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In Vitro Reconstitution of Formylglycine-Generating Enzymes Requires Copper(I).

  • Knop M Department of Chemistry, University of Basel, St. Johanns-Ring 19, 4056, Basel, Switzerland.
  • Engi P Department of Chemistry, University of Basel, St. Johanns-Ring 19, 4056, Basel, Switzerland.
  • Lemnaru R Department of Chemistry, University of Basel, St. Johanns-Ring 19, 4056, Basel, Switzerland.
  • Seebeck FP Department of Chemistry, University of Basel, St. Johanns-Ring 19, 4056, Basel, Switzerland. florian.seebeck@unibas.ch.
  • 2015-09-26
Published in:
  • Chembiochem : a European journal of chemical biology. - 2015
copper
formylglycine
kinetic isotope effects
oxygen activation
protein engineering
Actinobacteria
Biocatalysis
Copper
Glycine
Molecular Structure
Mycobacterium smegmatis
Oxygen
Sulfatases
English 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.
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  • English
Open access status
green
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https://folia.unifr.ch/global/documents/238435
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