VAAM-Jahrestagung 2012 18.–21. März in Tübingen

178PSP010Crystal structure of the electron-transferring flavoprotein (Etf)from Acidaminococcus fermentans involved in electronbifurcationN. Pal Chowdhury* 1,2 , A. Mohammed Hassan 1,2 , U. Demmer 3 , U. Ermler 3 ,W. Buckel 1,21 Philipps-Universität, Fachbereich Biologie, Marburg, Germany2 Max-Planck-Institut für terrestrische Mikrobiologie, Marburg, Germany3 Max-Planck-Institut für Biophysik, Frankfurt, GermanyAerobic organisms use electron-transferring flavoprotein (Etf) as electronacceptor for the oxidation of acyl-CoA to enoyl-CoA. The reduced form ofEtf is then reoxidized by quinone at the inner mitochondrial membrane ofeukaryotes or at the cytoplasmic membrane of bacteria. The structure ofthe human heterodimeric Etf () revealed three domains, two of which areformed by the -subunit (I and II) and one by the -subunit (III). -FADlocated at the surface of domain II interacts with acyl-CoA dehydrogenase.The center of domain III contains AMP with an enigmatic function. Theinterface between domains II and III appears to be flexible due to absenceof secondary structures [1].Anaerobic bacteria synthesize butyrate via the NADH-dependent reductionof crotonyl-CoA to butyryl-CoA mediated by butyryl-CoA dehydrogenaseand Etf. This exergonic reaction is coupled to the endergonic reduction offerredoxin by NADH, a process called electron bifurcation [2]. Whereas inClostridium klyuveri [3] and Clostridium tetanomorphum butyryl-CoAdehydrogenase and Etf form a tight complex, in A. fermentans bothcomponents separate during purification. Recombinant Etf from A.fermentans produced in Escherichia coli was crystallized and its structurehas been solved. The structure is closely related to that of the human Etf,but AMP is replaced by a second -FAD. We propose that NADH reduces-FAD to -FADH - . Then -FAD switches towards -FADH - and takesone electron to yield -FADH·and -FAD·- . Whereas -FAD·- is stabilizedby the flavodoxin-like domain II and transfers the electron further to thedehydrogenase, the remaining highly reactive -FADH·immediatelyreduces ferredoxin. Repetition of this process results in the reduction of 2ferredoxins and one crotonyl-CoA by 2 NADH. The reduced ferrdoxinsmay give rise to H 2 or to H + /Na + via a membrane bound NADferredoxinoxidoreductase also called Rnf.1. Roberts DL, Frerman FE & Kim JJ (1996) Proc Natl Acad Sci U S A 93, 14355-14360.2. Herrmann G, Jayamani E, Mai G & Buckel W (2008) J Bacteriol 190, 784-7913. Li F, Hinderberger J, Seedorf H, Zhang J, Buckel W & Thauer RK (2008) J Bacteriol 190, 843-850PSP011Nitrous oxide turnover in the nitrate-ammonifyingEpsilonproteobacterium Wolinella succinogenesM. Luckmann*, M. Kern, J. SimonTU-Darmstadt, Department of Biology, Darmstadt, GermanyGlobal warming is moving more and more to the public consciousness.Besides the commonly mentioned carbon dioxide, nitrous oxide (N 2O) isone of the most important greenhouse gases and accounts for about 10% ofthe anthropogenic greenhouse effect.In the environment N 2O is produced, for example, by nitrifying anddenitrifying microbial species. On the other hand, some respiratory nitrateammonifyingEpsilonproteobacteria are able to reduce nitrous oxide todinitrogen via an unconventional cytochrome c nitrous oxide reductase(cNosZ). The energy metabolism of one of these bacteria, Wolinellasuccinogenes, has been characterized thoroughly in the past. The cells areable to use either formate or hydrogen gas as electron donors together withtypical terminal electron acceptors like, for example, fumarate, nitrate,polysulfide or nitrous oxide. Despite utilizing nitrous oxide, it is notknown if these cells are producing N 2O in substantial amounts duringenergy substrate turnover or if they are acting only as N 2O sinks.The cytochromecnitrous oxide reductase of W. succinogenesis encoded bythe first gene of the nos gene cluster together with a unique electrontransport system that is predicted to connect the menaquinone/menaquinolpool with cNosZ. The involved electron transfer chain may comprise amenaquinol dehydrogenase of the unusual NapGH-type and the twomonohaem cytochromes c NosC1 and NosC2. Corresponding in-framegene deletion strains were constructed and characterized. Based on theresults, a model of nitrous oxide turnover in W. succinogenes will bepresented.PSP012Anaerobic n-hexane degradation in nitrate reducing strain HxN1A. Parthasarathy* 1,2 , M. Drozdowska 3 , J. Kahnt 2 , R. Rabus 4,5 , F. Widdel 5 ,B.T. Golding 3 , H. Wilkes 6 , W. Buckel 1,21 Philipps-Universität, Fachbereich Biologie, Marburg, Germany2 Max-Planck-Institut für terrestrische Mikrobiologie, Marburg, Germany3 University of Newcastle upon Tyne, Chemistry, Newcastle, UnitedKingdom4 Universität Oldenburg, Institut für Chemie und Biologie des Meeres,Oldenburg, Germany5 Max-Planck-Institut für Marine Mikrobiologie, Bremen, Germany6 Helmholtz-Zentrum Potsdam, Organische Geochemie, Potsdam, GermanyThe denitrifying Betaproteobacterium HxN1 grows on n-hexane [1]forming alkyl substituted succinates. The proposed pathway starts with theaddition of n-hexane to fumarate with the exclusive abstraction of the pro-S hydrogen of n-hexane via a glycyl-radical enzyme catalysed reaction [1],yielding a mixture of (2R,1'R) and (2S,1'R)-1'-methylpentylsuccinates mostlikely as CoA-thioesters [2]. These intermediates are proposed to bedegraded via intramolecular rearrangement to (4R)-(2-methylhexyl)malonyl-CoA and carboxyl group loss yielding (4R)-4-methyloctanoyl-CoA. Further degradation may occur via dehydrogenationand -oxidation [3]. If (4R)-(2-methylhexyl)malonyl-CoA, synthesised bya novel method, and propionyl-CoA were incubated with cell-free extractof strain HxN1, MALDI-TOF mass spectrometry revealed formation ofmethylmalonyl-CoA and 2-methylhex-2-enoyl-CoA (-oxidation product).Therefore, transcarboxylation (CO 2 exchange between substrates) occurs atthe CoA thioester level as predicted, linking the degradation of 1-methylpentylsuccinate to the generation of succinate via methylmalonyl-CoA.1) Rabus R, Wilkes H, Behrends A, Armstroff A, Fischer T, Widdel F (2001) J Bacteriol 183,1707-1715.2) Jarling R, Sadeghi M, Drozdowska M, Lahme S, Buckel W, Rabus R, Widdel F, Golding BT, Wilkes H(2011), Angew. Chem. in press.3) Wilkes H, Rabus R, Fischer T, Armstroff A, Behrends A, Widdel F (2002) Arch. Microbiol 177, 235.PSP013Streptomyces coelicolor A3(2) Spores are Prepared for an AbruptShift from Aerobic Respiration to Anaerobic Respiration withNitrateD. Falke*, M. Fischer, G. SawersMartin-Luther-University Halle, Biology/Microbiology, AG Sawers, Halle(Saale), GermanyThe filamentous actinobacterium Streptomyces coelicolor has a complexlife cycle including growth as vegetative hyphae, generation ofhydrophobic aerial hyphae and the production of exospores. Despite beingan obligate aerobe S. coelicolor is able to reduce nitrate to nitrite, probablyto help maintain a membrane potential during oxygen limitation. Thegenome of S. coelicolor has 3 copies of the narGHJI operon, eachencoding a nitrate reductase (Nar) [1]. Nars are multi-subunit, membraneassociatedenzymes that couple nitrate reduction to energy conservation.Each Nar enzyme is synthesized in S. coelicolor and is active in differentphases of growth and in different tissues: Nar1 is active in spores; Nar2 isactive in germinating spores and mycelium; while Nar3 is induced in thestationary phase correlating with the onset of secondary metabolism [2].The Nar enzymes are therefore not redundant but rather appear to havedistinct functions in the developmental program of the bacterium.In this study we focused on nitrate respiration in resting spores. Freshlyharvested spores of S. coelicolor wild type M145 could reduce nitrate at asignificant rate without addition of an exogenous electron donor. However,an exogenous electron donor was required to measure the activity in crudeextracts of spores. Moreover, activity could be visualized by direct stainingafter native PAGE. Analysis of defined knockout mutants demonstratedthat Nar activity in spores was due exclusively to Nar1. By using adiscontinuous assay to measure nitrite production by spores we coulddemonstrate that Nar1 was only capable of nitrate reduction in the absenceof oxygen. Addition of oxygen immediately prevented nitrate reduction.Since Nar1 activity in whole spores showed a reversible dependence onanaerobiosis, this finding suggests that spores regulate either nitratetransport or Nar1 activity in response to oxygen. Notably, studies usingprotein synthesis inhibitors revealed that Nar1 is always present and activein resting spores.[1] van Keulen et al. (2005) Nitrate respiration in the actinomycete Streptomyces coelicolor.Biochem Soc Trans. 33(Pt 1):210-2[2] Fischer et al. (2010) The obligate aerobe Streptomyces coelicolor A3(2) synthesizes three activerespiratory nitrate reductases. Microbiology. 156(Pt 10):3166-79PSP014New insights into acetate and glycerol metabolism ofSchizosaccharomyces pombeT. Klein*, K. Schneider, E. HeinzleSaarland University, Biochemical Engineering, Saarbruecken, GermanyThe fission yeast Schizosaccharomyces pombe has been a model organismof molecular biology for decades. However, little is known about itsBIOspektrum | Tagungsband 2012