Reconstitution of Formylglycine-generating Enzyme with Copper(II) for Aldehyde Tag Conversion

Abstract

To further our aim of synthesizing aldehyde-tagged proteins for research and biotechnology applications, we developed methods for recombinant production of aerobic formylglycine-generating enzyme (FGE) in good yield. We then optimized the FGE biocatalytic reaction conditions for conversion of cysteine to formylglycine in aldehyde tags on intact monoclonal antibodies. During the development of these conditions, we discovered that pretreating FGE with copper(II) is required for high turnover rates and yields. After further investigation, we confirmed that both aerobic prokaryotic (Streptomyces coelicolor) and eukaryotic (Homo sapiens) FGEs contain a copper cofactor. The complete kinetic parameters for both forms of FGE are described, along with a proposed mechanism for FGE catalysis that accounts for the copper-dependent activity.Background:Aerobic formylglycine-generating enzyme (FGE) converts cysteine to formylglycine in vivo.Results:Purified FGE requires preactivation with copper to convert cysteine to formylglycine in vitro.Conclusion:FGE is a metalloenzyme. It is also a useful biocatalyst for the production of proteins that contain aldehyde tags.Significance:Understanding FGE biochemistry informs research on sulfatases and enables expanded biotechnology applications of the aldehyde tag. To further our aim of synthesizing aldehyde-tagged proteins for research and biotechnology applications, we developed methods for recombinant production of aerobic formylglycine-generating enzyme (FGE) in good yield. We then optimized the FGE biocatalytic reaction conditions for conversion of cysteine to formylglycine in aldehyde tags on intact monoclonal antibodies. During the development of these conditions, we discovered that pretreating FGE with copper(II) is required for high turnover rates and yields. After further investigation, we confirmed that both aerobic prokaryotic (Streptomyces coelicolor) and eukaryotic (Homo sapiens) FGEs contain a copper cofactor. The complete kinetic parameters for both forms of FGE are described, along with a proposed mechanism for FGE catalysis that accounts for the copper-dependent activity. Aerobic formylglycine-generating enzyme (FGE) converts cysteine to formylglycine in vivo. Purified FGE requires preactivation with copper to convert cysteine to formylglycine in vitro. FGE is a metalloenzyme. It is also a useful biocatalyst for the production of proteins that contain aldehyde tags.