title: SdhE-dependent formation of a functional Acetobacter pasteurianus succinate dehydrogenase in Gluconobacter oxydans--a first step toward a complete tricarboxylic acid cycle.
abstract: The obligatory aerobic α-proteobacterium Gluconobacter oxydans 621H possesses an unusual metabolism in which the majority of the carbohydrate substrates are incompletely oxidized in the periplasm and only a small fraction is metabolized in the cytoplasm. The cytoplasmic oxidation capabilities are limited due to an incomplete tricarboxylic acid (TCA) cycle caused by the lack of succinate dehydrogenase (Sdh) and succinyl-CoA synthetase. As a first step to test the consequences of a functional TCA cycle for growth, metabolism, and bioenergetics of G. oxydans, we attempted to establish a heterologous Sdh in this species. Expression of Acetobacter pasteurianus sdhCDAB in G. oxydans did not yield an active succinate dehydrogenase. Co-expression of a putative sdhE gene from A. pasteurianus, which was assumed to encode an assembly factor for covalent attachment of flavin adenine dinucleotide (FAD) to SdhA, stimulated Sdh activity up to 400-fold to 4.0 ± 0.4 U (mg membrane protein)(‒1). The succinate/oxygen reductase activity of membranes was 0.68 ± 0.04 U (mg membrane protein)(‒1), indicating the formation of functional Sdh complex capable of transferring electrons from succinate to ubiquinone. A. pasteurianus SdhE could be functionally replaced by SdhE from the γ-proteobacterium Serratia sp. According to these results, the accessory protein SdhE was necessary and sufficient for heterologous synthesis of an active A. pasteurianus Sdh in G. oxydans. Studies with the Sdh-positive G. oxydans strain provided evidence for a limited functionality of the TCA cycle despite the absence of succinyl-CoA synthetase.
authors.affiliation: Institute of Bio- and Geosciences, IBG-1: Biotechnology, Forschungszentrum Jülich, 52425, Jülich, Germany.|The Bioeconomy Science Center (BioSC), c/o Forschungszentrum Jülich, 52425, Jülich, Germany.
authors.forename: Ines
authors.initials: I
authors.lastname: Kiefler
authors.affiliation: Institute of Bio- and Geosciences, IBG-1: Biotechnology, Forschungszentrum Jülich, 52425, Jülich, Germany. st.bringer-meyer@fz-juelich.de.|The Bioeconomy Science Center (BioSC), c/o Forschungszentrum Jülich, 52425, Jülich, Germany. st.bringer-meyer@fz-juelich.de.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Bringer
authors.affiliation: Institute of Bio- and Geosciences, IBG-1: Biotechnology, Forschungszentrum Jülich, 52425, Jülich, Germany. m.bott@fz-juelich.de.|The Bioeconomy Science Center (BioSC), c/o Forschungszentrum Jülich, 52425, Jülich, Germany. m.bott@fz-juelich.de.
authors.forename: Michael
authors.initials: M
authors.lastname: Bott
mesh_terms: D000091:Acetobacter / Q000201:enzymology* / Q000235:genetics; D002952:Citric Acid Cycle; D004734:Energy Metabolism; D020600:Gluconobacter oxydans / Q000201:enzymology / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013385:Succinate Dehydrogenase / Q000235:genetics* / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D013385:Succinate Dehydrogenase
keywords: Citrate cycle; Gluconobacter oxydans; Succinate dehydrogenase