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Structure and functional properties of Bacillus subtilis endospore biogenesis factor StoA.

Crow A, Liu Y, Möller MC, Le Brun NE, Hederstedt L - J. Biol. Chem. (2009)

Bottom Line: The CXXC active site of the crystallized protein was found to be in a mixture of oxidized and reduced states, illustrating that there is little conformational variation between redox states.Although Cys-68 is buried within the structure, both cysteines were found to be accessible to cysteine-specific alkylating reagents.In vivo studies of site-directed variants of StoA revealed that the active site cysteines are functionally important, as is Glu-71, which lies close to the active site and is conserved in many reducing extracytoplasmic TDORs.

View Article: PubMed Central - PubMed

Affiliation: Centre for Molecular and Structural Biochemistry, School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich NR4 7TJ, United Kingdom.

ABSTRACT
Bacillus subtilis StoA is an extracytoplasmic thiol-disulfide oxidoreductase (TDOR) important for the synthesis of the endospore peptidoglycan cortex protective layer. Here we demonstrate that StoA is membrane-associated in B. subtilis and report the crystal structure of the soluble protein lacking its membrane anchor. This showed that StoA adopts a thioredoxin-like fold with N-terminal and internal additions that are characteristic of extracytoplasmic TDORs. The CXXC active site of the crystallized protein was found to be in a mixture of oxidized and reduced states, illustrating that there is little conformational variation between redox states. The midpoint reduction potential was determined as -248 mV versus normal hydrogen electrode at pH 7 consistent with StoA fulfilling a reductive role in endospore biogenesis. pK(a) values of the active site cysteines, Cys-65 and Cys-68, were determined to be 5.5 and 7.8. Although Cys-68 is buried within the structure, both cysteines were found to be accessible to cysteine-specific alkylating reagents. In vivo studies of site-directed variants of StoA revealed that the active site cysteines are functionally important, as is Glu-71, which lies close to the active site and is conserved in many reducing extracytoplasmic TDORs. The structure and biophysical properties of StoA are very similar to those of ResA, a B. subtilis extracytoplasmic TDOR involved in cytochrome c maturation, raising important general questions about how these similar but non-redundant proteins achieve specificity. A detailed comparison of the two proteins demonstrates that relatively subtle differences, largely located around the active sites of the proteins, are sufficient to confer specificity.

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Redox titration of sStoA. A, fluorescence spectra of sStoA in 50 mm potassium phosphate, 5 mm oxidized DTT, pH 7.0 following incubation with increasing concentrations of reduced DTT at 25 °C. B, plot of fraction of reduced sStoA (calculated from the fluorescence (Fluor.) intensity at 344 nm as described in the supplemental data) as a function of the cell potential. The solid line shows a fit to supplemental Equation S1.
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fig5: Redox titration of sStoA. A, fluorescence spectra of sStoA in 50 mm potassium phosphate, 5 mm oxidized DTT, pH 7.0 following incubation with increasing concentrations of reduced DTT at 25 °C. B, plot of fraction of reduced sStoA (calculated from the fluorescence (Fluor.) intensity at 344 nm as described in the supplemental data) as a function of the cell potential. The solid line shows a fit to supplemental Equation S1.

Mentions: StoA Is a Low Potential TDOR—The reduction potential of the active site cysteines of sStoA was measured using the difference in tryptophan fluorescence intensity of oxidized and reduced sStoA to follow oxidation state as a function of reduction potential (see Fig. 5). The data fitted well to the Nernst equation, giving a midpoint reduction potential of -248 ± 2 mV versus normal hydrogen electrode at pH 7 with n = 2.18 ± 0.16, as expected for a two-electron reduction process. This value is similar to that measured for B. subtilis ResA (-256 mV at pH 7) (21) and E. coli thioredoxin (-270 mV at pH 7) (30, 31) and is entirely consistent with the structural similarity between these proteins and a role for StoA in the reduction of (as yet unidentified) proteins involved in endospore cortex synthesis (2, 9).


Structure and functional properties of Bacillus subtilis endospore biogenesis factor StoA.

Crow A, Liu Y, Möller MC, Le Brun NE, Hederstedt L - J. Biol. Chem. (2009)

Redox titration of sStoA. A, fluorescence spectra of sStoA in 50 mm potassium phosphate, 5 mm oxidized DTT, pH 7.0 following incubation with increasing concentrations of reduced DTT at 25 °C. B, plot of fraction of reduced sStoA (calculated from the fluorescence (Fluor.) intensity at 344 nm as described in the supplemental data) as a function of the cell potential. The solid line shows a fit to supplemental Equation S1.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2665060&req=5

fig5: Redox titration of sStoA. A, fluorescence spectra of sStoA in 50 mm potassium phosphate, 5 mm oxidized DTT, pH 7.0 following incubation with increasing concentrations of reduced DTT at 25 °C. B, plot of fraction of reduced sStoA (calculated from the fluorescence (Fluor.) intensity at 344 nm as described in the supplemental data) as a function of the cell potential. The solid line shows a fit to supplemental Equation S1.
Mentions: StoA Is a Low Potential TDOR—The reduction potential of the active site cysteines of sStoA was measured using the difference in tryptophan fluorescence intensity of oxidized and reduced sStoA to follow oxidation state as a function of reduction potential (see Fig. 5). The data fitted well to the Nernst equation, giving a midpoint reduction potential of -248 ± 2 mV versus normal hydrogen electrode at pH 7 with n = 2.18 ± 0.16, as expected for a two-electron reduction process. This value is similar to that measured for B. subtilis ResA (-256 mV at pH 7) (21) and E. coli thioredoxin (-270 mV at pH 7) (30, 31) and is entirely consistent with the structural similarity between these proteins and a role for StoA in the reduction of (as yet unidentified) proteins involved in endospore cortex synthesis (2, 9).

Bottom Line: The CXXC active site of the crystallized protein was found to be in a mixture of oxidized and reduced states, illustrating that there is little conformational variation between redox states.Although Cys-68 is buried within the structure, both cysteines were found to be accessible to cysteine-specific alkylating reagents.In vivo studies of site-directed variants of StoA revealed that the active site cysteines are functionally important, as is Glu-71, which lies close to the active site and is conserved in many reducing extracytoplasmic TDORs.

View Article: PubMed Central - PubMed

Affiliation: Centre for Molecular and Structural Biochemistry, School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich NR4 7TJ, United Kingdom.

ABSTRACT
Bacillus subtilis StoA is an extracytoplasmic thiol-disulfide oxidoreductase (TDOR) important for the synthesis of the endospore peptidoglycan cortex protective layer. Here we demonstrate that StoA is membrane-associated in B. subtilis and report the crystal structure of the soluble protein lacking its membrane anchor. This showed that StoA adopts a thioredoxin-like fold with N-terminal and internal additions that are characteristic of extracytoplasmic TDORs. The CXXC active site of the crystallized protein was found to be in a mixture of oxidized and reduced states, illustrating that there is little conformational variation between redox states. The midpoint reduction potential was determined as -248 mV versus normal hydrogen electrode at pH 7 consistent with StoA fulfilling a reductive role in endospore biogenesis. pK(a) values of the active site cysteines, Cys-65 and Cys-68, were determined to be 5.5 and 7.8. Although Cys-68 is buried within the structure, both cysteines were found to be accessible to cysteine-specific alkylating reagents. In vivo studies of site-directed variants of StoA revealed that the active site cysteines are functionally important, as is Glu-71, which lies close to the active site and is conserved in many reducing extracytoplasmic TDORs. The structure and biophysical properties of StoA are very similar to those of ResA, a B. subtilis extracytoplasmic TDOR involved in cytochrome c maturation, raising important general questions about how these similar but non-redundant proteins achieve specificity. A detailed comparison of the two proteins demonstrates that relatively subtle differences, largely located around the active sites of the proteins, are sufficient to confer specificity.

Show MeSH
Related in: MedlinePlus