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

185on acetophenone or acetate. It consists of 3 subunits (85, 75, and 20 kDa)in an () 2 composition and contains 2 Fe and 1 Zn per native complex.Interestingly, known acetone carboxylases from other organisms (e.g.Paracoccus denitrificans, Rhodobacter capsulatus, Geobacillusthermoglucosidasius) differ in in metal content and ATP hydrolysisstoichiometry, despite their high sequence similarities.Acetophenone carboxylase (Apc) is present in cells grown aerobically oranaerobically on acetophenone, but not in cells grown on acetone or 4-hydroxyacetophenone. Acetophenone is carboxylated to benzoylacetateconcomitant with hydrolysis of 2 ATP to 2 ADP and 2 Pi. 4-hydroxyacetophenone is not a substrate of Apc. The enzyme consists of 5subunits (87, 75, 70,34, and 15 kDa) in an (’) 2 2 composition. Four ofthe five subunits show high sequence similarity to the subunits of Acx,while the -subunit is unique. 2 Zn per native complex were identified ascofactors, but no Fe or biotin. The observed reaction mechanisms ofacetone carboxylase and acetophenone carboxylase represent novel ATPdependent,biotin-independent carboxylation mechanisms in bacterialketone catabolism, which likely involve the transient activation of bothsubstrates via phosphorylation.4-Hydroxyacetophenone carboxylase (Xcc) belongs to the class of biotindependentcarboxylases and consists of 3 subunits: a biotin carboxyl carrierprotein (18 kDa) and 2 carboxylase subunits (50, 55 kDa). Therefore, despitethe similarity of the respective substrates, completely different carboxylationmechanisms are employed for the carboxylation of acetophenone and 4-hydroxyacetophenone.PSP041Biosynthesis and attachment of open-chain tetrapyrroles incryptophytesK. Overkamp, N. Frankenberg-Dinkel, J. Schwach*Ruhr-University Bochum, Physiology of microorganisms, Bochum,GermanyPhycobiliproteins are light-harvesting proteins, which occur incyanobacteria, red algae and cryptophytes in addition to chlorophyllcontaining antenna complexes. They allow the organisms to efficientlyabsorb light in regions of the visible spectrum that are poorly covered bychlorophylls. Cryptophytes are unicellular, eukaryotic algae andwidespread in marine and limnic waters. Their phycobiliproteins consist ofan (‘) heterotetrameric apo-protein covalently associated withcharacteristic open chain tetrapyrroles, which act as light absorbingchromophores. Cryptophytes employ the six different chromophoresphycocyanobilin (PCB), phycoerythrobilin (PEB), 15,16-dihydrobiliverdin(15,16-DHBV), mesobiliverdin (MBV), bilin 584 and bilin 618 for lightharvesting.The biosynthetic pathway of open chain tetrapyrroles in cryptophytes isentirely unknown. The model organismGuillardia thetauses thephycobiliprotein PE545, which is associated with the chromophores 15,16-DHBV and PEB. This is an interesting fact, because 15,16-DHBV occursonly as an intermediate of PEB biosynthesis in cyanobacteria andcyanophages but not as a bound chromophore. This raises the question ofelucidating the chromophore biosynthesis and attachment in thecryptophyteG. theta. Extensive bioinformatic analyses and amino acidsequence alignments identified a putative heme oxygenase, two putativebilin reductases and different putative phycobiliprotein lyases inG. theta.Currently, the enzymatic activities of these putative bilin biosynthesisenzymes are analyzed. First results give some indications that the hemeoxygenase is able to cleave heme yielding the open-chain tetrapyrrolebiliverdin IX. Furthermore a bilin reductase reducing 15,16-DHBV to PEBcould be identified, which will be further investigated via crystallization studies.The enzymatic activity of a second bilin reductase will also be examined as wellas the attachment of the PEB molecules to the PE545- subunits and especiallythe 15,16 DHBV molecules to the PE545- subunits.PSP042Itaconate degradation may be important for pathogenesisJ. Sasikaran, M. Ziemski, P. Zadora, I. Berg*Albert-Ludwigs-University, Department of Microbiology, Freiburg,GermanyItaconate (methylenesuccinate) has recently been shown as a mammalianmetabolite whose production is induced during macrophage activation (1).This compound is a potent inhibitor of isocitrate lyase (2), which isimportant for survival of many pathogens inside the host (3). We haveshown that numerous pathogens includingYersinia pestisandPseudomonasaeruginosapossess genes for itaconate degradation, which were previouslyshown as pathogenesis-related in some species (4,5). Furthermore, weheterologously overproduced and characterized in detail a key enzyme ofthe itaconate degradation pathway, (S)-citramalyl-CoA lyase, fromY.pestisandP. aeruginosa. Besides bacteria, this enzyme is present inmammals. Interestingly, the corresponding gene was previously shown tobe highly expressed in some tumor cell lines with high metastaticpotential(6). Itaconate detoxification might be important for these cells,since this compound is an indirect inhibitor of phosphofructokinase (7) andtherefore of the glycolysis, the main bioenergetic process in tumor cells.Thus, itaconate degradation pathway may be considered as a perspectivetarget for the development of novel therapeutic agents.1. Strelko, C.L., et al. J. Am. Chem. Soc. 133, 16386-16389 (2011).2. Williams, J.O., et al. Biochemistry 10, 1384-1390 (1971).3. Dunn, M.F., et al. Microbiology 155, 3166-3175 (2009).4. Pujol, C., et al. Proc. Natl. Acad. Sci. USA 102, 12909-12914 (2005).5. Eriksson, S., et al. Mol. Microbiol. 47, 103-118 (2003).6. Morikawa, J., et al. Biochem. Biophys. Res. Commun. 289, 1282-1286 (2001).7. Sakai, A., et al. Nutrition 20, 997-1002 (2004).PSP043Cac0116 of Clostridium acetobutylicum - a carbon monoxidedehydrogenase?R. Uhlig*, R.-J. Fischer, H. BahlInstitute of Biological Sciences, Division of Microbiology, Rostock, GermanyCarbon monoxide dehydrogenases (CODHs) of anaerobic Organisms areenzymes with a special nickel, iron and sulphur containing cluster,enabling the reversible oxidation of CO to CO 2 [1]. CODHs are involvedin several metabolic functions like energy conservation, autotrophic CO 2-fixation or reductive regeneration of NADPH. Another function waspostulated for CODH-IV of the hydrogenogenic bacteriumCarboxydothermus hydrogenoformans. Based on the fact that the CODH-IV gene is located in a cluster of genes that might be necessary for thedetoxification of reactive oxygen species (ROS), a hydrogen peroxidereducing role was discussed [2].In Clostridium acetobutylicum the genes cac0116 and cac2498 are annotated asCODHs. Recent studies demonstrated a highly upregulation (24 fold) of thegene cac0116 under oxidative stress leading to the conclusion that its geneproduct is part of the ROS detoxification system [3].Here, we report on the purification of Cac0116 after overexpression in E.coli and C. acetobutylicum. So far, our results did not reveal any CODHactivity of this enzyme. Furthermore, a specific cac0116 knock out mutantwas constructed by using the ClosTron ® technology [4]. Comparativecharacterisation of the phenotypes (optical density, pH, product spectrum,produced gases) of the knock out mutant, the overexpression strain and thewild type strain of C. acetobutylicum indicated in the knock out mutant areduced glucose consumption. Interestingly, the H 2:CO 2 ratio seemed to bealtered, when the cac0116 gene was inactivated. This suggests a functionof Cac0116 in electron transfer processes, directly or indirectly coupledwith H 2 production in C. acetobutylicum.[1] James, G. F., 1995, Annu. Rev. Microbiol. 49:305-333.[2] Wu, M.et al., 2005, PLoS Genetics 1:563-574.[3] Hillmann, F., 2009, J. Bacteriol. 191:6082-6093.[4] Heap, J.et al., 2007, J. Microbiol. Methods. 70:452-464.PSP044Sulfur metabolism in the thermoacidophilic archaeonMetallosphaera cuprina: insights from genome analysis andgene expression studiesL. Liu* 1,2 , Y. Stockdreher 1 , M. Josten 3 , H.-G. Sahl 3 , C.-Y. Jiang 2 , S.-J. Liu 2 ,C. Dahl 11 Universität Bonn, Institut für Mikrobiologie & Biotechnologie, Bonn, Germany2 Chinese Academy of Sciences, Institute of Microbiology, Beijing, China3 Universität Bonn, Institut für Medizinische Mikrobiologie, Bonn, GermanyThe thermoacidophilic archaeon Metallosphaera cuprina Ar-4, originallyisolated from a sulfuric hot spring, Tengchong, Yunnan, China, has theability to oxidize reduced inorganic sulfur compounds (RISC) [1]. Thegenome has been completely sequenced and annotated. It consists of a1,840,348 bp circular chromosome (2029 ORFs) [2], including at least 35genes putatively related to sulfur metabolism.Genes potentially encoding a heterodisulfide reductase complex HdrABCare found in several archaeal and bacterial sulfur oxidizers. Correspondinggenes also exist in Metallospahera cuprina and are part of a gene cluster(mcup_0681-0689) that also comprises dsrE and sirA like genes. Inbacteria, rhodanese (thiosulfate:cyanide sulfurtransferase) encoding genesoften occur in immediate vicinity of dsrE-sirA homologous genes. Proteinsof the DsrE and SirA families have been implicated to be involved insulfur transfer reactions not only during biosynthesis of sulfur-containingcell constitutents like thiouridine [3] but also during oxidative sulfurmetabolism [4]. In both, the archaeon Metallosphaera sedula [5] and theproteobacterium Acidithiobacillus ferrooxidans [6], the hdr gene clusterincluding the sirA and dsrE homologs is highly upregulated by RISCfurther stressing a potential prominent role of the encoded proteins inoxidative sulfur metabolism.Mcup_0681 and Mcup_0682 from M. cuprina share 26% identity and bothpossess characteristic features of DsrE family proteins. Mcup_0683 is assignedas a SirA family protein. Mcup_0681-0683 were overproduced in E. coli. Both,Mcup_0681 and Mcup_0682, were identified as homotrimers by gelpermeation chromatography while Mcup_0683 is a monomer. Strong andspecific interaction between Mcup_0681 and Mcup_0683 was detected by cochromatographyof pairs of tagged and untagged proteins on Strep-Tactincolumns. All three proteins contain a strictly conserved cysteine residue in aBIOspektrum | Tagungsband 2012