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

93extremes in salinity (Halomonas elongata), pH (Alkalilimnicola ehrlichii;Acidiphilium cryptum) or temperature (Sphingopyxis alaskensis; Geobacillussp. Y412MC10). Although the kinetic parameters and catalytic properties of thecharacterized ectoine hydroxylases from these extremophiles are very similar,some of studied EctD proteins are very robust enzymes that makes theminteresting candidates as catalyst in recombinant-DNA based whole-cellbiotransformation processes and for structural analysis.(1) Bursy, J., Pierik, A.J., Pica, N. and Bremer, E. (2007). J. Biol. Chem. 282:31147-31155.(2) Reuter, K., Pittelkow, M., Bursy, J., Heine, A., Craan, T. and Bremer, E. (2010). PLoS ONE5(5):e10647.(3) Bursy, J., Kuhlmann, A.U., Pittelkow, M., Hartmann, H., Jebbar, M., Pierik, A.J. and Bremer, E. (2008).Appl. Env. Microbiol. 74:7286-7296.MEP003Structure-guided site-directed mutagenesis of the ectoinehydroxylase from the moderate halophile Virgibacillus salexigensM. Pittelkow 1 , N. Widderich* 1 , W. Buckel 1,2 , E. Bremer 11 Philipps-University Marburg, Biology, Marburg, Germany2 Max Planck Institute for Terrestrial Microbiology, Marburg, GermanyIncreases in the external salinity triggers water efflux from the microbialcell and the ensuing dehydration of the cytoplasm negatively affects cellgrowth and impairs survival. To balance the osmotic gradient across thecytoplasmic membrane, many microorganisms amass a selected class oforganic compounds, the "compatible solutes". One of the most widely usedcompatible solutes by members of the Bacteria is the tetrahydropyrimidineectoine and its derivative 5-hydroxyectoine. These two compatible soluteshave attracted considerable biotechnological attention, are produced inlarge-scale fermentation processes employing halotolerant microorganismsand are commercially used in skin-care products, as protein and cellstabilizers and medical applications of ectoines are envisioned. About athird of all microbial ectoine producers also synthesize 5-hydroxyectoinefrom ectoine. 5-hydroxyectoine is synthesized by a stereo-specifichydroxylase (EctD) that is a member of the non-heme iron (II) and 2-oxoglutarate-dependent dioxygenase super-family (1). Microbial EctDtypeproteins are closely related to each other and belong structurally to thePhyH-subgroup within the dioxygenase super-family. This was disclosedby the recently reported high-resolution crystal structure of the ectoinehydroxylase from the moderate halophile Virgibacillus salexigens (2). Thisstructure revealed the unambiguous positioning of the iron ligand withinthe active site of the EctD enzyme by an evolutionarily conserved ironbindingmotif, the so-called 2-His-1-carboxylase facial triad. However, theobtained crystal structure contained neither the substrate ectoine nor theco-substrate 2-oxoglutarate. Here we used the crystal structure of the V.salexigens EctD enzyme as a template to functionally probe, via sitedirectedmutagenesis, amino acid residues that seemed important for thecorrect positioning of the ligand ectoine and the co-substrate 2-oxoglutatewith respect to the catalytically critical iron-ligand. These studies allowedus to map the spatial organization of the active site of EctD that is buriedin a deep caveat formed by the monomeric EctD protein. A detailedreaction scheme for the stereo-chemical hydroxylation of ectoine to 5-hydroxyectoine catalyzed by the EctD enzyme will be presented.(1) Bursy, J., Pierik, A.J., Pica, N. and Bremer, E. (2007). J. Biol. Chem. 282:31147-31155.(2) Reuter, K. Pittelkow, M., Bursy, J., Heine, A., Craan, T. and Bremer, E. (2010). PLoS ONE 5(5):e10647MEP004Biosynthesis, Partial Purification and Characterization ofInvertase from Sacchromyces cerevisae by Solid-StateFermentation of Carrot PeelsZ.-E. Bilal*, H. AshrafUniversity of the Punjab, Agricultural sciences, Lahore, PakistanPotential of different Sacchromyces species,cultivated under solid-statefermentation (SSF) using carrot peels (Daucus carota L.) as substrate wasinvestigated. The highest productivity of invertase (7.95 U mL -1 ) wasachieved by using Sacchromyces cerevisae on 90% initial moisture contentwith 2.5 ml inoculum size after 72 h of incubation period. The enzyme waspurified about 1.42 fold by ammonium sulphate precipitation. It showedthermal stability from 20-40 o C over a pH range 5.5 to 6.5 with maximumactivity at pH 5.5 and 50° C. The enzyme was highly active towardssucrose at both concentrations viz: 0.1 M and 0.5 M, but it showed lessactivity towards glycerol. It was completely inhibited by Hg 2+ (1mM) andslightly stimulated by Co 2+ and Na +1 at the same concentration.a unique active site iron-guanylylpyridinol (FeGP) cofactor, in which alow-spin Fe II is coordinated by a pyridinol nitrogen, an acyl group, twocarbon monoxide, and the sulfur of the enzyme’s cysteine. Here, westudied the biosynthesis of the FeGP cofactor by following theincorporation of 13 C and 2 H from labeled precursors into the cofactor bygrowing methanogenic archaea and by subsequent NMR, MALDI-TOF-MS and/or ESI-FT-ICR-MS analysis [s1] of the isolated cofactor andreference compounds. The cofactors pyridinol moiety was found to besynthesized from three C-1 of acetate, two C-2 of acetate, two C-1 ofpyruvate, one carbon from the methyl group of l-methionine, and onecarbon directly from CO 2. The metabolic origin of the two CO- ligandswas CO 2 rather than C-1 or C-2 of acetate or pyruvate excluding that the twoCO are derived from dehydroglycine as has previously been shown for the COligandsin [FeFe]-hydrogenases. A formation of the CO from CO 2 via directreduction catalyzed by a nickel-dependent CO dehydrogenase or from formatecould also be excluded. When the cells were grown in the presence of 13 CO thetwo CO-ligands and the acyl group became 13 C labeled, indicating that free COis either an intermediate in their synthesis or that free CO can exchange withthese iron-bound ligands. Based on these findings, we propose pathways ofhow the FeGP cofactor might be synthesized.MEP006A recombinant system for the biotransfomation of ectoine intothe chemical chaperone 5-hydroxyectoineN. Stöveken*, N. Widderich, M. Pittelkow, E. BremerPhilipps University Marburg, Laboratory of Microbiology, Marburg,GermanyEctoine and 5-hydroxyectoine are an important class of compatible solutesthat are synthesized by many microorganisms in response to high salinity.Some ectoine producers transform part of the newly formed ectoine into 5-hydroxyectoine through the enzymatic action of the ectoine hydroxylase(EctD), a non-heme iron (II)- and 2-oxoglutarate dependent dioxygenase(1, 2). Ectoine and 5-hydroxyectoine have attracted considerablybiotechnological interest since they possess interesting stabilizingproperties for proteins, nucleic acids, membranes and whole cells.Although closely related in chemical structure, ectoine and 5-hydroxyectoine have different properties, with 5-hydroxyectoine beingoften the more effective stabilizing compound and the more potent cellularstress protectant. Currently, ectoine and 5-hydroxyectoine arebiotechnologically produced by large-scale fermentation of halotolerantmicroorganisms using the bacterial milking process. Synthesis of 5-hydroxyectoine depends on the prior production of ectoine, a processwhose efficiency depends on various environmental conditions and thegrowth phase of the culture. As a consequence, ectoine/5-hydroxyectoineproducers often contain a mixture of these compounds and this requirestime-consuming and costly separation procedures during the downstreamprocesses for the biotechnological production of pure ectoine and 5-hydroxyectoine. Recombinant-DNA based biotransformation processesmight be an interesting alternative to produce 5-hydroxyectoine.Escherichia coli can import ectoine under osmotic stress conditions (viathe ProP and ProU transporters) but it cannot synthesize it. We set up a cellfactory of an E. coli strain that is unable to synthesize its naturalcompatible solute trehalose and that carries on a plasmid heterologousectD genes whose expression can be triggered by adding an inducer to thegrowth medium. This biotransformation process was optimized by usingdifferent expression strains, various cultivation conditions and byemploying EctD proteins from various extremophiles. We found that ectoine iseffectively taken up by these recombinant E. coli cells, converted efficientlyinto 5-hydroxyectoine and that a substantial portion of the newly produced 5-hydroxyectoine is secreted into the growth medium.(1)Bursy, J., Pierik, A.J., Pica, N. and Bremer, E.(2007) Osmotically induced synthesis of thecompatible solute hydroxyectoine is mediated by an evolutionarily conserved ectoine hydroxylase.J. Biol. Chem.282:31147-31155.(2)Reuter, K., Pittelkow, M., Bursy, J., Heine, A., Craan, T. and Bremer, E.(2010) Synthesis of 5-hydroxyectoine from ectoine: crystal structure of the non-heme iron (II) and 2-oxoglutaratedependentdioxygenase EctD. PLoS ONE 5(5):e10647.MEP005Biosynthesis of the iron-guanylylpyridinol cofactor of [Fe]-hydrogenase in methanogenic archaea as elucidated by stableisotopelabelingM. Schick*, X. Xie, U. Linne, J. Kahnt, S. ShimaMPI für terrestrische Mikrobiologie, Biochemie, Marburg, Germany[Fe]-hydrogenase catalyzes the reversible hydride transfer from H 2 tomethenyltetrahydromethanoptherin, which is an intermediate in methaneformation from H 2 and CO 2 in methanogenic archaea. The enzyme harborsBIOspektrum | Tagungsband 2012