Formylglycine-generating enzymes are of increasing interest in the field of bioconjugation chemistry. They catalyze the site-specific oxidation of a cysteine residue to the aldehyde-containing amino acid C -formylglycine (FGly). This non-canonical residue can be generated within any desired target protein and can subsequently be used for bioorthogonal conjugation reactions. The prototypic formylglycine-generating enzyme (FGE) and the iron-sulfur protein AtsB display slight variations in their recognition sequences. We designed specific tags in peptides and proteins that were selectively converted by the different enzymes. Combination of the different tag motifs within a single peptide or recombinant protein enabled the independent and consecutive introduction of two formylglycine residues and the generation of heterobifunctionalized protein conjugates.

Formylglycin-generierende Enzyme haben in den letzten Jahren breite Anwendung in der bioorthogonalen Chemie gefunden. Sie katalysieren die ortsspezifische Oxidation eines Cysteinrestes zur Aldehyd-haltigen Aminosäure C a -Formylglycin (FGly). FGly kann in jedem beliebigen Zielprotein generiert und anschließend fürb ioorthogonale Konjugationsreaktionen genutzt werden. Das prototypische Formylglycin-generierende Enzym (FGE) und das Eisen-Schwefel-Protein AtsB zeigen leichte Variationen in ihren Erkennungssequenzen. Spezifische Motive in Peptiden und Proteinen wurden entworfen, die selektiv von diesen Enzymen adressiert werden. Die Kombination dieser Motive innerhalb eines Peptids oder rekombinanten Proteins ermçglichte die unabhängige und sukzessive Einführung von zwei FGly-Resten und die Generierung hetero-difunktionalisierter Proteinkonjugate.

Formylglycine‐generating enzymes provide a convenient tool for site‐specific protein derivatization. Their ability to oxidize cysteine or serine residues within a defined consensus sequence to Cα‐formylglycine (FGly) allows for the targeted introduction of a unique chemical handle for various bioconjugation reactions. In recent years, oxygen‐dependent FGly‐generating enzyme saw broad use in protein functionalization and the generation of protein conjugates. Yet, the FGly‐generating system AtsB, along with its capability to convert unusual aldehyde tag sequences, remains mostly unused. Herein, the ability of AtsB from Methanosarcina mazei to convert nonclassical aldehyde tags of the SX(A/P)XR‐type and its potential use in bioconjugation chemistry are demonstrated.