Anaerobic sulfatase-maturating enzyme - A mechanistic link with glycyl radical-activating enzymes?

Sulfatases form a major group of enzymes present in prokaryotes and eukaryotes. This class of hydrolases is unique in requiring essential post-translational modification of a critical active-site cysteinyl or seryl residue to C-alpha-formylglycine (FGly). Herein, we report mechanistic investigations of a unique class of radical-S-adenosyl-L-methionine (AdoMet) enzymes, namely anaerobic sulfatase-maturating enzymes (anSMEs), which catalyze the oxidation of Cys-type and Ser-type sulfatases and possess three [4Fe-4S]2+,+ clusters. We were able to develop a reliable quantitative enzymatic assay that allowed the direct measurement of FGly production and AdoMet cleavage. The results demonstrate stoichiometric coupling of AdoMet cleavage and FGly formation using peptide substrates with cysteinyl or seryl active-site residues. Analytical and EPR studies of the reconstituted wild-type enzyme and cysteinyl cluster mutants indicate the presence of three almost isopotential [4Fe-4S]2+,+ clusters, each of which is required for the generation of FGly in vitro. More surprisingly, our data indicate that the two additional [4Fe-4S]2+,+ clusters are required to obtain efficient reductive cleavage of AdoMet, suggesting their involvement in the reduction of the radical AdoMet [4Fe-4S]2+,+ center. These results, in addition to the recent demonstration of direct abstraction by anSMEs of the C-beta H-atom from the sulfatase active-site cysteinyl or seryl residue using a 5'-deoxyadenosyl radical, provide new insights into the mechanism of this new class of radical-AdoMet enzymes.