A New Type of Bacterial Sulfatase Reveals a Novel Maturation Pathway in Prokaryotes

Olivier Berteau,Alain Guillot,Alhosna Benjdia,Sylvie Rabot

doi:10.1074/jbc.m602504200

Abstract

Sulfatases are a highly conserved family of enzymes found in all three domains of life. To be active, sulfatases undergo a unique post-translational modification leading to the conversion of either a critical cysteine ("Cys-type" sulfatases) or a serine ("Ser-type" sulfatases) into a Calpha-formylglycine (FGly). This conversion depends on a strictly conserved sequence called "sulfatase signature" (C/S)XPXR. In a search for new enzymes from the human microbiota, we identified the first sulfatase from Firmicutes. Matrix-assisted laser desorption ionization time-of-flight analysis revealed that this enzyme undergoes conversion of its critical cysteine residue into FGly, even though it has a modified (C/S)XAXR sulfatase signature. Examination of the bacterial and archaeal genomes sequenced to date has identified many genes bearing this new motif, suggesting that the definition of the sulfatase signature should be expanded. Furthermore, we have also identified a new Cys-type sulfatase-maturating enzyme that catalyzes the conversion of cysteine into FGly, in anaerobic conditions, whereas the only enzyme reported so far to be able to catalyze this reaction is oxygen-dependent. The new enzyme belongs to the radical S-adenosyl-l-methionine enzyme superfamily and is related to the Ser-type sulfatase-maturating enzymes. This finding leads to the definition of a new enzyme family of sulfatase-maturating enzymes that we have named anSME (anaerobic sulfatase-maturating enzyme). This family includes enzymes able to maturate Cys-type as well as Ser-type sulfatases in anaerobic conditions. In conclusion, our results lead to a new scheme for the biochemistry of sulfatases maturation and suggest that the number of genes and bacterial species encoding sulfatase enzymes is currently underestimated.

Highlights

At neutral pH, the extract exhibited a weak sulfatase activity of 0.25 nmol1⁄7minϪ11⁄7mgϪ1, which almost vanished when we grew C. perfringens in presence of 50 mM ammonium sulfate. This result suggested that C. perfringens possesses at least one sulfatase enzyme regulated by the sulfate content of the medium
A search for genes annotated as sulfatases in the C. perfringens genome did not show any obvious candidates [15], whereas a blast search with sulfatase sequences led to the identification of one gene, cpe0231, annotated as a “probable phosphonate monoester hydrolase” [21, 22]
All the sequenced sulfatases studied to date share a consensus motif (C/S)XPXR, the protein encoded by the cpe0231 gene was predicted to be devoid of the strictly conserved proline (Fig. 1)

Summary

EXPERIMENTAL PROCEDURES

Cloning and Construction of the pET-6His-CPE0231 Overexpressing Plasmid—C. perfringens ATCC 13124 was grown anaerobically in BHI medium, pH 7.0, and the cells were harvested to extract the genomic DNA using the Wizard genomic kit from Promega. The PCR product was digested with NdeI and XhoI and ligated with T4 DNA ligase into pET28(a) plasmid digested previously with the same restriction enzymes. The PCR product was digested with NdeI and XhoI and ligated with T4 DNA ligase into pRSF plasmid digested previously with the same restriction enzymes. The entire sequence of the cloned gene was sequenced to ensure that no errors were introduced during the PCR, and the plasmid was named pRSF-CPE0635. The phylogenetic tree was drawn by using the neighbor-joining method [20] with the Kimura two-parameter calculation model

RESULTS

DISCUSSION

Until now it was unclear why