The Role of Cysteine Residues in Redox Regulation and Protein Stability of Arabidopsis thaliana Starch Synthase 1
Starch biosynthesis in Arabidopsis thaliana is strictly regulated. In leaf extracts, starch synthase 1 (AtSS1) responds to the redox potential within a physiologically relevant range. This study presents data testing two main hypotheses: 1) that specific thiol-disulfide exchange in AtSS1 influences...
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| description | Starch biosynthesis in Arabidopsis thaliana is strictly regulated. In leaf extracts, starch synthase 1 (AtSS1) responds to the redox potential within a physiologically relevant range. This study presents data testing two main hypotheses: 1) that specific thiol-disulfide exchange in AtSS1 influences its catalytic function 2) that each conserved Cys residue has an impact on AtSS1 catalysis. Recombinant AtSS1 versions carrying combinations of cysteine-to-serine substitutions were generated and characterized in vitro. The results demonstrate that AtSS1 is activated and deactivated by the physiological redox transmitters thioredoxin f1 (Trxf1), thioredoxin m4 (Trxm4) and the bifunctional NADPH-dependent thioredoxin reductase C (NTRC). AtSS1 displayed an activity change within the physiologically relevant redox range, with a midpoint potential equal to -306 mV, suggesting that AtSS1 is in the reduced and active form during the day with active photosynthesis. Cys164 and Cys545 were the key cysteine residues involved in regulatory disulfide formation upon oxidation. A C164S_C545S double mutant had considerably decreased redox sensitivity as compared to wild type AtSS1 (30% vs 77%). Michaelis-Menten kinetics and molecular modeling suggest that both cysteines play important roles in enzyme catalysis, namely, Cys545 is involved in ADP-glucose binding and Cys164 is involved in acceptor binding. All the other single mutants had essentially complete redox sensitivity (98-99%). In addition of being part of a redox directed activity "light switch", reactivation tests and low heterologous expression levels indicate that specific cysteine residues might play additional roles. Specifically, Cys265 in combination with Cys164 can be involved in proper protein folding or/and stabilization of translated protein prior to its transport into the plastid. Cys442 can play an important role in enzyme stability upon oxidation. The physiological and phylogenetic relevance of these findings is discussed. |
| doi_str_mv | 10.1371/journal.pone.0136997 |
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In leaf extracts, starch synthase 1 (AtSS1) responds to the redox potential within a physiologically relevant range. This study presents data testing two main hypotheses: 1) that specific thiol-disulfide exchange in AtSS1 influences its catalytic function 2) that each conserved Cys residue has an impact on AtSS1 catalysis. Recombinant AtSS1 versions carrying combinations of cysteine-to-serine substitutions were generated and characterized in vitro. The results demonstrate that AtSS1 is activated and deactivated by the physiological redox transmitters thioredoxin f1 (Trxf1), thioredoxin m4 (Trxm4) and the bifunctional NADPH-dependent thioredoxin reductase C (NTRC). AtSS1 displayed an activity change within the physiologically relevant redox range, with a midpoint potential equal to -306 mV, suggesting that AtSS1 is in the reduced and active form during the day with active photosynthesis. Cys164 and Cys545 were the key cysteine residues involved in regulatory disulfide formation upon oxidation. A C164S_C545S double mutant had considerably decreased redox sensitivity as compared to wild type AtSS1 (30% vs 77%). Michaelis-Menten kinetics and molecular modeling suggest that both cysteines play important roles in enzyme catalysis, namely, Cys545 is involved in ADP-glucose binding and Cys164 is involved in acceptor binding. All the other single mutants had essentially complete redox sensitivity (98-99%). In addition of being part of a redox directed activity "light switch", reactivation tests and low heterologous expression levels indicate that specific cysteine residues might play additional roles. Specifically, Cys265 in combination with Cys164 can be involved in proper protein folding or/and stabilization of translated protein prior to its transport into the plastid. Cys442 can play an important role in enzyme stability upon oxidation. The physiological and phylogenetic relevance of these findings is discussed.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0136997</identifier><identifier>PMID: 26367870</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Activation ; Adenosine diphosphate ; Antioxidants ; Arabidopsis ; Arabidopsis - chemistry ; Arabidopsis - enzymology ; Arabidopsis - physiology ; Arabidopsis Proteins - chemistry ; Arabidopsis Proteins - genetics ; Arabidopsis Proteins - metabolism ; Arabidopsis thaliana ; Binding ; Biochemistry ; Biodegradable materials ; Biosynthesis ; Carbohydrates ; Catalysis ; Catalytic Domain ; Chloroplasts ; Cysteine ; Cysteine - genetics ; Cysteine - metabolism ; Deactivation ; Environmental science ; Enzyme Stability ; Enzymes ; Gene Expression Regulation, Plant ; Glucosyltransferases - chemistry ; Glucosyltransferases - genetics ; Glucosyltransferases - metabolism ; Kinetics ; Laboratories ; Metabolism ; Metabolites ; Models, Molecular ; Molecular modelling ; Mutants ; NADP ; Neural networks ; Oxidation ; Oxidation-Reduction ; Phosphorylation ; Photosynthesis ; Phylogeny ; Physiology ; Plant sciences ; Plastids ; Protein folding ; Protein transport ; Proteins ; Reaction kinetics ; Redox potential ; Reductase ; Residues ; Sensitivity ; Serine ; Stability ; Starch ; Starch synthase ; Sucrose ; Thioredoxin ; Thioredoxins - metabolism ; Transmitters</subject><ispartof>PloS one, 2015-09, Vol.10 (9), p.e0136997-e0136997</ispartof><rights>2015 Skryhan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>2015 Skryhan et al 2015 Skryhan et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c526t-b9eaf71ec41230573f1aa7eb4ec826bfae300df41801a1e0d4afdf6e365d64e13</citedby><cites>FETCH-LOGICAL-c526t-b9eaf71ec41230573f1aa7eb4ec826bfae300df41801a1e0d4afdf6e365d64e13</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4569185/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4569185/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,860,881,2096,2915,23845,27901,27902,53766,53768,79343,79344</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/26367870$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Skryhan, Katsiaryna</creatorcontrib><creatorcontrib>Cuesta-Seijo, Jose A</creatorcontrib><creatorcontrib>Nielsen, Morten M</creatorcontrib><creatorcontrib>Marri, Lucia</creatorcontrib><creatorcontrib>Mellor, Silas B</creatorcontrib><creatorcontrib>Glaring, Mikkel A</creatorcontrib><creatorcontrib>Jensen, Poul E</creatorcontrib><creatorcontrib>Palcic, Monica M</creatorcontrib><creatorcontrib>Blennow, Andreas</creatorcontrib><title>The Role of Cysteine Residues in Redox Regulation and Protein Stability of Arabidopsis thaliana Starch Synthase 1</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>Starch biosynthesis in Arabidopsis thaliana is strictly regulated. In leaf extracts, starch synthase 1 (AtSS1) responds to the redox potential within a physiologically relevant range. This study presents data testing two main hypotheses: 1) that specific thiol-disulfide exchange in AtSS1 influences its catalytic function 2) that each conserved Cys residue has an impact on AtSS1 catalysis. Recombinant AtSS1 versions carrying combinations of cysteine-to-serine substitutions were generated and characterized in vitro. The results demonstrate that AtSS1 is activated and deactivated by the physiological redox transmitters thioredoxin f1 (Trxf1), thioredoxin m4 (Trxm4) and the bifunctional NADPH-dependent thioredoxin reductase C (NTRC). AtSS1 displayed an activity change within the physiologically relevant redox range, with a midpoint potential equal to -306 mV, suggesting that AtSS1 is in the reduced and active form during the day with active photosynthesis. Cys164 and Cys545 were the key cysteine residues involved in regulatory disulfide formation upon oxidation. A C164S_C545S double mutant had considerably decreased redox sensitivity as compared to wild type AtSS1 (30% vs 77%). Michaelis-Menten kinetics and molecular modeling suggest that both cysteines play important roles in enzyme catalysis, namely, Cys545 is involved in ADP-glucose binding and Cys164 is involved in acceptor binding. All the other single mutants had essentially complete redox sensitivity (98-99%). In addition of being part of a redox directed activity "light switch", reactivation tests and low heterologous expression levels indicate that specific cysteine residues might play additional roles. Specifically, Cys265 in combination with Cys164 can be involved in proper protein folding or/and stabilization of translated protein prior to its transport into the plastid. Cys442 can play an important role in enzyme stability upon oxidation. The physiological and phylogenetic relevance of these findings is discussed.</description><subject>Activation</subject><subject>Adenosine diphosphate</subject><subject>Antioxidants</subject><subject>Arabidopsis</subject><subject>Arabidopsis - chemistry</subject><subject>Arabidopsis - enzymology</subject><subject>Arabidopsis - physiology</subject><subject>Arabidopsis Proteins - chemistry</subject><subject>Arabidopsis Proteins - genetics</subject><subject>Arabidopsis Proteins - metabolism</subject><subject>Arabidopsis thaliana</subject><subject>Binding</subject><subject>Biochemistry</subject><subject>Biodegradable materials</subject><subject>Biosynthesis</subject><subject>Carbohydrates</subject><subject>Catalysis</subject><subject>Catalytic Domain</subject><subject>Chloroplasts</subject><subject>Cysteine</subject><subject>Cysteine - genetics</subject><subject>Cysteine - metabolism</subject><subject>Deactivation</subject><subject>Environmental science</subject><subject>Enzyme Stability</subject><subject>Enzymes</subject><subject>Gene Expression Regulation, Plant</subject><subject>Glucosyltransferases - chemistry</subject><subject>Glucosyltransferases - genetics</subject><subject>Glucosyltransferases - metabolism</subject><subject>Kinetics</subject><subject>Laboratories</subject><subject>Metabolism</subject><subject>Metabolites</subject><subject>Models, Molecular</subject><subject>Molecular modelling</subject><subject>Mutants</subject><subject>NADP</subject><subject>Neural networks</subject><subject>Oxidation</subject><subject>Oxidation-Reduction</subject><subject>Phosphorylation</subject><subject>Photosynthesis</subject><subject>Phylogeny</subject><subject>Physiology</subject><subject>Plant sciences</subject><subject>Plastids</subject><subject>Protein folding</subject><subject>Protein transport</subject><subject>Proteins</subject><subject>Reaction kinetics</subject><subject>Redox potential</subject><subject>Reductase</subject><subject>Residues</subject><subject>Sensitivity</subject><subject>Serine</subject><subject>Stability</subject><subject>Starch</subject><subject>Starch synthase</subject><subject>Sucrose</subject><subject>Thioredoxin</subject><subject>Thioredoxins - 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In leaf extracts, starch synthase 1 (AtSS1) responds to the redox potential within a physiologically relevant range. This study presents data testing two main hypotheses: 1) that specific thiol-disulfide exchange in AtSS1 influences its catalytic function 2) that each conserved Cys residue has an impact on AtSS1 catalysis. Recombinant AtSS1 versions carrying combinations of cysteine-to-serine substitutions were generated and characterized in vitro. The results demonstrate that AtSS1 is activated and deactivated by the physiological redox transmitters thioredoxin f1 (Trxf1), thioredoxin m4 (Trxm4) and the bifunctional NADPH-dependent thioredoxin reductase C (NTRC). AtSS1 displayed an activity change within the physiologically relevant redox range, with a midpoint potential equal to -306 mV, suggesting that AtSS1 is in the reduced and active form during the day with active photosynthesis. Cys164 and Cys545 were the key cysteine residues involved in regulatory disulfide formation upon oxidation. A C164S_C545S double mutant had considerably decreased redox sensitivity as compared to wild type AtSS1 (30% vs 77%). Michaelis-Menten kinetics and molecular modeling suggest that both cysteines play important roles in enzyme catalysis, namely, Cys545 is involved in ADP-glucose binding and Cys164 is involved in acceptor binding. All the other single mutants had essentially complete redox sensitivity (98-99%). In addition of being part of a redox directed activity "light switch", reactivation tests and low heterologous expression levels indicate that specific cysteine residues might play additional roles. Specifically, Cys265 in combination with Cys164 can be involved in proper protein folding or/and stabilization of translated protein prior to its transport into the plastid. Cys442 can play an important role in enzyme stability upon oxidation. The physiological and phylogenetic relevance of these findings is discussed.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>26367870</pmid><doi>10.1371/journal.pone.0136997</doi><oa>free_for_read</oa></addata></record> |
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| recordid | cdi_plos_journals_1719284367 |
| source | Public Library of Science (PLoS) Journals Open Access; MEDLINE; DOAJ Directory of Open Access Journals; EZB-FREE-00999 freely available EZB journals; PubMed Central; Free Full-Text Journals in Chemistry |
| subjects | Activation Adenosine diphosphate Antioxidants Arabidopsis Arabidopsis - chemistry Arabidopsis - enzymology Arabidopsis - physiology Arabidopsis Proteins - chemistry Arabidopsis Proteins - genetics Arabidopsis Proteins - metabolism Arabidopsis thaliana Binding Biochemistry Biodegradable materials Biosynthesis Carbohydrates Catalysis Catalytic Domain Chloroplasts Cysteine Cysteine - genetics Cysteine - metabolism Deactivation Environmental science Enzyme Stability Enzymes Gene Expression Regulation, Plant Glucosyltransferases - chemistry Glucosyltransferases - genetics Glucosyltransferases - metabolism Kinetics Laboratories Metabolism Metabolites Models, Molecular Molecular modelling Mutants NADP Neural networks Oxidation Oxidation-Reduction Phosphorylation Photosynthesis Phylogeny Physiology Plant sciences Plastids Protein folding Protein transport Proteins Reaction kinetics Redox potential Reductase Residues Sensitivity Serine Stability Starch Starch synthase Sucrose Thioredoxin Thioredoxins - metabolism Transmitters |
| title | The Role of Cysteine Residues in Redox Regulation and Protein Stability of Arabidopsis thaliana Starch Synthase 1 |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-02-01T09%3A38%3A38IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-proquest_plos_&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=The%20Role%20of%20Cysteine%20Residues%20in%20Redox%20Regulation%20and%20Protein%20Stability%20of%20Arabidopsis%20thaliana%20Starch%20Synthase%201&rft.jtitle=PloS%20one&rft.au=Skryhan,%20Katsiaryna&rft.date=2015-09-14&rft.volume=10&rft.issue=9&rft.spage=e0136997&rft.epage=e0136997&rft.pages=e0136997-e0136997&rft.issn=1932-6203&rft.eissn=1932-6203&rft_id=info:doi/10.1371/journal.pone.0136997&rft_dat=%3Cproquest_plos_%3E3828132691%3C/proquest_plos_%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=1719284367&rft_id=info:pmid/26367870&rft_doaj_id=oai_doaj_org_article_3d5842fc4f8842d3b74aaa3ac90ad80f&rfr_iscdi=true |