title: In Vitro Reconstitution of Formylglycine-Generating Enzymes Requires Copper(I).
abstract: Formylglycine-generating enzymes (FGEs) catalyze O2 -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 O2 -utilizing enzyme.
authors.affiliation: Department of Chemistry, University of Basel, St. Johanns-Ring 19, 4056, Basel, Switzerland.
authors.forename: Matthias
authors.initials: M
authors.lastname: Knop
authors.affiliation: Department of Chemistry, University of Basel, St. Johanns-Ring 19, 4056, Basel, Switzerland.
authors.forename: Pascal
authors.initials: P
authors.lastname: Engi
authors.affiliation: Department of Chemistry, University of Basel, St. Johanns-Ring 19, 4056, Basel, Switzerland.
authors.forename: Roxana
authors.initials: R
authors.lastname: Lemnaru
authors.affiliation: Department of Chemistry, University of Basel, St. Johanns-Ring 19, 4056, Basel, Switzerland. florian.seebeck@unibas.ch.
authors.forename: Florian P
authors.initials: FP
authors.lastname: Seebeck
mesh_terms: D039903:Actinobacteria / Q000201:enzymology; D055162:Biocatalysis; D003300:Copper / Q000737:chemistry / Q000378:metabolism*; D005998:Glycine / Q000031:analogs & derivatives* / Q000096:biosynthesis; D015394:Molecular Structure; D020102:Mycobacterium smegmatis / Q000201:enzymology; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism; D013429:Sulfatases / Q000737:chemistry / Q000378:metabolism*
chemical_list: C032736:N-formylglycine; D003300:Copper; D013429:Sulfatases; D010100:Oxygen; D005998:Glycine
keywords: copper; formylglycine; kinetic isotope effects; oxygen activation; protein engineering