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

Tyler L. Grove, Jessica H. Ahlum, Rosie M. Qin, Nicholas D. Lanz, Matthew I. Radle, Carsten Krebs, Squire J. Booker

Research output: Contribution to journalArticle

30 Citations (Scopus)

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) Biochemistry49, 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.

Original languageEnglish (US)
Pages (from-to)2874-2887
Number of pages14
JournalBiochemistry
Volume52
Issue number17
DOIs
StatePublished - Apr 30 2013

Fingerprint

Sulfatases
Catalysis
Clostridium
Clostridium perfringens
S-Adenosylmethionine
Enzymes
Proteins
Electrons
Peptides
Substitution reactions
Flavodoxin
Arylsulfatases
Oxidation
Mutagenesis
Size exclusion chromatography
Site-Directed Mutagenesis
Ketones
Solubility
Gel Chromatography
Hydrogen

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Grove, Tyler L. ; Ahlum, Jessica H. ; Qin, Rosie M. ; Lanz, Nicholas D. ; Radle, Matthew I. ; Krebs, Carsten ; Booker, Squire J. / Further characterization of Cys-type and Ser-type anaerobic sulfatase maturating enzymes suggests a commonality in the mechanism of catalysis. In: Biochemistry. 2013 ; Vol. 52, No. 17. pp. 2874-2887.
@article{80b60e94e4974dcebcf54af91b66819e,
title = "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) Biochemistry49, 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.",
author = "Grove, {Tyler L.} and Ahlum, {Jessica H.} and Qin, {Rosie M.} and Lanz, {Nicholas D.} and Radle, {Matthew I.} and Carsten Krebs and Booker, {Squire J.}",
year = "2013",
month = "4",
day = "30",
doi = "10.1021/bi400136u",
language = "English (US)",
volume = "52",
pages = "2874--2887",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "17",

}

Further characterization of Cys-type and Ser-type anaerobic sulfatase maturating enzymes suggests a commonality in the mechanism of catalysis. / Grove, Tyler L.; Ahlum, Jessica H.; Qin, Rosie M.; Lanz, Nicholas D.; Radle, Matthew I.; Krebs, Carsten; Booker, Squire J.

In: Biochemistry, Vol. 52, No. 17, 30.04.2013, p. 2874-2887.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Further characterization of Cys-type and Ser-type anaerobic sulfatase maturating enzymes suggests a commonality in the mechanism of catalysis

AU - Grove, Tyler L.

AU - Ahlum, Jessica H.

AU - Qin, Rosie M.

AU - Lanz, Nicholas D.

AU - Radle, Matthew I.

AU - Krebs, Carsten

AU - Booker, Squire J.

PY - 2013/4/30

Y1 - 2013/4/30

N2 - 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) Biochemistry49, 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.

AB - 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) Biochemistry49, 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.

UR - http://www.scopus.com/inward/record.url?scp=84877062815&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84877062815&partnerID=8YFLogxK

U2 - 10.1021/bi400136u

DO - 10.1021/bi400136u

M3 - Article

C2 - 23477283

AN - SCOPUS:84877062815

VL - 52

SP - 2874

EP - 2887

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 17

ER -