• Identification of persulfide-binding and disulfide-forming cysteine residues in the NifS-like domain of the molybdenum cofactor sulfurase ABA3 by cysteine-scanning mutagenesis.

      Lehrke, Markus; Rump, Steffen; Heidenreich, Torsten; Wissing, Josef; Mendel, Ralf R; Bittner, Florian; Department of Plant Biology, Braunschweig University of Technology, Humboldtstrasse 1, 38023 Braunschweig, Germany. (2012-02-01)
      The Moco (molybdenum cofactor) sulfurase ABA3 from Arabidopsis thaliana catalyses the sulfuration of the Moco of aldehyde oxidase and xanthine oxidoreductase, which represents the final activation step of these enzymes. ABA3 consists of an N-terminal NifS-like domain that exhibits L-cysteine desulfurase activity and a C-terminal domain that binds sulfurated Moco. The strictly conserved Cys430 in the NifS-like domain binds a persulfide intermediate, which is abstracted from the substrate L-cysteine and finally needs to be transferred to the Moco of aldehyde oxidase and xanthine oxidoreductase. In addition to Cys⁴³⁰, another eight cysteine residues are located in the NifS-like domain, with two of them being highly conserved among Moco sulfurase proteins and, at the same time, being in close proximity to Cys⁴³⁰. By determination of the number of surface-exposed cysteine residues and the number of persulfide-binding cysteine residues in combination with the sequential substitution of each of the nine cysteine residues, a second persulfide-binding cysteine residue, Cys²⁰⁶, was identified. Furthermore, the active-site Cys⁴³⁰ was found to be located on top of a loop structure, formed by the two flanking residues Cys⁴²⁸ and Cys⁴³⁵, which are likely to form an intramolecular disulfide bridge. These findings are confirmed by a structural model of the NifS-like domain, which indicates that Cys⁴²⁸ and Cys⁴³⁵ are within disulfide bond distance and that a persulfide transfer from Cys⁴³⁰ to Cys²⁰⁶ is indeed possible.
    • Plastid gene expression and plant development require a plastidic protein of the mitochondrial transcription termination factor family.

      Babiychuk, Elena; Vandepoele, Klaas; Wissing, Josef; Garcia-Diaz, Miguel; De Rycke, Riet; Akbari, Hana; Joubès, Jérôme; Beeckman, Tom; Jänsch, Lothar; Frentzen, Margrit; et al. (2011-04-19)
      Plastids are DNA-containing organelles unique to plant cells. In Arabidopsis, one-third of the genes required for embryo development encode plastid-localized proteins. To help understand the role of plastids in embryogenesis and postembryonic development, we characterized proteins of the mitochondrial transcription termination factor (mTERF) family, which in animal models, comprises DNA-binding regulators of mitochondrial transcription. Of 35 Arabidopsis mTERF proteins, 11 are plastid-localized. Genetic complementation shows that at least one plastidic mTERF, BELAYA SMERT' (BSM), is required for embryogenesis. The main postembryonic phenotypes of genetic mosaics with the bsm mutation are severe abnormalities in leaf development. Mutant bsm cells are albino, are compromised in growth, and suffer defects in global plastidic gene expression. The bsm phenotype could be phenocopied by inhibition of plastid translation with spectinomycin. Plastid translation is essential for cell viability in dicotyledonous species such as tobacco but not in monocotyledonous maize. Here, genetic interactions between BSM and the gene encoding plastid homomeric acetyl-CoA carboxylase ACC2 suggest that there is a functional redundancy in malonyl-CoA biosynthesis that permits bsm cell survival in Arabidopsis. Overall, our results indicate that biosynthesis of malonyl-CoA and plastid-derived systemic growth-promoting compounds are the processes that link plant development and plastid gene expression.