Two‐fold Bioorthogonal Derivatization by Different Formylglycine‐Generating Enzymes

Abstract

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.

Conversion of Serine‐Type Aldehyde Tags by the Radical SAM Protein AtsB from Methanosarcina mazei

“…Because AtsB proteins are predicted to contain three [4 Fe−4 S] 2+ ‐type clusters, a maximum of 12 Fe atoms per molecule can be expected . We observed that AtsB required additional N‐ and C‐terminal auxiliary sequences to efficiently convert cysteine within its endogenous substrate sequence in vitro (peptide P1 ) . Peptide P2 , which carries merely one additional amino acid residue on either end of the core motif, was not converted in detectable amounts by MM‐AtsB in a standard 5:1 substrate/enzyme ratio and an incubation time of 60 min at 37 °C in the presence of excess SAM under argon (Figure S2).…”

“…As described in our previous study for MM‐AtsB, both enzymes were purified anaerobically and we were able to isolate KP‐AtsB and MM‐AtsB with an average iron content of 4.0±0.6 and 7.9±0.7 atoms per molecule, respectively (see the Supporting Information and Figure S1). Because AtsB proteins are predicted to contain three [4 Fe−4 S] 2+ ‐type clusters, a maximum of 12 Fe atoms per molecule can be expected .…”

“…Aldehyde tag technology offers an efficient route for site‐specific protein conjugation to enable enzyme immobilization, protein fusion, or the production of antibody–drug conjugates . This strategy is based on the conversion of a cysteine or serine residue within the conserved (C/S)X(P/A)XR motif to C α ‐formylglycine (FGly) by formylglycine‐generating enzymes (FGEs; Scheme A, B) . The aldehyde moiety of FGly can then react with specialized hydrazine or enolic nucleophiles to form hydrolytically stable protein–payload linkages (Scheme C) …”

“…The prototypic FGE is a copper‐dependent metallo‐oxidase responsible for type I sulfatase maturation and strongly prefers CXPXR‐type motifs as a substrate (Scheme A) . It has been widely used for in vitro and in vivo FGly generation; thus allowing for efficient production of protein aldehydes …”

“…It belongs to the radical S ‐adenosyl methionine (SAM) protein superfamily and converts cysteine and serine within (C/S)X(P/A)XR‐type motifs into FGly (Scheme B). It can be shown that AtsB can be used orthogonally to FGE with two different cysteine‐type tags, namely, CTAGR and CTPSR …”