Eukaryotic formylglycine‐generating enzyme catalyses a monooxygenase type of reaction

Abstract

C a-formylglycine (FGly) is the catalytic residue of sulfatases in eukaryotes. It is generated by a unique post-translational modification catalysed by the FGly-generating enzyme (FGE) in the endoplasmic reticulum. FGE oxidizes a cysteine residue within the conserved CxPxR sequence motif of nascent sulfatase polypeptides to FGly. Here we show that this oxidation is strictly dependent on molecular oxygen (O 2 ) and consumes 1 mol O 2 per mol FGly formed. For maximal activity FGE requires an O 2 concentration of 9% (105 lM). Sustained FGE activity further requires the presence of a thiol-based reductant such as DTT. FGly is also formed in the absence of DTT, but its formation ceases rapidly. Thus inactivated FGE accumulates in which the cysteine pair Cys336/Cys341 in the catalytic site is oxidized to form disulfide bridges between either Cys336 and Cys341 or Cys341 and the CxPxR cysteine of the sulfatase. These results strongly suggest that the Cys336/Cys341 pair is directly involved in the O 2 -dependent conversion of the CxPxR cysteine to FGly. The available data characterize eukaryotic FGE as a monooxygenase, in which Cys336/Cys341 disulfide bridge formation donates the electrons required to reduce one oxygen atom of O 2 to water while the other oxygen atom oxidizes the CxPxR cysteine to FGly. Regeneration of a reduced Cys336/Cys341 pair is accomplished in vivo by a yet unknown reductant of the endoplasmic reticulum or in vitro by DTT. Remarkably, this monooxygenase reaction utilizes O 2 without involvement of any activating cofactor.

Copper is a Cofactor of the Formylglycine‐Generating Enzyme

“…The combination of catalytic and structural analyses of FGE described here and elsewhere– strongly implicates FGE as a copper‐metalloenzyme: the active form strongly binds one equivalent of Cu I in the active site; the Cu I :protein complex remains intact throughout the entire catalytic cycle (Figure ); other transition metals cannot complement FGE, thus suggesting that the cofactor engages in redox chemistry; FGE reduces O 2 by using two electrons from the substrate and two electrons from an auxiliary reducing agent such as DTT; in the presence of DTT the rate‐limiting step is hydrogen‐atom abstraction from the substrate; the accumulating species during catalysis is an EPR‐silent species (Figure ); in the absence of an appropriate reducing agent turnover is much slower, and a Cu II ‐containing species accumulates (Figure ); this oxidized species can slowly turn over by using the substrate thiol as an electron source; and finally, addition of a proper reducing agent to this slow reaction immediately reactivates the enzyme …”

Two‐fold Bioorthogonal Derivatization by Different Formylglycine‐Generating Enzymes

“…[17] This recognition sequence can be introduced into recombinant proteins and converted by FGE in vivo or in vitro. [19][20][21][22] In addition, the iron-sulfur protein AtsB of the radical S-adenosyl methionine (radical-SAM) protein superfamily is another FGly-generating system that is exclusively found in prokaryotes. [19][20][21][22] In addition, the iron-sulfur protein AtsB of the radical S-adenosyl methionine (radical-SAM) protein superfamily is another FGly-generating system that is exclusively found in prokaryotes.…”

Dealing with oxygen using bare hands

“…Peng et al . correctly point out that their data leave this problem open for further investigation. X‐ray crystallographic studies on human FGE have shown that, upon aging, the crystalline enzyme oxidizes so that the peroxysulfenic form of the catalytic Cys336 (Cys‐S‐O‐O − ) accumulates.…”

“…In this issue of FEBS Journal , Peng et al . report interesting experimental data that provide insight into the unusual and intriguing FGE‐catalyzed reaction. Their starting point is that FGE enzymatic activity is known to depend on molecular oxygen as a co‐substrate as well as on two essential catalytic residues, Cys336 and Cys341 (with reference to the human FGE sequence).…”

“…Peng et al . demonstrated that the reaction also requires an electron donor as additional substrate. Moreover, they confirmed that no additional cofactors (such as metals) are indispensable for the reaction, i.e.…”