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

and THNCoA reductase activities in extracts from the sulphate reducingenrichment culture N47 grown on naphthalene. The activity (0,7 nmol min -1mg -1 ) was sufficiently high for the growth rate of cells. Evidence wasobtained that two different dearomatizing reductases were involved inanaerobic naphthalene degradation: while the first reduction step of the nonactivatedring was independent of ATP hydrolysis, reduction of THNCoAwas only observed in the presence of ATP.[1] Annweiler, E. et al (2002): Identical ring cleavage products during anaerobic degradation ofnaphthalene, 2-methylnaphthalene and tetralin indicate a new metabolic pathway. Appl. Environ.Microbiol. 68:852-858.[2] Musat, F. et al (2009): Anaerobic degradation of naphthalene and 2-methylnaphthalene by strainsof marine sulfate-reducing bacteria. Environ Microbiol. 11:209-19.[3] Safinowski, M. and R.U. Meckenstock (2006): Methylation is the initial reaction in anaerobicnaphthalene degradation by a sulfate-reducing enrichment culture. Environ. Microbiol. 8:347-352.[4] Selesi, D. et al (2010): Combined Genomic and Proteomic Approaches Identify Gene ClustersInvolved in Anaerobic 2-Methylnaphthalene Degradation in the Sulfate-Reducing Enrichment CultureN47. Journal of Bact. 192:295-306.AMP023Structure and function of the F 420 -reducing [NiFe]-hydrogenases (Frh) from methanogensS. Vitt* 1 , J. Vonck 2 , D. Mills 2 , M. Strauss 2 , U. Ermler 3 , S. Shima 1,31 Max Planck Institute for Biochemistry, Marburg, Germany2 Max Planck Institute for Structural Biology, Frankfurt am Main, Germany3 Max Planck Institute for Molecular Membrane Biology, Frankfurt amMain, GermanyF 420-reducing [NiFe]-hydrogenase (Frh) is a cytoplasmic enzyme, whichcatalyzes the reversible reduction of coenzyme F 420 with H 2. Coenzyme F 420,a 5-deazaflavin, structurally resembles a flavin. However, it functionallybehaves more like the pyridine nucleotides NAD(P) + in transferring twoelectrons plus a proton (a hydride) rather than single electrons. F 420 isinvolved as a hydride donor/acceptor in the central methanogenic pathway,in which F 420 is used in the reversible redox reactions between methenylandmethylene-H 4MPT and between methylene- and methyl-H 4MPT. Frh inthe hydrogenotrophic methanogens regenerates the reduced form of F 420.Architecturally Frh forms a huge complex with a molecular mass of > 1200-kDa composed of 12 Frh protomers. Each protomer consists of the 47-kDa„large subunit” (FrhA) with the [NiFe]-center, the 26-kDa „small subunit”(FrhG) with three [4Fe4S]-clusters and the 31-kDa iron-sulfur flavoprotein(FrhB) with one [4Fe4S]-cluster and one FAD, which functions as oneelectron/two electron switch. The Frh-complex from Methanothermobactermarburgensis was purified under strictly anaerobic conditions to apparenthomogeneity. The Frh complex forms unspecific aggregates with otherproteins, which constrain the purification of this enzyme complex. Toovercome this problem we used a detergent to solve this aggregates.Structure analysis of the purified enzyme by single particle electron cryomicroscopyand x-ray crystallography are in progress.AMP024Differential expression of reductive dehalogenase geneclusters in Desulfitobacterium hafniense DCB-2 duringgrowth in the presence of different aromaticorganohalidesA. MacNelly*, T. Schubert, G. DiekertInstitute of Microbiology, Department of Applied and EcologicalMicrobiology, Friedrich Schiller University, Jena, GermanyLignin-degrading fungi of boreal forests show the ability to producechlorinated organic compounds while growing on wood. The organohalidescan be subsequently dechlorinated under anoxic conditions by aheterogeneous group of soil bacteria including Desulfitobacteriumsubspecies. Recently, a Desulfitobacterium hafniense strain was isolatedfrom a soil sample, in which ligninolytic enzyme activities were detected[1].D. hafniense strain DCB-2 harbors seven genes encoding reductivedehalogenases [2]. The organism was shown to degrade 3-chloro-4-hydroxyphenylacetate, a model compound for products of fungal lignindegradation, to 4-hydroxyphenylacetate with pyruvate as the electron donor.A 3-chloro-4-hydroxyphenylacetate reductive dehalogenase was purifiedfrom D. hafniense DCB-2 cells [3]. In the present study we tested theorganism for the ability to dechlorinate different ortho- and meta-chlorinatedphenols. Results will be presented that elucidate the effect of the differentaromatic organohalides on the reductive dehalogenase (Rdh) geneexpression in D. hafniense DCB-2. The transcript level of the different rdhgenes was tested via RT-PCR and the formation of enzymes was examinedvia activity measurements. Experiments are underway to investigate theinfluence of fungal exudates and soil extracts on the set of reductivedehalogenases formed in resting cells of D. hafniense DCB-2.[1] Ye, Lidan (2010): PhD thesis. Friedrich-Schiller-University Jena.[2] These sequence data were produced by the US Department of Energy Joint Genome Institute(http://www.jgi.doe.gov/).[3] Christiansen, N. et al. (1998): FEBS Letters 436:159-162.AMP025The electron transport chain of nitrous oxide respirationin Wolinella succinogenesM. Luckmann*, M. Kern, J. SimonInstitute of Microbiology and Genetics, University of Technology,Darmstadt, GermanyLaughing gas (nitrous oxide) is one of the most important greenhouse gasesand accounts for about 10% of the global warming effect. It is commonlyproduced in the environment by denitrifying and nitrifying microbialspecies. In addition to denitrifiers, some respiratory nitrate-ammonifyingEpsilonproteobacteria also reduce nitrous oxide to dinitrogen although theseorganisms probably do not produce substantial amounts of endogenousnitrous oxide in energy substrate turnover. The energy metabolism of one ofthese bacteria, Wolinella succinogenes, has been thoroughly characterized inthe past. These cells use either hydrogen or formate as electron donortogether with typical terminal electron acceptors of anaerobic respirationlike fumarate, nitrate or polysulfide. Here, we show that W. succinogenesgrows efficiently with formate and nitrous oxide as sole energy substrates tohigh optical densities. Nitrous oxide is reduced by an unconventionalcytochrome c nitrous oxide reductase (cNosZ) whose presence seems to belargely restricted to Epsilonproteobacteria. The corresponding nos genecluster predicts the presence of a unique electron transport system that ispredicted to connect the menaquinone/menaquinol pool with cNosZ. Theinvolved electron transport chain may comprise a menaquinoldehydrogenase of the unusual NapGH-type and one or two monohaemcytochromes c. Various nos gene cluster mutants were constructed andcharacterized with regard to growth behaviour and enzyme activity. Basedon these data, a model of the respiratory cNos system in W. succinogeneswill be presented.AMP026Molybdo- and tungstoenzymes in the anaerobicmetabolism of aromatics in Aromatoleum aromaticum:Ethylbenzene dehydrogenase and phenylacetaldehyde:ferredoxin oxidoreductaseC. Debnar-Daumler*, D. Knack*, J. HeiderLaborartory for Microbiology, Philipps-University, Marburg, GermanyAromatoleum aromaticum contains several molybdo- or tungstoenzymes of3 different families: DMSO reductase, xanthin oxidase andaldehyde:ferredoxin oxidoreductase (AOR). Among these are enzymesinvolved in the degradation of different aromatics such as the DSMOreductase type enzyme ethylbenzene dehydrogenase and the AOR typeenzyme phenylacetaldehyde:ferredoxin oxidoreductase. These two enzymeswill be presented here.Ethylbenzene dehydrogenase (EbDH) catalyzes the first step of anaerobicethylbenzene degradation, namely the oxygen independent hydroxylation ofethylbenzene to (S)-phenylethanol. EbDH is a heterotrimeric (αβγ)periplasmic enzyme of 160 kDa. The large α subunit contains a bismolybdopterincofactor as the active site of the enzyme (MoCo enzyme).The α- and β subunits contain 5 [Fe 4S 4] clusters which are involved in thetransport of electrons. The smallest subunit (γ) contains a heme b whichaccepts the electrons from the iron-sulfur clusters of the β subunit. The basicbiochemical and structural properties of the enzyme were investigatedrecently. New insights into the catalytic mechanism will be shown on ourposter.In contrast to EbDH, AOR enzymes contain a tungsten cofactor and mostrepresentatives described play important roles in peptide fermentation inhyperthermophlic archaea. However, more and more AOR type enzymes arealso found in anaerobic mesophilic bacteria. When grown on phenylalanineas sole carbon source, A. aromaticum produces an enzyme homologous tothese thermophilic tungsten enzymes. Simultaneously, an inducedphenylacetaldehyde:ferredoxin oxidoreductase activity has been observed inspektrum | Tagungsband 2011