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

131OTV003First crenarchaeal chitinase detected in Sulfolobus tokodaiiT. Staufenberger*, J.F. Imhoff, A. LabesGEOMAR, KiWiZ, Kiel, GermanyChitin is after cellulose the second most abundant biopolymer on earth,consisting of beta 1,4-glycosidic bonded N-acetyl-glucosamine subunitswith various grades of acetylation. It is wide spread from deserts to thedeep sea, generated mostly by arthropoda and fungi with a production andsteady state amount of an estimated 10 10 to 10 11 tons per year [1]. Chitindegradation is an extremely important step in nutrient cycling especially inthe oceans [2] and comprises the combined action of several enzymes.During the degradation process chitinases (EC3.2.4.14) mainly hydrolysethe beta 1,4-glycosidic bonds within the chitin polymer. Althoughchitinases are widely distributed in all domains of life, only little is knownabout archaeal chitinases. Within the domain of archaea, only teneuryarchaeal chitinases were found so far in terms of genetic or molecularinformation. Until now, no chitinases or chitinase genes were described orannotated from crenarchaea.Here we show that the ORF BAB65950 from Sulfolobus tokodaii str. 7encodes for the first functional crenarchaeal chitinase. The ORF wasexpressed in E. coli and the resulting protein degraded chitin. It was henceclassified as a chitinase (EC 3.2.4.14). The protein characterisationrevealed a specific activity of 75 mU/mg when incubated with colloidalchitin as substrate. The optimal activity of the enzyme was at pH 2.5 and70°C. A dimeric enzyme configuration is proposed. The derived aminoacid sequence of the enzyme could neither be attributed to the glycosidehydrolase family 18 nor 19. However, within a phylogenetic sequence tree,the deduced amino acid sequence of the ORF clustered into closeproximity of members of the glycoside hydrolase family 18 [3].[1] Patil et al. 2000 Enzyme and Microbial Technology; 26: 473-483[2] Poulicek et al. 1991 Biochemical Systematics and Ecology; 19: 385-394[3] Staufenberger et al. 2011 Microbiological Research; in pressOTV004A novel biosynthetic pathway for the synthesis of Archaeatypeether lipids in BacteriaH. Guldan 1 , F.-M. Matysik 2 , M. Bocola 1 , R. Sterner 1 , B. Patrick* 11 University of Regensburg, Biophysics and physical Biochemistry,Regensburg, Germany2 University of Regensburg, Analytical Chemistry, Chemo- and Biosensors,Regensburg, GermanyThe universal tree of life divides all organisms into the phylogeneticsuperkingdoms Eukarya, Bacteria and Archaea, which differ by thechemical composition of their membrane lipids. Lipids from Bacteria andEukarya are composed of a sn-glycerol-3-phosphate core to which fattyacids are bound via ester linkages, while lipids from Archaea consist of snglycerol-1-phosphate(G1P) to which polyprenyl chains are attached byether bonds.This difference has suggested that the emergence of the Archaea duringevolution was linked to the advent of glycerol-1-phosphate dehydrogenase(G1PDH) and geranylgeranylglyceryl phosphate synthase (GGGPS).These enzymes catalyze the first two steps leading to G1P-based etherlipids, the reduction of dihydroxyacetone phosphate to G1P, and thecondensation of G1P with geranylgeranyl pyrophosphate togeranylgeranylglyceryl phosphate.We were interested to elucidate the function of the hithertouncharacterized AraM and PcrB proteins, which show a significantsequence similarity to the archaeal G1PDH and GGGPS, respectively, butoccur in gram-positive bacteria such as Bacillus subtilis. We first showedthat AraM is a Ni 2+ -dependent G1PDH [1] . We then analyzed the functionof PcrB, which is a homologue of the archaeal GGGPS and therefore wasassumed to link G1P generated by AraM with an unknown polyprenylpyrophosphate substrate, yielding a specific ether lipid. We developed aprotocol for the identification of this substrate of PcrB which is based onits reaction with 14 C-G1P and the subsequent isolation of the formed radiolabeledether lipid product from B. subtilis cells. The results showed thatPcrB catalyzes the reaction of G1P with heptaprenyl pyrophosphate toheptaprenylglyceryl phosphate, which is subsequently dephosphorylatedand acetylated.The functional assignment of AraM and PcrB has allowed us to identify ahitherto unknown pathway for the biosynthesis of archaea-type ether lipidsin gram-positive bacteria. Moreover, we show that the different substratespecificities of the archaeal GGGPS and the bacterial PcrB, which bindpolyprenyl moieties containing 20 and 35 carbon atoms, respectively, arecaused by a single amino acid difference at the bottom of the active site [2] .[1] H. Guldan, R. Sterner, P. Babinger, Biochemistry 2008, 47, 7376-7384.[2] H. Guldan, F. M. Matysik, M. Bocola, R. Sterner, P. Babinger, Angewandte Chemie Int. Ed. 2011, 50,8188-8191.OTV005De novo structure of the membrane anchor domain of thetrimeric autotransporter YadA by solid-state NMR spectroscopyD. Linke* 1 , S. Shahid 1 , M. Habeck 1 , B. Bardiaux 2 , B. van Rossum 21 MPI Entwicklungsbiologie, Protein Evolution, Tübingen, Germany2 FMP Berlin, Berlin, GermanyQuestion: Solid-state magic-angle spinning (MAS) NMR spectroscopyhas long been discussed as the emerging method of choice for membraneprotein structural biology (1,2). MAS NMR does not necessarily needhighly and macroscopically ordered material and is not hampered by slowtumbling. Moreover, solid-state NMR is a unique tool to study bothdynamics and structure of proteins simultaneously at atomic resolution (3-5).YadA is a trimeric autotransporter adhesin (TAA (6)). Many members ofthe TAA family are important pathogenicity factors that mediate adhesionto host cells and tissues in such diverse diseases as diarrhea, urinary tractinfections, or airway infections. The common structural features of TAAsare trimeric domains with a high content of alpha-helical coiled coils andof beta-helical or beta-trefoil structures (6). These domains occur invarying order and repeat number in different bacterial TAAs, but thedefining element of the family is the membrane anchor (or translocator)domain which hosts two important functions. It anchors the adhesin in thebacterial outer membrane and exports all other, extracellular domains tothe cell surface - hence the name, autotransporter. The mechanism of thisautotransport is poorly understood.Methods: solid-state magic angle spinning NMRResults: Here, we present the first structure of a membrane protein, thetransmembrane domain of the Yersinia Adhesin A (YadA), solvedexclusively with solid-state MAS NMR data, using a single, uniformly13C/15N labeled sample.Conlusions: The first partial structure of a TAA was obtained for thecollagen-binding, extracellular head domain of YadA from theenteropathogenYersinia enterocolitica (7). Thus far, only for one TAAanchor domain, of Haemophilus Hia, an x-ray structure has been obtained(11). We applied solid-state MAS NMR to crystalline YadA-M to collecthigh-resolution structural data. In addition, NMR allowed us to acquireinformation on flexibility and other mechanistic detail that cannot betransferred from the x-ray structure of Hia (11).1. A. McDermott, Annual Review of Biophysics 38, 385 (2009).2. P. J. Judge, A. Watts, Current Opinion in Chemical Biology 15, 690 (2011).3. W. T. Frankset al., Journal of the American Chemical Society 127, 12291 (2005).4. S. Jehleet al., Nature Structural & Molecular Biology 17, 1037 (2010).5. C. Wasmeret al., Science 319, 1523 (2008).6. D. Linke et al., Trends in Microbiology 14, 264 (2006).7. H. Nummelinet al., The EMBO Journal 23, 701 (2004).8. U. Grosskinskyet al., Journal of Bacteriology 189, 9011 (2007).9. U. Lehr et al., Molecular Microbiology 78, 932 (2010).10. A. Roggenkampet al., Journal of Bacteriology 185, 3735 (2003).11. G. Meng et al., The EMBO journal 25, 2297 (2006).OTV006Biochemical and structural analysis of FlaH, a component ofthe crenarchaeal flagellumT. Neiner* 1 , K. Lassak 1 , A. Ghosh 1 , S. Hartung 1,2 , J.A. Tainer 2 , S.-V. Albers 11 Max Planck Institut for terrestrial Microbiology , Molecular biology ofArchaea, Marburg, Germany2 Lawrence Berkeley National Lab, Life Sciences Division, Berkeley, UnitedStatesMotility is a very important attribute of live. It allows the organisms fromall three domains of life to adapt to a changing environment, which iscrucial for the survival of various species. The two most commonly usedmotility structures in bacteria are flagella and type IV pili. Flagella areinvolved in swimming motility whereas type IV pili are mostly involved intwitching motility. Both modes of movements have been extensivelystudied and the assembly systems and functions are well characterized.This is not the case for archaeal motility. Numerous cell appendages suchas flagella and pili have been already identified, but still not much isknown about their assembly mechanisms and functions. We are mostlyinterested in the archaeal flagellum, which is a unique motility apparatusthat performs the same function as bacterial flagella; although it isstructurally more related to bacterial type IV pili. In most knownflagellated archaea the flagella-associated genes are organized in a singlefla gene cluster, consisting of flagellin encoding genes (flaA, flaB), somevariations of genes encoding accessory proteins (flaCDEGFH), genesencoding an ATPase (flaI) and a polytopic membrane protein (flaJ).Using Sulfolobus acidocaldarius as a model organism we want to analyzethe crenarchaeal flagella assembly system and its function. We couldalready show that all seven genes encoded in the fla gene cluster ofSulfolobus acidocaldarius are essential for crenarchaeal flagella assemblyand for swimming motility in liquid environments. My project is mainlyfocused on the in vivo and in vitro study of S. acidocaldarius flagellacomponent FlaH. FlaH is an incomplete ATPase, which contains a welldefined Walker A, but lacks the Walker B motive. As we determined thestructure of FlaH and using this, we constructed defined point mutants.Combining mutant analysis with biochemical studies will help us toBIOspektrum | Tagungsband 2012