In Vitro Reconstitution of Formylglycine-Generating Enzymes Requires Copper(I).
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.
['Knop|Matthias|M|', 'Engi|Pascal|P|', 'Lemnaru|Roxana|R|', 'Seebeck|Florian P|FP|']
D039903:Actinobacteria D055162:Biocatalysis D003300:Copper D005998:Glycine D015394:Molecular Structure D020102:Mycobacterium smegmatis D010100:Oxygen D013429:Sulfatases
formylglycine protein engineering copper kinetic isotope effects oxygen activation