalyzed activation of a tyrosine-containing peptide tag.
abstract: Protein modification methods represent fundamental techniques that are applicable in many fields. In this study, a site-specific protein cross-linking based on the oxidative tyrosine coupling reaction was demonstrated. In the presence of horseradish peroxidase (HRP) and H(2)O(2), tyrosine residues undergo one-electron oxidation reactions and form radicals in their phenolic moieties, and these species subsequently react with each other to form dimers or further react to generate polymers. Here, a peptide-tag containing a tyrosine residue(s) (Y-tag, of which the amino acid sequences were either GGGGY or GGYYY) was genetically introduced at the C-terminus of a model protein, Escherichia coli alkaline phosphatase (BAP). Following the incubation of recombinant BAPs with HRP and H(2)O(2), Y-tagged BAPs were efficiently cross-linked with each other, whereas wild-type BAP did not undergo cross-linking, indicating that the tyrosine residues in the Y-tags were recognized by HRP as the substrates. To determine the site-specificity of the cross-linking reaction, the Y-tag was selectively removed by thrombin digestion. The resultant BAP without the Y-tag showed no reactivity in the presence of HRP and H(2)O(2). Conversely, Y-tagged BAPs cross-linked by HRP treatment were almost completely digested into monomeric BAP units following incubation with the protease. Moreover, cross-linked Y-tagged BAPs retained ∼95% of their native enzymatic activity. These results show that HRP catalyzed the site-specific cross-linking of BAPs through tyrosine residues positioned in the C-terminal Y-tag. The site-selective enzymatic oxidative tyrosine coupling reaction should offer a practical option for site-specific and covalent protein modifications.
authors.affiliation: Department of Applied Chemistry, Graduate School of Engineering, Kyushu University.
authors.forename: Kosuke
authors.initials: K
authors.lastname: Minamihata
authors.forename: Masahiro
authors.initials: M
authors.lastname: Goto
authors.forename: Noriho
authors.initials: N
authors.lastname: Kamiya
mesh_terms: D000469:Alkaline Phosphatase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001665:Binding Sites; D055162:Biocatalysis; D002850:Chromatography, Gel; D004926:Escherichia coli / Q000201:enzymology; D006735:Horseradish Peroxidase / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013917:Thrombin / Q000378:metabolism; D014443:Tyrosine
chemical_list: D009842:Oligopeptides; D014443:Tyrosine; D006735:Horseradish Peroxidase; D000469:Alkaline Phosphatase; D013917:Thrombin