VAAM-Jahrestagung 2011 Karlsruhe, 3.–6. April 2011

eaction mechanism. While Apc consists of 5 subunits in an (αββ′γ) 2ε 2composition, Acx is a (αβγ) 2-heterohexamer. The catalytic properties of bothenzymes and their respective reaction mechanisms were investigated andcompared. Acetophenone carboxylase converts a variety of aromaticketones, while acetone carboxylase shows a very narrow substrate spectrumand carboxylates only acetone and butanone. Also, the products of ATPhydrolysisdiffer: per carboxylated substrate acetophenone carboxylasehydrolyses 2 ATP to 2 ADP, while acetone carboxylase hydrolyses 2 ATP to2 AMP.The observed reaction mechanisms of acetone carboxylase andacetophenone carboxylase represent novel ATP-dependent, biotinindependentcarboxylation mechanisms in bacterial ketone catabolism,which likely involve the activation of both substrates via phosphorylation.AMV004The W-/Se-containing class II benzoyl-CoA reductasecomplex in obligately anaerobic bacteriaC. Löffler* 1 , J.W. Kung 1 , T. Weinert 2 , U. Ermler 2 , M. Boll 11 Institute of Biochemistry, University of Leipzig, Leipzig, Germany2 Max Planck Institute of Biophysics, Frankfurt am Main, GermanyBenzoyl-Coenzyme A (CoA) is a central intermediate in the anaerobicdegradation of aromatic compounds which is dearomatized to cyclohexa-1,5-diene-1-carbonyl-CoA by benzoyl-CoA reductases (BCRs). There aretwo completely different classes of BCRs which both yield the identicalproduct [1,2]. ATP-dependent class I BCRs, referred to as BcrABCD are[4Fe-4S] clusters containing enzymes that are present in facultativeanaerobes. In contrast, obligately anaerobic bacteria are proposed to employa W-/Zn-/FeS-/Flavin-/Se-containing, ATP-independent BamBCDEFGHIcomplex. The active site harbouring BamBC components were characterizedfrom the aromatic compound degrading Deltaproteobacterium Geobactermetallireducens [1]. BamB is similar to aldehyde:ferredoxinoxidoreductases and is supposed to contain a W-pterin cofactor at the activesite. We present kinetic and molecular properties of BamBC and provideevidence that class II BCRs are composed of the predicted high molecularBamBCDEFGHI complex. Initial data indicate that the exergonic electrontransfer to the aromatic ring is driven by an electron bifurcation process.AMV005Nitrogen oxides involved in anaerobic alkane activationby strain HdN1J. Zedelius* 1 , R. Rabus 2 , M.M.M. Kuypers 3 , F. Schreiber 3 , F. Widdel 11 Department of Microbiology, Max Planck Institute for MarineMicrobiology, Bremen, Germany2 Institute for Chemistry and Biology of the Marine Environment (ICBM),Carl von Ossietzky University, Oldenburg, Germany3 Department of Biogeochemistry, Max Planck Institute for MarineMicrobiology, Bremen, GermanyAlkanes are naturally wide-spread hydrocarbons, originating from petroleumor synthesized by living organisms. Their degradation by microorganismshas been studied extensively in the past century. Only a small number ofbacterial strains have been described so far with the ability to activatesaturated alkanes under anaerobic conditions, employing unique biochemicalreactions to overcome the inertia of C-H bonds. TheGammaproteobacterium strain HdN1 degrades linear alkanes between C 6H 14and C 30H 62 under denitrifying conditions. Genetic, proteomic and metabolicanalyses did not yield any evidence for the well-described fumarate-additionmechanism for anaerobic alkane activation. Surprisingly for a denitrifier,N 2O did not sustain growth of strain HdN1 with alkanes, while it supportedfast growth with fatty acids or long-chain alcohols [1]. Cultures that grew ontetradecane formed N 2O and N 2 in short-term experiments from nitrite ornitric oxide, as detected by membrane-inlet mass-spectrometry (MIMS).Monooxygenases presumably involved in alkane-activation were found to beexpressed in cells grown on tetradecane and nitrate in anoxic medium, butnot in cells grown with tetradecanoate and nitrate. A mechanism based onthe dismutation of two NO molecules to O 2 and N 2 and the immediate use ofthe produced O 2 for „intra-aerobic” hydrocarbon-activation can be envisagedfrom these observations. A similar pathway has been suggested for theanaerobic methane oxidation by a denitrifying bacterium [2].[1] Zedelius, J. et al: Env Microbiol Rep, DOI: 10.1111/j.1758-2229.2010.00198.x.[2] Ettwig, K.F. et al. (2010): Nature 464: 543-548.AMV006The biochemistry of anaerobic ammonium oxidationW.J. Maalcke* 1 , C. Ferousi 1 , T.R. Barends 2 , W.J. Geerts 1 , J.T. Keltjens 1 ,M.S.M. Jetten 1 , B. Kartal 11 Research Group Microbiology, Darwin Center for Biogeosciences,Radboud University Nijmegen , Nijmegen, Netherlands2 Department of Biomolecular Mechanisms, Max Planck Institute forMedical Research, Heidelberg, GermanyKuenenia stuttgartiensis is a planctomycete capable of the anaerobicoxidation of ammonium to dinitrogen gas, with nitrite as electron acceptor[1]. Anaerobic ammonium oxidation (anammox) is one of the latestadditions to the nitrogen cycle, and found to play a major role in removingfixed nitrogen from oceanic oxygen minimum zones. In addition, thediscovery of anammox led to innovative new ways of treating waste water[2].Although the physiology of anaerobic ammonium oxidation is wellunderstood, the biochemistry is less clear. Based on physiological studiesand the genome sequence of K. stuttgartiensis [3], a metabolic pathway waspredicted. This pathway involves the synthesis and subsequent oxidation ofhydrazine, a toxic compound rarely found in biological systems. In thegenome sequence, candidate gene clusters for these reactions wereidentified.To provide biochemical evidence for this pathway, single cell anammoxbacteria were cultivated in a membrane bioreactor. Several highly expressedhaem-containing protein complexes were purified by FPLC and identifiedby MALDI-TOF spectroscopy. The activity of these enzymes was assayedusing colorimetric assays, and gaseous end products were analyzed by usingstable isotope labeled substrates and GC/MS.Novel multihaem protein complexes were purified and their catalyticproperties with respect to hydroxylamine and hydrazine conversion areinvestigated. Several of these had high sequence identity to hydroxylamineoxidoreductase. The detailed biochemical characterization and elucidation ofthe crystal structures of these complexes are currently in progress.[1] Strous et al. (1999): Nature 400, 446-449.[2] Kartal et al. (2010): Science 328, 702-703.[3] Strous et al. (2006): Nature 440, 790-794.AMV007The Explanation for the Hydrogenase-NegativePhenotype of Escherichia coli B Strain BL21(DE3)C. Pinske* 1 , S. Krüger 1 , M. Bönn 2 , G. Sawers 11 Institute for Biology/Microbiology, Martin-Luther-University Halle-Wittenberg, Halle(Saale), Germany2 Institute of Computer Science, Martin-Luther-University Halle-Wittenberg,Halle(Saale), GermanyUnder anaerobic conditions Escherichia coli K-12 synthesizes 3 membraneassociated[NiFe]-hydrogenases (Hyd). Hyd 1 and 2 are uptakehydrogenases that face the periplasm and transfer electrons from molecularhydrogen to the electron transport chain. Hyd 3, together with the formatedehydrogenase H, forms the hydrogen-evolving formate hydrogenlyase(FHL) complex, which uses formate as substrate. The E. coli B strainBL21(DE3) is phenotypically Hyd - when grown anaerobically. Analysis ofthe genome sequence of BL21(DE3) revealed that all of the genes encodingstructural and maturation proteins necessary for the synthesis of active[NiFe]-hydrogenases are present; however, many exhibit amino acidexchanges. In particular, the structural proteins of the FHL complex showmultiple substitutions, which correlates with the strain’s inability to producehydrogen gas. Through a series of complementation analyses we could showthat BL21(DE3) is able to produce active Hyd 1 and 2 when grown in thepresence of high concentrations of nickel ions or when the fnr gene wasintroduced on a plasmid. Immunological evidence for an Fnr protein couldnot be found in strain BL21(DE3) consistent with the finding that the fnrgene of BL21(DE3) has an amber (UAG) mutation at codon 141. Nickeltransport is known to be FNR-dependent [1]. Neither introduction of fnr noraddition of Ni 2+ ions restored FHL activity, indicating that the amino acidexchanges in the structural proteins have inactivated at least one componentof the complex. Surprisingly, introduction of the fnr gene into BL21(DE3)impaired anaerobic growth, suggesting that selective pressure for rapidlygrowing strains may have led to the inactivation of the fnr gene.[1] Wu, L.-F. and Mandrand-Berthelot, M.-A. (1986): Genetic and physiological characterization ofnew Escherichia coli mutants impaired in hydrogenase activity. Biochimie 68:167-79.spektrum | Tagungsband 2011