title: Redox regulation of Arabidopsis mitochondrial citrate synthase.
abstract: Citrate synthase has a key role in the tricarboxylic (TCA) cycle of mitochondria of all organisms, as it catalyzes the first committed step which is the fusion of a carbon-carbon bond between oxaloacetate and acetyl CoA. The regulation of TCA cycle function is especially important in plants, since mitochondrial activities have to be coordinated with photosynthesis. The posttranslational regulation of TCA cycle activity in plants is thus far almost entirely unexplored. Although several TCA cycle enzymes have been identified as thioredoxin targets in vitro, the existence of any thioredoxin-dependent regulation as known for the Calvin cycle, yet remains to be demonstrated. Here we have investigated the redox regulation of the Arabidopsis citrate synthase enzyme by site-directed mutagenesis of its six cysteine residues. Our results indicate that oxidation inhibits the enzyme activity by the formation of mixed disulfides, as the partially oxidized citrate synthase enzyme forms large redox-dependent aggregates. Furthermore, we were able to demonstrate that thioredoxin can cleave diverse intra- as well as intermolecular disulfide bridges, which strongly enhances the activity of the enzyme. Activity measurements with the cysteine variants of the enzyme revealed important cysteine residues affecting total enzyme activity as well as the redox sensitivity of the enzyme.
authors.affiliation: Botany, Department I of Biology, Ludwig-Maximilians-University Munich, Grosshaderner Strasse 2, 82152 Planegg-Martinsried, Germany.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Schmidtmann
authors.forename: Ann-Christine
authors.initials: AC
authors.lastname: König
authors.forename: Anne
authors.initials: A
authors.lastname: Orwat
authors.forename: Dario
authors.initials: D
authors.lastname: Leister
authors.forename: Markus
authors.initials: M
authors.lastname: Hartl
authors.forename: Iris
authors.initials: I
authors.lastname: Finkemeier
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000166:cytology* / Q000201:enzymology* / Q000378:metabolism; D002950:Citrate (si)-Synthase / Q000737:chemistry / Q000378:metabolism*; D017124:Conserved Sequence; D003545:Cysteine; D004220:Disulfides / Q000737:chemistry; D004789:Enzyme Activation / Q000187:drug effects; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011487:Protein Conformation; D013879:Thioredoxins / Q000378:metabolism
chemical_list: D004220:Disulfides; D007527:Isoenzymes; D013879:Thioredoxins; D006861:Hydrogen Peroxide; D002950:Citrate (si)-Synthase; D003545:Cysteine
keywords: Arabidopsis.; TCA cycle; citrate synthase; cysteine residues; mitochondria; redox regulation; thioredoxin