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Tetramerization reinforces the dimer interface of MnSOD.

Sheng Y, Durazo A, Schumacher M, Gralla EB, Cascio D, Cabelli DE, Valentine JS - PLoS ONE (2013)

Bottom Line: Although CaMnSODc was found to crystallize as a tetramer, there is no indication from the solution properties that the functionality of CaMnSODc in vivo depends upon the formation of the tetrameric structure.Dimeric CaMnSODc was found to be significantly more subject to thermal or denaturant-induced unfolding than tetrameric ScMnSOD.We conclude that the tetrameric assembly strongly reinforces the dimer interface, which is critical for MnSOD activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of California Los Angeles, Los Angeles, California, United States of America.

ABSTRACT
Two yeast manganese superoxide dismutases (MnSOD), one from Saccharomyces cerevisiae mitochondria (ScMnSOD) and the other from Candida albicans cytosol (CaMnSODc), have most biochemical and biophysical properties in common, yet ScMnSOD is a tetramer and CaMnSODc is a dimer or "loose tetramer" in solution. Although CaMnSODc was found to crystallize as a tetramer, there is no indication from the solution properties that the functionality of CaMnSODc in vivo depends upon the formation of the tetrameric structure. To elucidate further the functional significance of MnSOD quaternary structure, wild-type and mutant forms of ScMnSOD (K182R, A183P mutant) and CaMnSODc (K184R, L185P mutant) with the substitutions at dimer interfaces were analyzed with respect to their oligomeric states and resistance to pH, heat, and denaturant. Dimeric CaMnSODc was found to be significantly more subject to thermal or denaturant-induced unfolding than tetrameric ScMnSOD. The residue substitutions at dimer interfaces caused dimeric CaMnSODc but not tetrameric ScMnSOD to dissociate into monomers. We conclude that the tetrameric assembly strongly reinforces the dimer interface, which is critical for MnSOD activity.

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Related in: MedlinePlus

RP-mutant CaMnSODc is inactivated by heat like EcMnSOD.Rate constants as a function of pH were determined by fitting the disappearances of low doses of O2− ([O2−]:[MnSOD] from 1–3) to first-order processes. The enzymes were EcMnSOD (grey rectangle), WT ScMnSOD (solid triangle), K182R, A183P ScMnSOD (hollow triangle), WT CaMnSODc (solid circle) and K184R, L185P CaMnSODc (hollow circle). The data points indicated with an arrow were obtained before the sample solution reached the desired temperature. All other data points were obtained after the sample solution was equilibrated to the desired temperature. The sample solutions contained 1 µM (in Mn) MnSOD in 10 mM potassium phosphate (pH 7), 10 mM sodium formate and 10 µM EDTA.
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pone-0062446-g005: RP-mutant CaMnSODc is inactivated by heat like EcMnSOD.Rate constants as a function of pH were determined by fitting the disappearances of low doses of O2− ([O2−]:[MnSOD] from 1–3) to first-order processes. The enzymes were EcMnSOD (grey rectangle), WT ScMnSOD (solid triangle), K182R, A183P ScMnSOD (hollow triangle), WT CaMnSODc (solid circle) and K184R, L185P CaMnSODc (hollow circle). The data points indicated with an arrow were obtained before the sample solution reached the desired temperature. All other data points were obtained after the sample solution was equilibrated to the desired temperature. The sample solutions contained 1 µM (in Mn) MnSOD in 10 mM potassium phosphate (pH 7), 10 mM sodium formate and 10 µM EDTA.

Mentions: Yeast MnSODs showed full activity until the protein unfolding temperature. ScMnSOD was fully active up to 75°C, the highest temperature allowed in pulse radiolysis measurements. CaMnSODc, with a much lower thermostability than ScMnSOD (see below), was fully active as long as the enzyme stayed folded in solution. Protein aggregation was noticeable when the sample became more opaque to light at 260 nm. Aggregation of as-isolated CaMnSODc occurred at 50°C (see below), and the enzyme stayed fully active up to 49°C (Figure 5).


Tetramerization reinforces the dimer interface of MnSOD.

Sheng Y, Durazo A, Schumacher M, Gralla EB, Cascio D, Cabelli DE, Valentine JS - PLoS ONE (2013)

RP-mutant CaMnSODc is inactivated by heat like EcMnSOD.Rate constants as a function of pH were determined by fitting the disappearances of low doses of O2− ([O2−]:[MnSOD] from 1–3) to first-order processes. The enzymes were EcMnSOD (grey rectangle), WT ScMnSOD (solid triangle), K182R, A183P ScMnSOD (hollow triangle), WT CaMnSODc (solid circle) and K184R, L185P CaMnSODc (hollow circle). The data points indicated with an arrow were obtained before the sample solution reached the desired temperature. All other data points were obtained after the sample solution was equilibrated to the desired temperature. The sample solutions contained 1 µM (in Mn) MnSOD in 10 mM potassium phosphate (pH 7), 10 mM sodium formate and 10 µM EDTA.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3646814&req=5

pone-0062446-g005: RP-mutant CaMnSODc is inactivated by heat like EcMnSOD.Rate constants as a function of pH were determined by fitting the disappearances of low doses of O2− ([O2−]:[MnSOD] from 1–3) to first-order processes. The enzymes were EcMnSOD (grey rectangle), WT ScMnSOD (solid triangle), K182R, A183P ScMnSOD (hollow triangle), WT CaMnSODc (solid circle) and K184R, L185P CaMnSODc (hollow circle). The data points indicated with an arrow were obtained before the sample solution reached the desired temperature. All other data points were obtained after the sample solution was equilibrated to the desired temperature. The sample solutions contained 1 µM (in Mn) MnSOD in 10 mM potassium phosphate (pH 7), 10 mM sodium formate and 10 µM EDTA.
Mentions: Yeast MnSODs showed full activity until the protein unfolding temperature. ScMnSOD was fully active up to 75°C, the highest temperature allowed in pulse radiolysis measurements. CaMnSODc, with a much lower thermostability than ScMnSOD (see below), was fully active as long as the enzyme stayed folded in solution. Protein aggregation was noticeable when the sample became more opaque to light at 260 nm. Aggregation of as-isolated CaMnSODc occurred at 50°C (see below), and the enzyme stayed fully active up to 49°C (Figure 5).

Bottom Line: Although CaMnSODc was found to crystallize as a tetramer, there is no indication from the solution properties that the functionality of CaMnSODc in vivo depends upon the formation of the tetrameric structure.Dimeric CaMnSODc was found to be significantly more subject to thermal or denaturant-induced unfolding than tetrameric ScMnSOD.We conclude that the tetrameric assembly strongly reinforces the dimer interface, which is critical for MnSOD activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of California Los Angeles, Los Angeles, California, United States of America.

ABSTRACT
Two yeast manganese superoxide dismutases (MnSOD), one from Saccharomyces cerevisiae mitochondria (ScMnSOD) and the other from Candida albicans cytosol (CaMnSODc), have most biochemical and biophysical properties in common, yet ScMnSOD is a tetramer and CaMnSODc is a dimer or "loose tetramer" in solution. Although CaMnSODc was found to crystallize as a tetramer, there is no indication from the solution properties that the functionality of CaMnSODc in vivo depends upon the formation of the tetrameric structure. To elucidate further the functional significance of MnSOD quaternary structure, wild-type and mutant forms of ScMnSOD (K182R, A183P mutant) and CaMnSODc (K184R, L185P mutant) with the substitutions at dimer interfaces were analyzed with respect to their oligomeric states and resistance to pH, heat, and denaturant. Dimeric CaMnSODc was found to be significantly more subject to thermal or denaturant-induced unfolding than tetrameric ScMnSOD. The residue substitutions at dimer interfaces caused dimeric CaMnSODc but not tetrameric ScMnSOD to dissociate into monomers. We conclude that the tetrameric assembly strongly reinforces the dimer interface, which is critical for MnSOD activity.

Show MeSH
Related in: MedlinePlus