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In Vitro Reconstitution of Formylglycine‐Generating Enzymes Requires Copper(I)
Author(s) -
Knop Matthias,
Engi Pascal,
Lemnaru Roxana,
Seebeck Florian P.
Publication year - 2015
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201500322
Subject(s) - enzyme , cysteine , chemistry , cofactor , biochemistry , protein engineering , phosphatase , in vitro , recombinant dna , stereochemistry , substrate (aquarium) , bond cleavage , catalysis , combinatorial chemistry , biology , ecology , gene
Formylglycine‐generating enzymes (FGEs) catalyze O 2 ‐dependent conversion of specific cysteine residues of arylsulfatases and alkaline phosphatases into formylglycine. The ability also to introduce unique aldehyde functions into recombinant proteins makes FGEs a powerful tool for protein engineering. One limitation of this technology is poor in vitro activity of reconstituted FGEs. Although FGEs have been characterized as cofactor‐free enzymes we report that the addition of one equivalent of Cu I increases catalytic efficiency more than 20‐fold and enables the identification of stereoselective C−H bond cleavage at the substrate as the rate‐limiting step. These findings remove previous limitations of FGE‐based protein engineering and also pose new questions about the catalytic mechanism of this O 2 ‐utilizing enzyme.