Identification and characterization of a novel copper dependent enzyme

Abstract

The Formylglycine generating enzyme (FGE) catalyses the oxygen dependent oxidation of a highly conserved cysteine residue in sulfatases and phosphatases to formylglycine (fGly). In sulfatases the hydrated form of fGly serves as the catalytic residue to cleave sulfate esters of their substrates. After the first identification of FGE as the key enzyme in sulfatase activation, it has been quickly associated with multiple sulfatase deficiency (MSD), a rare but fatal lysosomal storage disease. Various mutations in the FGE encoding SUMF1 gene have been identified in patients suffering from MSD. Alongside the medicinal importance, FGE has gained significant attention due to the fact, that it has been identified as a cofactor-independent oxidase with an unkown mechanism of oxygen activation. The absence of metals and cofactors in published crystal structures and enzyme preperations raised the question of how oxygen activation can occur. We hereby show that FGE is a copper dependent oxidase. Recombinant FGE from Thermomonospora curvata contains a disulfide bond but is readily reduced in the presence of reducing agents such as DTT or cysteamine. Reduced FGE shows a near atto molar affinity to copper(I) and binds copper throughout multiple turnovers. Copper binds to two highly conserved cysteine residues in the active site of FGE where it is involved in oxygen activation. While copper can be replaced by other metals such as silver, only copper facilitates the reaction, which supports its participation in redox chemistry. Furthermore, we describe the dependence of FGE on a peptidyl reducing agent containing a thioredoxin like CxC amino acid motif. FGE from Homo sapiens is linked to an N-terminal domain bearing the CxC sequence. This peptide tag is believed to be involved in retention of FGE to the ER. We additionally suggest that this domain serves as the immediate reducing agent of FGE and is then reduced by other pathways. Sequence similarities of this domain to the substrate polypeptide indicate that the N-terminal domain binds to the substrate binding groove of FGE. This suggests a ping-pong mechanism of product formation and subsequent reduction of the oxidized enzyme intermediate. In addition to the mechanistic interest, FGE has been used as a tool to site-specifically introduce aldehyde functionalities into recombinant proteins for further bioconjugation. We now identify a set of five mutations which increases the catalytic efficiency of FGE by 38 fold. The resulting variant completely oxidizes a specific cysteine residue in a target protein within 90 min with only 0.3 % of catalyst loading at room temperature. This high reactivity is maintained even when the recognition motif is installed into an internal loop region of the target protein. We envision that these findings will not only drive the mechanistic discussion about the previously mysterious oxygen activation in FGE but will also support the usage of fGly in bioconjugation research.