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.

Mutation of Conserved Residues Increases in Vitro Activity of the Formylglycine‐Generating Enzyme

“…Recent work, however, showed that FGE is a copper‐dependent oxidase that might take an approach to O 2 activation and C−H cleavage similar to that of the copper‐dependent lytic polysaccharide monooxygenases (LPMOs), the peptidylglycine α‐hydroxylating monooxygenase, or dopamine/tyramine‐β‐monooxygenases . In the course of isolating the catalytic principle of FGE from T. curvata we constructed a variant in which all cysteine residues outside the active site were mutated . We converted the three buried Cys residues (187, 231, 298) into alanine and the surface‐exposed Cys284 into serine.…”

“…The proteins were produced in Escherichia coli in lysogeny broth (LB) medium at 37 °C. The catalytic activities of the purified enzymes were assayed as previously described . Each reaction mixture contained 2 μ m enzyme, 2 μ m CuSO 4 , 2 m m dithiothreitol (DTT), 50 m m EDTA, 50 m m NaCl and 50 m m Tris ⋅ HCl at pH 8.…”

“…As a FGE substrate we used 200 μ m of a peptide (sequence Abz‐SALCSPTRA‐NH 2 ) that includes the CxPXR motif and a N‐terminal ortho ‐aminobenzoic acid (Abz) residue as chromophore. Time‐dependent oxidation of this peptide to the formylglycine‐containing product was monitored by RP‐HPLC …”

“…Protein production : Variants FGE 1–7 and loop_GFP were generated by side‐directed mutagenesis by using synthetic oligonucleotides (Table S1). The construction of ald_GFP has been described previously . Purified PCR fragments were ligated into pET28a(+) expression vectors by use of NdeI and XhoI restriction sites.…”

“…Kinetics : Catalytic activities were quantified in reaction mixtures containing substrate (Abz‐SALCSPTRA‐NH 2 , 200 μ m ), DTT (2 m m ), CuSO 4 (0 or 2 μ m ), EDTA (50 m m ), and NaCl (50 m m ) in Tris buffer (pH 8, 50 m m ) at 25 °C . The reactions were initiated by addition of FGE (2 μ m ).…”