Specific and Reversible Inactivation of Phycomyces blakesleeanus Isocitrate Lyase by Ascorbate-Iron: Role of Two Redox-Active Cysteines

Rúa, Javier; Soler, Joaquı́n; Busto, Félix; Arriaga, Dolores de

doi:10.1006/fgbi.2001.1324

Cited by 8 publications

(2 citation statements)

References 51 publications

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“…1A). Cys 301 is only conserved in bacterial-type ICLs, whereas Cys 178 is conserved in all organisms, consistent with its role in catalysis (29,30) Because two cysteine residues of ICL have been previ-ously suggested to be sensitive to redox modifications (10,11,17), based on sequence conservation, Cys 178 and Cys 301 appear as the most likely candidates. Three-dimensional modeling of CrICL suggests that these two residues are the two closest cysteines in the enzyme but are too distant to allow formation of a disulfide bridge (Fig.…”

Section: Sequence and Phylogenetic Analysis Of Chlamydomonas Icl-mentioning

confidence: 79%

“…These results strongly suggest that CrICL, and therefore the assimilation of acetate in Chlamydomonas, are redox-regulated. The existence of two redox active cysteines has also been suggested for Phycomyces blakasleeanus ICL (17). In the case of CrICL, it is clear that the enzyme contains one or more redox-sensitive cysteines whose oxidation state could affect protein activity.…”

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confidence: 90%

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Regulation by Glutathionylation of Isocitrate Lyase from Chlamydomonas reinhardtii

Bedhomme

Zaffagnini

Marchand

et al. 2009

Journal of Biological Chemistry

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Post-translational modification of protein cysteine residues is emerging as an important regulatory and signaling mechanism. We have identified numerous putative targets of redox regulation in the unicellular green alga Chlamydomonas reinhardtii. One enzyme, isocitrate lyase (ICL), was identified both as a putative thioredoxin target and as an S-thiolated protein in vivo. ICL is a key enzyme of the glyoxylate cycle that allows growth on acetate as a sole source of carbon. The aim of the present study was to clarify the molecular mechanism of the redox regulation of Chlamydomonas ICL using a combination of biochemical and biophysical methods. The results clearly show that purified C. reinhardtii ICL can be inactivated by glutathionylation and reactivated by glutaredoxin, whereas thioredoxin does not appear to regulate ICL activity, and no inter-or intramolecular disulfide bond could be formed under any of the conditions tested. Glutathionylation of the protein was investigated by mass spectrometry analysis, Western blotting, and site-directed mutagenesis. The enzyme was found to be protected from irreversible oxidative inactivation by glutathionylation of its catalytic Cys 178 , whereas a second residue, Cys 247 , becomes artifactually glutathionylated after prolonged incubation with GSSG. The possible functional significance of this post-translational modification of ICL in Chlamydomonas and other organisms is discussed.

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Section: Sequence and Phylogenetic Analysis Of Chlamydomonas Icl-mentioning

confidence: 79%

mentioning

confidence: 90%

Regulation by Glutathionylation of Isocitrate Lyase from Chlamydomonas reinhardtii

Bedhomme

Zaffagnini

Marchand

et al. 2009

Journal of Biological Chemistry

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Oxidation of buried cysteines is slow and an insignificant factor in the structural destabilization of staphylococcal nuclease caused by H2O2 exposure

Kim

Stites

2004

Amino Acids

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The oxidation of buried cysteine or methionine residues can destroy the enzyme activity of a protein by disrupting structure. Engineering in such an oxidatively triggered switch for enzyme activity would only be useful if the effects of substitution are relatively minor, while the effects of the oxidized side chain upon structure are significant and the oxidation relatively easy. To assess the feasibility of this strategy for controlling enzyme activity, the effects of such substitutions and their oxidation were studied in a well characterized model protein, staphylococcal nuclease. Stability and enzyme activity of the oxidized proteins was assessed and compared to the stability and enzyme activity of the unoxidized proteins. Cysteines were found to be generally well tolerated in buried positions but these mutants were not more destabilized than wild-type when oxidized. This shows that buried cysteines are difficult enough to oxidize that this is not likely to be a useful protein engineering strategy or a commonly used regulatory modification. Similar effects were observed for methionine.

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A protective association between catalase and isocitrate lyase in peroxisomes

Yanik

Donaldson

2005

Archives of Biochemistry and Biophysics

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Specific and Reversible Inactivation of Phycomyces blakesleeanus Isocitrate Lyase by Ascorbate-Iron: Role of Two Redox-Active Cysteines

Cited by 8 publications

References 51 publications

Regulation by Glutathionylation of Isocitrate Lyase from Chlamydomonas reinhardtii

Regulation by Glutathionylation of Isocitrate Lyase from Chlamydomonas reinhardtii

Oxidation of buried cysteines is slow and an insignificant factor in the structural destabilization of staphylococcal nuclease caused by H2O2 exposure

A protective association between catalase and isocitrate lyase in peroxisomes

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