In Vitro Characterization of AtsB, a Radical SAM Formylglycine-Generating Enzyme That Contains Three [4Fe-4S] Clusters

Abstract

Sulfatases catalyze the cleavage of a variety of cellular sulfate esters via a novel mechanism that requires the action of a protein-derived formylglycine cofactor. Formation of the cofactor is catalyzed by an accessory protein and involves the two-electron oxidation of a specific cysteinyl or seryl residue on the relevant sulfatase. Although some sulfatases undergo maturation via mechanisms in which oxygen serves as an electron acceptor, AtsB, the maturase from Klebsiella pneumoniae, catalyzes the oxidation of Ser72 on AtsA, its cognate sulfatase, via an oxygen-independent mechanism. Moreover, it does not make use of pyridine or flavin nucleotide cofactors as direct electron acceptors. In fact, AtsB has been shown to be a member of the radical S-adenosylmethionine superfamily of proteins, suggesting that it catalyzes this oxidation via an intermediate 5′-deoxyadenosyl 5′-radical that is generated by a reductive cleavage of S-adenosyl-L-methionine. In contrast to AtsA, very little in vitro characterization of AtsB has been conducted. Herein we show that coexpression of the K. pneumoniae atsB gene with a plasmid that encodes genes that are known to be involved in ironsulfur cluster biosynthesis yields soluble protein that can be characterized in vitro. The as-isolated protein contained 8.7 ± 0.4 irons and 12.2 ± 2.6 sulfides per polypeptide, which existed almost entirely in the [4Fe-4S] 2+ configuration, as determined by Mössbauer spectroscopy, suggesting that it contained at least two of these clusters per polypeptide. Reconstitution of the as-isolated protein with additional iron and sulfide indicated the presence of 12.3 ± 0.2 irons and 9.9 ± 0.4 sulfides per polypeptide. Subsequent characterization of the reconstituted protein by Mössbauer spectroscopy indicated the presence of only [4Fe-4S] clusters, suggesting that reconstituted AtsB contains three per polypeptide. Consistent with this stoichiometry, an as-isolated AtsB triple variant containing Cys → Ala substitutions at each of the cysteines in its CX 3 CX 2 C radical SAM motif contained 7.3 ± 0.1 irons and 7.2 ± 0.2 sulfides per polypeptide while the reconstituted triple variant contained 7.7 ± 0.1 irons and 8.4 ± 0.4 sulfides per polypeptide, indicating that it was unable to incorporate an additional cluster. UV-visible and Mössbauer spectra of both samples indicated the presence of only clusters. AtsB was capable of catalyzing multiple turnovers and exhibited a V max /[E T ] of ~0.36 min −1 for an 18-amino acid peptide substrate using dithionite to supply the requisite electron and a value of ~0.039 min −1 for the same substrate using the physiologically relevant flavodoxin reducing system. Simultaneous quantification of formylglycine and 5′-deoxyadenosine as a function of time indicates an approximate 1:1 stoichiometry. Use of a peptide substrate in which the target serine is † This work was supported by NIH Grant GM-63847 (S.J.B.), the Dreyfus Foundation (Teacher Scholar Award to C.K.), and the Beckman Foundation (Young Investigator Award to...

X-ray structure of an AdoMet radical activase reveals an anaerobic solution for formylglycine posttranslational modification

“…The maturation of these sulfatases involves two classes of enzymes, one that requires molecular oxygen and another that can function in its absence. FGly generating enzymes (FGEs), found in eukaryotes or aerobically living prokaryotes, generate FGly by oxidizing a cysteine residue on the target sulfatase using molecular oxygen (6,7), whereas anaerobic sulfatase maturating enzymes (anSMEs) generate FGly from either cysteine or serine residues on their target sulfatases using S-adenosyl-L-methionine (AdoMet) radical chemistry (8)(9)(10)(11). In addition to their importance for sulfatase chemistry, FGEs have commercial applications for generating site-specific "aldehyde tags" to use in proteinlabeling technology (12).…”

“…Differentiation among the family members is a result of the action of this substrate radical. In anSMEs, the removal of a proton and an electron from the radical intermediate completes catalysis (10,17) (Fig. 1).…”

“…Interestingly, both enzymes harbor additional [4Fe-4S] clusters that are necessary for turnover (20). In the case of BtrN, one auxiliary cluster has been identified (21), while anSMEs have two (10,17). For anSME, the sequence surrounding these two clusters, including a previously identified 7-cysteine motif (CX 9-15 GX 4 C-gap-CX 2 CX 5 CX 3 C-gap-C) (17), places it in a ∼1,400-membered AdoMet radical subfamily that was recently described by Haft and Basu through bioinformatic analysis and thought to function in the modification of ribosomally translated peptides (22).…”

“…This subfamily has been designated TIGR04085 and named SPASM for its biochemically characterized founding members AlbA, PqqE, anSMEs, and MtfC, which are involved in subtilosin A, pyrroloquinoline quinone, anaerobic sulfatase, and mycofactocin maturation, respectively (22,23). While the function of these auxiliary clusters is unknown, the 7-cysteine motif prompted speculation that members of the SPASM subfamily, including anSMEs, use an available ligation site on one of the [4Fe-4S] clusters for substrate binding (10). Direct binding of substrate to an auxiliary [4Fe-4S] cluster has been observed in the molybdenum cofactor biosynthetic enzyme MoaA, another AdoMet radical enzyme (24,25).…”

EPR-kinetic isotope effect study of the mechanism of radical-mediated dehydrogenation of an alcohol by the radical SAM enzyme DesII

“…In addition to the primary [4Fe-4S] 1+ cluster, which reduces SAM (1) to methionine (2) and the 5′-deoxyadenosyl radical (17; see Fig. 4) to initiate radical catalysis, BtrN (13,14) and anSMEs (15)(16)(17)(18) possess one or more auxiliary [4Fe-4S] aux clusters, which have been proposed to coordinate the hydroxyl group to be oxidized in the substrate.…”