Anaerobic Sulfatase-maturating Enzymes, First Dual Substrate Radical S-Adenosylmethionine Enzymes

Abstract

Sulfatases are a major group of enzymes involved in many critical physiological processes as reflected by their broad distribution in all three domains of life. This class of hydrolases is unique in requiring an essential post-translational modification of a critical active-site cysteine or serine residue to Cα-formylglycine. This modification is catalyzed by at least three nonhomologous enzymatic systems in bacteria. Each enzymatic system is currently considered to be dedicated to the modification of either cysteine or serine residues encoded in the sulfatase-active site and has been accordingly categorized as Cys-type and Ser-type sulfatase-maturating enzymes. We report here the first detailed characterization of two bacterial anaerobic sulfatase-maturating enzymes (anSMEs) that are physiologically responsible for either Cys-type or Ser-type sulfatase maturation. The activity of both enzymes was investigated in vivo and in vitro using synthetic substrates and the successful purification of both enzymes facilitated the first biochemical and spectroscopic characterization of this class of enzyme. We demonstrate that reconstituted anSMEs are radical S-adenosyl-l-methionine enzymes containing a redox active [4Fe-4S]2+,+ cluster that initiates the radical reaction by binding and reductively cleaving S-adenosyl-l-methionine to yield 5 ′-deoxyadenosine and methionine. Surprisingly, our results show that anSMEs are dual substrate enzymes able to oxidize both cysteine and serine residues to Cα-formylglycine. Taken together, the results support a radical modification mechanism that is initiated by hydrogen abstraction from a serine or cysteine residue located in an appropriate target sequence. Sulfatases are a major group of enzymes involved in many critical physiological processes as reflected by their broad distribution in all three domains of life. This class of hydrolases is unique in requiring an essential post-translational modification of a critical active-site cysteine or serine residue to Cα-formylglycine. This modification is catalyzed by at least three nonhomologous enzymatic systems in bacteria. Each enzymatic system is currently considered to be dedicated to the modification of either cysteine or serine residues encoded in the sulfatase-active site and has been accordingly categorized as Cys-type and Ser-type sulfatase-maturating enzymes. We report here the first detailed characterization of two bacterial anaerobic sulfatase-maturating enzymes (anSMEs) that are physiologically responsible for either Cys-type or Ser-type sulfatase maturation. The activity of both enzymes was investigated in vivo and in vitro using synthetic substrates and the successful purification of both enzymes facilitated the first biochemical and spectroscopic characterization of this class of enzyme. We demonstrate that reconstituted anSMEs are radical S-adenosyl-l-methionine enzymes containing a redox active [4Fe-4S]2+,+ cluster that initiates the radical reaction by binding and reductively cleaving S-adenosyl-l-methionine to yield 5 ′-deoxyadenosine and methionine. Surprisingly, our results show that anSMEs are dual substrate enzymes able to oxidize both cysteine and serine residues to Cα-formylglycine. Taken together, the results support a radical modification mechanism that is initiated by hydrogen abstraction from a serine or cysteine residue located in an appropriate target sequence. Bacterial sulfatases are widely distributed among the major bacterial phyla. Nevertheless, they have attracted only few studies compared with their eukaryotic counterparts (1Hanson S.R. Best M.D. Wong C.H. Angew. Chem. Int. Ed. Engl. 2004; 43: 5736-5763Crossref PubMed Scopus (287) Google Scholar). One of the difficulties in studying sulfatases is the requirement for post-translational modification of a critical active-site cysteine or serine residue to generate an active enzyme. Indeed, despite high homologies in the sulfatase domain (PFAM domain PF00884), sulfatases are divided into two sub-classes, Cys-type and Ser-type, according to the genetically encoded active site residue. In eukaryotes, only Cys-type sulfatases have been identified so far, whereas in bacteria, both types of sulfatases exist. Nevertheless, eukaryotic and prokaryotic sulfatases undergo the same post-translational modification involving oxidation of cysteine or serine to oxoalanine, also called Cα-formylglycine (FGly). 3The abbreviations used are: FGly, Cα-formylglycine; AdoMet, S-adenosylmethionine; anSME, anaerobic sulfatase-maturating enzyme; DNPH, 2,4-dinitrophenylhydrazine; Ni-NTA, nickel-nitrilotriacetic acid; HPLC, high pressure liquid chromatography; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; MS, mass spectrometry. To date, two distinct enzymatic systems have been identified for catalyzing this reaction. One is the formylglycine generating enzyme system, an oxygen-dependent oxidoreductase that has been extensively studied in humans and is functional only on Cys-type sulfatases (2Roeser D. Preusser-Kunze A. Schmidt B. Gasow K. Wittmann J.G. Dierks T. von Figura K. Rudolph M.G. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 81-86Crossref PubMed Scopus (84) Google Scholar). The other is AtsB, a putative member of the S-adenosyl-l-methionine (AdoMet)-dependent family of radical enzymes, which is responsible for maturation of Ser-type sulfatases and was first identified in Klebsiella aeruginosa (3Szameit C. Miech C. Balleininger M. Schmidt B. von Figura K. Dierks T. J. Biol. Chem. 1999; 274: 15375-15381Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 4Fang Q. Peng J. Dierks T. J. Biol. Chem. 2004; 279: 14570-14578Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Thus, the maturation of Cys-type sulfatases, the most abundant type, in anaerobic environments such as the human gut, has long remained unknown. We recently identified an enzyme related to the AtsB enzyme from K. aeruginosa in the obligate anaerobe Clostridum perfringens, which is surprisingly involved in Cys-type sulfatase maturation (5Berteau O. Guillot A. Benjdia A. Rabot S. J. Biol. Chem. 2006; 281: 22464-22470Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Although the K. aeruginosa enzyme, AtsB, appeared to be restricted to Ser-type sulfatases harboring a signal peptide (4Fang Q. Peng J. Dierks T. J. Biol. Chem. 2004; 279: 14570-14578Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 6Marquordt C. Fang Q. Will E. Peng J. von Figura K. Dierks T. J. Biol. Chem. 2003; 278: 2212-2218Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar), we grouped these enzymes into a family called anaerobic sulfatase-maturating enzymes (anSMEs) (5Berteau O. Guillot A. Benjdia A. Rabot S. J. Biol. Chem. 2006; 281: 22464-22470Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). These results raised the possibility that anSMEs could be divided into two subgroups that differ in specificity for sulfatase FGly generation via oxidation of an active-site cysteine or serine residue. Subsequent purification and characterization of C. perfringens anSME showed it to be a member of the radical-AdoMet superfamily of iron-sulfur enzymes (7Benjdia A. Leprince J. Guillot A. Vaudry H. Rabot S. Berteau O. J. Am. Chem. Soc. 2007; 129: 3462-3463Crossref PubMed Scopus (58) Google Scholar). All the members of this superfamily share a strictly conserved cysteine motif CX3CX2C, which coordinates an unconventional [4Fe-4S] cluster. The [4Fe-4S] cluster is directly involved in the reductive cleavage of AdoMet to generate a 5′-deoxyadenosyl radical, which then initiates various radical-based reactions according to the functional specificity of the enzyme. In addition to the CX3CX2C motif, all the anSMEs identified to date have two other strictly conserved cysteine clusters (Fig. 1), which could also be involved in the coordination of iron-sulfur centers. However, no experimental data have been produced to substantiate the existence of additional iron-sulfur clusters and, other than UV-visible absorption, no spectroscopic data have been reported for these enzymes. To get insights into this group of enzymes, we have investigated the in vitro and in vivo activity of bacterial anSMEs that are responsible for modification of either Cys-type or Ser-type sulfatases: anSMEcpe cloned from C. perfringens, which is responsible for maturation of a Cys-type sulfatase and anSMEbt cloned from Bacteroides thetaiotaomicron, a prominent gut symbiont, which possesses only Ser-type sulfatases. We report here the first detailed characterization of purified anSMEs and demonstrate that both enzymes can maturate cysteine and serine residues in vitro and in vivo. Furthermore, the ability to purify these unstable enzymes allowed us to provide the first spectroscopic characterization of their iron-sulfur centers. Chemicals—p-Nitrophenylsulfate was purchased from Sigma. Enzymes for from purchased from and from and of Bacterial and B. used in this was the was used for E. was used for enzyme The and used to the various was from The purification and from from and purified with the and system was by and of the and was in and the to using the The the putative anSME was by a using as a The site in to the at the of the and site to the was as in the of the was with the and of at at at by a at The was with and and then with into with the same enzymes. The of the cloned was to that no The was The containing the from C. perfringens has been reported (5Berteau O. Guillot A. Benjdia A. Rabot S. J. Biol. Chem. 2006; 281: 22464-22470Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). anSMEcpe and anSMEbt and with or then at in with was then used to with the same and bacterial at the The by and at in the by and at at for The was then a with The was extensively with the same of the by a with and and the was by containing the anSMEcpe or anSMEbt in with a of of on anSMEbt and was in a purified anSMEs with 5 and with a of both and at The was using a and the on by the using as a using of the in of C. perfringens or with anSMEbt or C. perfringens Cys-type sulfatase was by a using as a The site and in which to the of the and site to the of the was as in the of the was with the and of at at and at by a at The was with and and into site The was The anSMEbt or anSMEcpe cloned into by the and enzymes. Each was with The of the cloned to that no the The containing the C. perfringens sulfatase and anSMEbt or anSMEcpe and of the of C. perfringens Cys-type was on to cysteine into The cysteine to serine of the C. perfringens sulfatase was with the using the and a 1999; PubMed Scopus Google Scholar). to and in of the by the was The anSMEbt or anSMEcpe cloned into by the and enzymes and, a the and the critical residue in and by the on a using a peptide and the from the The synthetic purified by on a using a of at a of HPLC, on a showed that the of the was The purified by mass on a in the with as a containing reconstituted anSMEbt or anSMEcpe with and AdoMet in The reactions in an anaerobic and at for and divided in and was used to the maturation activity by mass whereas the other was in of and then the to the reductive cleavage of enzyme to the and AdoMet of AdoMet of AdoMet and a at with the a of a from to with was used to the was at on a with an by a using an with a with using and in by signal using the and and are to from a laser used for and using a of laser was and to was from the of a of using a anaerobic Scopus Google to the of an This to be to which and and with or to the Cys-type sulfatase or with the anSMEs also with or to the Ser-type sulfatases. The in an at in with The was then used to with the same and bacterial at the The by and at in the by and at at for The then with The extensively with the same two with and the by activity was using the substrate as at using in containing of was at at for sulfatases into then with in at 5 of sulfatase was with 5 of in in the at then and purified on mass was at in The was at in of and the target was then on a in the and a of B. reported the of the C. perfringens anaerobic sulfatase-maturating enzyme, anSMEcpe (5Berteau O. Guillot A. Benjdia A. Rabot S. J. Biol. Chem. 2006; 281: 22464-22470Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, A. Leprince J. Guillot A. Vaudry H. Rabot S. Berteau O. J. Am. Chem. Soc. 2007; 129: 3462-3463Crossref PubMed Scopus (58) Google Scholar). as a only enzyme with anSMEcpe and the three strictly conserved cysteine clusters was identified by the B. which only Ser-type sulfatases J. J. S. 2003; PubMed Scopus Google (Fig. This encoded by the as a and for Q. J. PubMed Google Scholar), was by and and of anSMEbt and anSMEcpe with an anSMEcpe and anSMEbt into the and the and used to E. for of anSMEbt and enzymes as and was to of the of from a as from which was with the of iron-sulfur and according to mass and for anSMEcpe and on (Fig. with and the iron-sulfur cluster as by the in at to the at in the UV-visible of the reconstituted anSMEs and both the with iron-sulfur The and of the iron-sulfur clusters in as and reconstituted anSMEcpe by and and the of UV-visible absorption, and and of with studies (7Benjdia A. Leprince J. Guillot A. Vaudry H. Rabot S. Berteau O. J. Am. Chem. Soc. 2007; 129: 3462-3463Crossref PubMed Scopus (58) Google Scholar), data for the anSMEcpe used for spectroscopic for purified and for reconstituted with and The and of UV-visible are with and cluster in the as and reconstituted anSMEcpe has at and and at and that are of clusters and at is the high of the with a cluster M.G. PubMed Scopus Google Scholar). anSMEcpe broad at and that are of a cluster and the at is the with a cluster C. J. PubMed Scopus Google Scholar). the of the iron-sulfur clusters in as and reconstituted anSMEcpe and for AdoMet binding to the cluster in the reconstituted enzyme was by (Fig. The of as anSMEcpe broad at and the possibility of an additional the at (Fig. and is of a cluster that is from of a cluster. have been in the as purified enzyme and in of to K. J. Am. Chem. Soc. Scopus Google Scholar, C. 2004; 43: PubMed Scopus Google Scholar). we that the cluster in the as anSMEcpe is or from of a radical-AdoMet cluster anSMEcpe has a that is by at and that are of a cluster C. 2004; 43: PubMed Scopus Google (Fig. with the cluster that is in the as enzyme are at they to the cluster as of clusters are than that of clusters with C. 2004; 43: PubMed Scopus Google Scholar). on and studies of a of radical-AdoMet enzymes with of clusters and their cluster we the cluster of reconstituted anSMEcpe to be clusters and the of the clusters in the as anSMEcpe have been by clusters anaerobic the clusters have been in the radical-AdoMet cluster binding as by in of reconstituted anSMEcpe on addition of AdoMet (Fig. studies have that AdoMet to the unique of the active site cluster in radical-AdoMet enzymes via the and E. D. J. J. Biol. Chem. and this is by in both in the the of the [4Fe-4S] and the of the J. Am. Chem. Soc. PubMed Scopus Google Scholar). These are at and in reconstituted anSMEcpe and to and on addition of was used to the and of the of anSMEcpe that by anaerobic with (Fig. The of reconstituted anSMEcpe of a cluster in a radical-AdoMet enzyme PubMed Scopus Google Scholar, S. C. E. J. A. T. M. M. J. Am. Chem. Soc. 1999; Scopus Google (Fig. system with a by the broad at in the and a for with and was on of the as enzyme (Fig. We this to clusters either by of clusters in the as or to [4Fe-4S] clusters that in the radical-AdoMet cluster binding site on reductive of the cluster. The cluster in the reconstituted enzyme with AdoMet as by of the signal to a with on addition of AdoMet (Fig. in the signal on addition of AdoMet have been with many radical-AdoMet enzymes S. E. Schmidt J. C. T. A. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. D. J. Am. Chem. Soc. PubMed Scopus Google Scholar, M. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). the radical-AdoMet active site can on addition of in the of AdoMet, it is to get the cluster in a Taken together, the spectroscopic and data are with the anSMEcpe investigated in this only clusters in the radical-AdoMet cluster binding site involving the conserved CX3CX2C have a [4Fe-4S]2+,+ cluster in this site that can with AdoMet in both the and In the [4Fe-4S]2+,+ cluster to an cluster that is and on Nevertheless, be to the of a iron-sulfur cluster. In of anSMEbt and and reconstituted anSMEs in vitro anaerobic for their ability to AdoMet and to two the Cys-type and Ser-type sulfatases. Each reconstituted was with AdoMet in the or of using as and, in was by In the of anSMEcpe and anSMEbt (Fig. a anSMEcpe was able to only of whereas anSMEbt produced In both enzymes in the of enzymes to the of the first residue in the motif, of in the of the peptide for anSMEcpe and for (Fig. compared with the peptide and (Fig. Furthermore, of the cysteine residue by in of AdoMet cleavage for both enzymes and for In MS, peptide maturation was both by in a and with J. Schmidt B. von Figura K. Dierks T. J. 2003; PubMed Scopus Google Scholar). anSMEcpe the cysteine of peptide as by the of a peptide with an mass (Fig. The of the modification was using DNPH, which with the FGly to a with a mass (Fig. and the with the we to directly a peptide that have a from the peptide (Fig. Nevertheless, with allowed us to a with the mass for the of the peptide (Fig. and only a mass also peptide ionization that is then To demonstrate the of an from the the from the purified using and the by In our the peptide at as by and in a we able to the peptide as by mass (Fig. with the peptide to peptide despite the of serine residues the peptide (Fig. and on the with B. enzyme results and anSMEbt and anSMEcpe active on cysteine and serine Nevertheless, the the of maturation. the peptide than the and the to peptide maturation using the peptide to be We an in vivo to anSME maturation on both In of C. perfringens Cys-type used the Cys-type sulfatase from C. perfringens as a and three containing the Cys-type sulfatase into or with anSMEbt or anSMEcpe into These and these we three the Cys-type sulfatase as a or with anSMEbt or These anaerobic as our have that E. Cys-type sulfatases this maturation was anaerobic A. Rabot S. Berteau O. 2007; PubMed Scopus Google Scholar). with the sulfatases purified and their maturation by mass spectrometry. The of cysteine into FGly is to in a mass of which could be in the of the sulfatases (7Benjdia A. Leprince J. Guillot A. Vaudry H. Rabot S. Berteau O. J. Am. Chem. Soc. 2007; 129: 3462-3463Crossref PubMed Scopus (58) Google Scholar). We also the activity of the purified sulfatases using the substrate as the sulfatase activity is directly to the maturation in the Cys-type sulfatase was anaerobic peptide was containing the critical and residues from to mass This peptide with an cysteine residue to the sulfatase was with anSMEcpe (Fig. the peptide and was by a peptide with a mass of This peptide mass to Surprisingly, we the Cys-type sulfatase with the of sulfatase (Fig. The sulfatase activity of enzyme the results by (Fig. in the sulfatase activity was the Cys-type sulfatase or in the of whereas anSMEcpe the sulfatase activity from to The sulfatase activity in the of maturation enzyme was to an of the E. sulfatase maturation system as maturation could be in all the of the of C. perfringens Cys-type the activity of both anSMEs the serine we the C. perfringens Cys-type sulfatase into a Ser-type sulfatase by We three harboring the and These three also anaerobic (Fig. results The of purified sulfatases, the of the into as a peptide was with the (Fig. in the in vitro the ionization of this peptide from of the cysteine and a this peptide the and the of maturation by The sulfatase activity of the serine in E. was (Fig. 1), with on Ser-type sulfatases (3Szameit C. Miech C. Balleininger M. Schmidt B. von Figura K. Dierks T. J. Biol. Chem. 1999; 274: 15375-15381Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, J. PubMed Scopus Google Scholar). This that the E. sulfatase maturation system is restricted to the Cys-type sulfatases A. Rabot S. Berteau O. 2007; PubMed Scopus Google Scholar). Nevertheless, the serine was with anSMEcpe or we sulfatase activity which was than in the anSMEbt and anSMEcpe are able to Ser-type sulfatases with an than on Cys-type Sulfatases are widely distributed in and (1Hanson S.R. Best M.D. Wong C.H. Angew. Chem. Int. Ed. Engl. 2004; 43: 5736-5763Crossref PubMed Scopus (287) Google to their and their high substrate Sulfatases are involved in the of various from to such as and (1Hanson S.R. Best M.D. Wong C.H. Angew. Chem. Int. Ed. Engl. 2004; 43: 5736-5763Crossref PubMed Scopus (287) Google Scholar). are considered to be restricted to they also have other that to be sulfatase are identified in many bacterial and from from and gut environments J. J. S. 2003; PubMed Scopus Google Scholar, S.R. M. 2006; PubMed Scopus Google Scholar, M. M. H. T. D. A. K. K. H. Proc. Natl. Acad. Sci. U. S. A. 2003; PubMed Scopus Google Scholar, J. M. B. H. A. K. Biol. 2007; PubMed Scopus Google Scholar). their functional is sulfatase maturation. it was that an enzymatic system, formylglycine generating enzyme, was dedicated to the maturation of Cys-type sulfatases T. Schmidt B. Peng A. M. von Figura K. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, S. M. A. 2003; Full Text Full Text PDF PubMed Scopus Google and AtsB, to the Ser-type sulfatases (3Szameit C. Miech C. Balleininger M. Schmidt B. von Figura K. Dierks T. J. Biol. Chem. 1999; 274: 15375-15381Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). These on The specificity of formylglycine generating enzyme is on that eukaryotic are to maturate a serine sulfatase M. T. Dierks T. Schmidt B. von Figura K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In the data are for with a serine sulfatase from the Cys-type sulfatase of aeruginosa to an active enzyme. Nevertheless, an active sulfatase was this serine a signal peptide in addition of the critical serine residue C. Fang Q. Will E. Peng J. von Figura K. Dierks T. J. Biol. Chem. 2003; 278: 2212-2218Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). This to the that AtsB only Ser-type sulfatases with the of a signal peptide (4Fang Q. Peng J. Dierks T. J. Biol. Chem. 2004; 279: 14570-14578Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 6Marquordt C. Fang Q. Will E. Peng J. von Figura K. Dierks T. J. Biol. Chem. 2003; 278: 2212-2218Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). We recently that the C. perfringens AtsB is able to cysteine residue into In of the homologies the enzyme from K. and C. perfringens, these enzymes the of anSME (5Berteau O. Guillot A. Benjdia A. Rabot S. J. Biol. Chem. 2006; 281: 22464-22470Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). This raised the possibility of enzymes with substrate the anSME enzymes dedicated to maturation of Ser-type sulfatases and to the Cys-type sulfatases. To this we to the specificity of the anSMEs from C. perfringens and B. thetaiotaomicron, a prominent gut J. J. S. 2003; PubMed Scopus Google Scholar), which possesses only Ser-type sulfatases and only enzyme related to the anSME The B. anSME is currently called and has been as a as the to various substrates Q. J. PubMed Google Scholar). We cloned this enzyme and it We both enzymes and in and in the of the of anSMEcpe to characterization of the iron-sulfur in and reconstituted using the of UV-visible absorption, and The results that anaerobic a [4Fe-4S]2+,+ cluster in the radical-AdoMet cluster binding site that is able to AdoMet in both oxidation The enzyme of a reductively cluster with to for clusters that as a of of radical-AdoMet active-site it that the active-site cluster in anSME is in and to yield an cluster in the radical-AdoMet cluster binding site Although the spectroscopic and results in this can be in of a or cluster by the radical-AdoMet CX3CX2C motif, it is to the possibility that or both of the two other cysteine clusters in anSMEs (Fig. are involved in iron-sulfur cluster coordination as (4Fang Q. Peng J. Dierks T. J. Biol. Chem. 2004; 279: 14570-14578Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). the cysteine cluster is also strictly conserved in the AdoMet radical enzyme involved in K. T. M. S. D. K. J. Biol. Chem. 2006; 281: Full Text Full Text PDF PubMed Scopus Google Scholar). studies with spectroscopic be to the of these cysteine The in vitro with both enzymes that anSMEs able to from Furthermore, in the of the activity of both enzymes was and, by the first in the target sulfatase motif a cysteine or serine residue was both enzymes produced from AdoMet at according to the target residue. However, this residue was with the was a of the anSME of the showed that the serine residue of the peptide and the cysteine residue of the peptide both into FGly by anSMEbt and This that both enzymes able to oxidize serine or cysteine residues to Furthermore, our results also that the enzyme encoded by the is a a radical-AdoMet enzyme involved in sulfatase maturation. We also showed that an peptide which in was by either enzyme the specificity of anSMEs for the sulfatase target sequence. Nevertheless, all critical peptide that the of these enzymes are and are currently these in vitro we able to the maturation of both enzymes the cysteine and serine We an in vivo to their activity on cysteine and serine the C. perfringens sulfatase and serine we the in vivo catalyzed by anSMEbt and enzymes able to the Ser-type sulfatase with sulfatase activity to Nevertheless, only anSMEcpe the Cys-type sulfatase This that despite dual substrate enzymes, substrate These in vivo also that to (4Fang Q. Peng J. Dierks T. J. Biol. Chem. 2004; 279: 14570-14578Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 6Marquordt C. Fang Q. Will E. Peng J. von Figura K. Dierks T. J. Biol. Chem. 2003; 278: 2212-2218Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar), able to maturate Ser-type sulfatases the of a signal In our data support the that anSMEs are dual substrate radical-AdoMet enzymes than a class of enzymes divided into two subgroups dedicated to the maturation of either Ser-type or Cys-type sulfatases. with our experimental we the first mechanism for this class of enzymes (Fig. The cluster in anSME with AdoMet and reductively AdoMet to yield and a 5′-deoxyadenosyl radical which a from either a cysteine or serine residue to the of and to the of In this it is to AdoMet is a as is the for the of radical-AdoMet enzymes, or a as in the of or Sci. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar, J. J. Am. Chem. Soc. PubMed Scopus Google Scholar, M. M. E. Sci. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). be to the mechanism of this class of radical-AdoMet enzymes. We E. for at the with