The Iron Sulfur Protein AtsB Is Required for Posttranslational Formation of Formylglycine in the Klebsiella Sulfatase

Abstract

The catalytic residue of eukaryotic and prokaryotic sulfatases is a ␣-formylglycine. In the sulfatase of Klebsiella pneumoniae the formylglycine is generated by posttranslational oxidation of serine 72. We cloned the atsBA operon of K. pneumoniae and found that the sulfatase was expressed in inactive form in Escherichia coli transformed with the structural gene (atsA). Coexpression of the atsB gene, however, led to production of high sulfatase activity, indicating that the atsB gene product plays a posttranslational role that is essential for the sulfatase to gain its catalytic activity. This was verified after purification of the sulfatase from the periplasm of the cells. Peptide analysis of the protein expressed in the presence of AtsB revealed that half of the polypeptides carried the formylglycine at position 72, while the remaining polypeptides carried the encoded serine. The inactive sulfatase expressed in the absence of AtsB carried exclusively serine 72, demonstrating that the atsB gene is required for formylglycine modification. This gene encodes a 395-amino acid residue iron sulfur protein that has a cytosolic localization and is supposed to directly or indirectly catalyze the oxidation of the serine to formylglycine.

Crystal Structure of Quinohemoprotein Amine Dehydrogenase from Pseudomonas putida

“…However, sequencing of the gene cluster encoding QH-AmDH of P. putida has already provided a clue for how the biogenesis could take place (10). In the stretch containing the structural genes for the three subunits, a hypothetical 53-kDa protein was found whose sequence is homologous to that of "sulfatase-activating enzyme," containing an iron/sulfur cluster and participating in the oxidative formation of the formylglycine cofactor in the active site of sulfatase (33). By analogy, the hypothetical 53-kDa protein could play a role in the post-translational formation of CTQ and/or the thioether bonds in the proenzyme of QH-AmDH.…”

Structure of a quinohemoprotein amine dehydrogenase with an uncommon redox cofactor and highly unusual crosslinking

“…Moreover, the genetic locus of Klebsiella aslB, separated by only 52 nt 5Ј-upstream from the structural gene for sulfatase (aslA) contained in the same Asl operon, is also very similar to that of ORF2 relative to the ␥ subunit gene (ORF3), which is separated by 83 nt. It has been speculated that the AslB protein, which lacks a signal peptide, oxidizes the critical serine of the unfolded sulfatase during or shortly after synthesis and that, after cofactor formation the sulfatase polypeptide with a signal peptide, is translocated into the periplasm (34). Interestingly, neither the ORF2 nor the ORF3 gene codes for signal peptides, suggesting that the interaction of the Asl-like protein and the ␥ subunit occurs within the cytoplasm before the oxidized ␥ subunit associates with the periplasmic ␣ and ␤ subunits.…”

“…A likely candidate is the AslB-like [FeOS] protein encoded in ORF2 of the enzyme genes (Fig. 5), which may oxidize the tryptophan residue (Trp-43␥) to Trq in the ␥ subunit polypeptide, analogous to the Radical SAM AslB protein oxidizing a Cys or Ser residue to formylglycine in sulfatases (34). Although there is no precedent for a Radical SAM protein to be involved in the biogenesis of protein quinone cofactors, the PqqE protein functioning in PQQ biosynthesis has been recognized as a Radical SAM protein (33).…”

“…All these proteins are included in a recently described protein superfamily, named ''Radical SAM'' (S-adenosylmethionine) proteins, catalyzing diverse reactions, such as unusual methylations, isomerization, sulfur insertion, ring formation, anaerobic oxidation, and protein radical formation (33). AslB is required for the oxidation of a specific serine or cysteine residue in sulfatases to posttranslationally generate a formylglycine (2-oxoalanine) cofactor (34), hence designated as an arylsulfatase-activating enzyme. The Cys-rich region predicted to serve as a binding motif for the [FeOS] cluster and a Gly-rich sequence likely involved in binding of SAM (33) are both conserved in the ORF2 protein (Fig.…”

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.…”