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Further characterization of Cys-type and Ser-type anaerobic sulfatase maturating enzymes suggests a commonality in the mechanism of catalysis.

ABSTRACT: The anaerobic sulfatase-maturating enzyme from Clostridium perfringens (anSMEcpe) catalyzes the two-electron oxidation of a cysteinyl residue on a cognate protein to a formylglycyl residue (FGly) using a mechanism that involves organic radicals. The FGly residue plays a unique role as a cofactor in a class of enzymes termed arylsulfatases, which catalyze the hydrolysis of various organosulfate monoesters. anSMEcpe has been shown to be a member of the radical S-adenosylmethionine (SAM) family of enzymes, [4Fe-4S] cluster-requiring proteins that use a 5'-deoxyadenosyl 5'-radical (5'-dA(•)) generated from a reductive cleavage of SAM to initiate radical-based catalysis. Herein, we show that anSMEcpe contains in addition to the [4Fe-4S] cluster harbored by all radical SAM (RS) enzymes, two additional [4Fe-4S] clusters, similar to the radical SAM protein AtsB, which catalyzes the two-electron oxidation of a seryl residue to a FGly residue. We show by size-exclusion chromatography that both AtsB and anSMEcpe are monomeric proteins, and site-directed mutagenesis studies of AtsB reveal that individual Cys ? Ala substitutions at seven conserved positions result in an insoluble protein, consistent with those residues acting as ligands to the two additional [4Fe-4S] clusters. Ala substitutions at an additional conserved Cys residue (C291 in AtsB and C276 in anSMEcpe) afford proteins that display intermediate behavior. These proteins exhibit reduced solubility and drastically reduced activity, behavior that is conspicuously similar to that of a critical Cys residue in BtrN, another radical SAM dehydrogenase [Grove, T. L., et al. (2010) Biochemistry 49, 3783-3785]. We also show that wild-type anSMEcpe acts on peptides containing other oxidizable amino acids at the target position. Moreover, we show that the enzyme will convert threonyl peptides to the corresponding ketone product, and also allo-threonyl peptides, but with a significantly reduced efficiency, suggesting that the pro-S hydrogen atom of the normal cysteinyl substrate is stereoselectively removed during turnover. Lastly, we show that the electron generated during catalysis by AtsB and anSMEcpe can be utilized for multiple turnovers, albeit through a reduced flavodoxin-mediated pathway.

SUBMITTER: Grove TL

PROVIDER: S-EPMC3897223 | biostudies-literature | 2013 Apr

REPOSITORIES: biostudies-literature

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Further characterization of Cys-type and Ser-type anaerobic sulfatase maturating enzymes suggests a commonality in the mechanism of catalysis.

Grove Tyler L TL Ahlum Jessica H JH Qin Rosie M RM Lanz Nicholas D ND Radle Matthew I MI Krebs Carsten C Booker Squire J SJ

Biochemistry 20130416 17

The anaerobic sulfatase-maturating enzyme from Clostridium perfringens (anSMEcpe) catalyzes the two-electron oxidation of a cysteinyl residue on a cognate protein to a formylglycyl residue (FGly) using a mechanism that involves organic radicals. The FGly residue plays a unique role as a cofactor in a class of enzymes termed arylsulfatases, which catalyze the hydrolysis of various organosulfate monoesters. anSMEcpe has been shown to be a member of the radical S-adenosylmethionine (SAM) family of ...[more]

PMID: 23477283

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Further characterization of Cys-type and Ser-type anaerobic sulfatase maturating enzymes suggests a commonality in the mechanism of catalysis.

Publications

Further characterization of Cys-type and Ser-type anaerobic sulfatase maturating enzymes suggests a commonality in the mechanism of catalysis.

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