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

intersubunit electron transfer. Circular dichroism spectroscopic experimentsindicated nucleotide-dependent conformational changes for ChlL 2 after ATPbinding. A nucleotide-dependent switch mechanism triggering ternarycomplex formation and electron transfer was concluded. The crystalstructure of the (ChlN/ChlB) 2 complex revealed three cysteine residues and ahighly unusual aspartate residue for the coordination of the redox active[4Fe-4S] cluster of this catalytic subcomplex.PSP026Serratia odorifera emits a complex bouquet of volatilesM. Kai* 1 , E. Crespo 2 , S.M. Cristescu 2 , F.J. Harren 2 , W. Francke 3 ,B. Piechulla 11 Department of Biochemistry, University of Rostock, Rostock, Germany2 Life Science Trace Gas Facility, Radboud University Nijmegen, Nijmegen,Netherlands3 Institute of Organic Chemistry, University of Hamburg, Hamburg,GermanyOnly very recently it was realized that bacteria, including aboveground andbelowground living species, are able to emit enourmous spectra of volatiles.The physiological and ecological functions of compound mixtures and/orindividual compounds are presently far from being understood. Todetermine the complete volatile spectrum of the rhizobacterium Serratiaodorifera 4Rx13 a combination of different techniques including coupledgas chromatography/mass spectrometry (GC/MS), proton-transfer-reactionmass spectrometry (PTR-MS), laser photoacoustic spectroscopy, midinfraredlaser based spectroscopy and different analytical chemistry methodswere applied [1]. More than 100 compounds were emitted from S. odorifera4Rx13 comprising one of the most comprehensive bacterial volatile profilesknown to date. Two main components methanethiol and ´sodorifen`, a novelbicyclic multiple methylated octadien, were accompanied by dimethyldisulfide (DMDS), dimethyl trisulfide (DMTS), 2-phenylethanol, severalterpenoids and methanol. In addition to organic volatiles ammonia wasreleased, while ethylene, nitric oxid (NO) and hydrogen cyanide (HCN)could not be detected. Experiments showed that the composition of thebouquet did not alter during the growth of S. odorifera. The highestemission was detected at the beginning of the stationary phase.We are presently investigating which role these volatiles play in organismicinteractions (e.g. communication, defence, attraction).[1] Kai, M. et al (2010): Serratia odorifera:analysis of volatile emission and biological impact ofvolatile compounds on Arabidopsis thaliana.Applied Microbiology and Biotechnology 88:965-976.PSP027The phototrophic bacterium Chloroflexus aurantiacusforms acetate from acetyl-CoA via an „archaeal” ADPformingacetyl-CoA synthetaseM. Schmidt*, C. Bräsen, P. SchönheitInstitute for General Microbiology, Christian-Albrechts-University, Kiel,GermanyIn prokaryotes, the mechanism of acetate formation from acetyl-CoA andthe concomitant synthesis of ATP from ADP and phosphate appear to bedomain specific. In archaea, this reaction is catalyzed by an unusual Acetyl-CoA Synthetase (ADP-forming) (ACD), (acetyl-CoA + ADP + P ↔ acetate+ ATP + CoA) (1) whereas bacteria utilize the classical two-enzymemechanism involving phosphotransacetylase (PTA) and acetate kinase (AK).Here we studied the mechanism of acetate formation in the bacteriumChloroflexus aurantiacus, which excrete significant amounts of acetateduring phototrophic growth on glucose. In acetate-forming cells, activities ofPTA and AK could not be detected; however, the cells contained inducibleactivity of an ACD. The ACD was purified and the encoding gene identifiedvia MALDI-TOF analysis. The acd gene was expressed in E. coli and therecombinant enzyme biochemically characterized. The enzyme is ahomotetrameric protein composed of 70 kDa-subunits. Substratespecificitiesfor acetyl-CoA/acetate and other acyl-CoA esters/acids weredetermined, defining the Chloroflexus enzyme as ACD-isoenzyme I. Thisisoenzyme has been reported to be the predominant ACD in sugarfermentation of archaea. It is concluded that the bacterium C. aurantiacusutilizes an ACD, i.e. the „archaeal mechanism” for conversion of acetyl-CoA to acetate. This is the first report of a functional acetate forming ACDin the domain of bacteria.[1] Bräsen, C. (2008): J Biol Chem. 283:15409-18.PSP028Requirement of the proteins CoxE and CoxF for theassembly of the [CuSMoO 2 ] cluster in the active site ofCO dehydrogenaseA. Pelzmann*, F. Mickoleit, O. MeyerDepartment for Microbiology, University of Bayreuth, Bayreuth, GermanyCO dehydrogenase of the chemolithoautotrophic α-proteobacteriumOligotropha carboxidovorans OM5 is a structurally characterizedmolybdenum containing iron-sulfur flavoenzyme which catalyzes theoxidation of CO (CO + H 2O → CO 2 + 2 e - + 2 H + ) [1]. The [CuSMoO 2]cluster in its active site is subject to posttranslational assembly. The proteinsCoxD, CoxE, and CoxF are assumed to form a complex, which introduces Sand Cu + into [MoO 3] using apo-CO dehydrogenase as a scaffold. The threeCox-proteins resemble BchI, BchD and BchH of Mg-chelatase whichcatalyses the introduction of Mg 2+ into protoporphyrin IX [2]. CoxD is anovel AAA+ ATPase which is required for the sulfuration of [MoO 3][2].CoxE is a Von Willebrand Factor A (VWA) protein with a VWA domainand a MIDAS motif. BchD (which is analogous to CoxE) serves as aplatform for the assembly of the Mg-chelatase complex [4]. CoxF ispredicted as a histidine acid phosphatase with a VWA binding motif and thecopper binding motif MCxxHxxM [5].To get information on the functions of CoxE and CoxF, the correspondinggenes have been inactivated by insertion of a kanamycin resistance cassettewhich led to the mutants O. carboxidovorans OM5 E::km and F::km,respectively. Both mutants were unable to utilize CO underchemolithoautotrophic conditions, but they could be cultivated with H 2 plusCO 2 in the presence of CO to induce the transcription of cox genes. Themutant in coxE formed a fully assembled, but completely inactive apo-COdehydrogenase, whereas the mutant in coxF was leaky to some extentbecause its apo-CO dehydrogenase showed roughly 1% of the holo-enzymeactivity. The CO-oxidizing activity in both apo-CO dehydrogenases couldbe restored through reconstitution with the [Cu + (thiourea) 3] complex, whichsuggests the presence of a [MoO 2S] site. However, this applied only to afraction of the entire apo-CO dehydrogenase population, which might beexplained by chemical modifications at the Mo-SH; this aspect is subject tocurrent research. The presence of [MoO 2S] in the apo-CO dehydrogenasesof the two mutants is further corroborated by EPR spectroscopy whichshowed Mo(V) resting signals. Based on this data a model on the assemblyof the [CuSMoO 2] cluster is proposed.[1] Dobbek, H., L. et al (2002): Proc. Natl. Acad. Sci. USA 99: 15971-15976.[2] Fodje, M. N. et al (2001): J. Mol. Biol. 311: 111-122.[3] Pelzmann, A. et al (2009): J. Biol. Chem. 284: 9578-9586.[4] Lundqvist, J. et al (2010): Structure 18: 354-365.[5] Kaufman Katz, A. et al (2003): Helvetica Chimica Acta, 86: 1320-1338.PSP029A novel (S)-citramalyl-CoA/(R)-3-hydroxy-3-methylglutaryl-CoA lyase in Archaea, Bacteria andEukaryaM. Ziemski, P. Zadora 1 , Ö. Bukmez, I. Berg*Department of Microbiology, Albert-Ludwigs-University, Freiburg,GermanyThe genomes of many actino- and proteobacteria as well as of haloarchaeaand animals possess homologues of a gene encoding citrate lyase β-subunit,although a gene for the α-subunit of this protein is absent. Examples of suchorganisms are Pseudomonas aeruginosa, Mycobacterium tuberculosis,Haloarcula marismortui as well as Homo sapiens. The corresponding genesfrom these organisms were cloned, overexpressed in Escherichia coli. Theencoded enzymes were identified as bifunctional (S)-citramalyl-CoA/ (R)-3-hydroxy-3-methylglutaryl-CoA lyases catalyzing the following reactions:(S)-citramalyl-CoA → acetyl-CoA + pyruvate(R)-3-hydroxy-3-methylglutaryl-CoA → acetyl-CoA + acetoacetateFurthermore, we showed that in M. tuberculosis and H. marismortui thisenzyme is involved in a modified leucine degradation pathway. In theclassical pathway, leucine is first converted to 3-methylglutaconyl-CoA,which is further hydrated to (S)-3-hydroxy-3-methylglutaryl-CoA and thencleaved to acetyl-CoA and acetoacetate. In the novel modified pathway, 3-methylglutaconyl-CoA hydratation is catalyzed by an (R)-specific enoyl-CoA hydratase and leads to (R)-stereoisomer of 3-hydroxy-3-methylglutaryl-CoA. As in the classical pathway, this compound is furthersplit into acetyl-CoA and acetoacetate in the CitE catalyzed reaction. Incontrast to haloarchaea and mycobacteria, in P. aeruginosa this enzymefunctions in vivo as (S)-citramalyl-CoA lyase in itaconate catabolism.spektrum | Tagungsband 2011