Biological transformations of hydrocarbons without oxygen: from the

BIOLOGICAL TRANSFORMATIONS OF HYDROCARBONS

DFG – Priority Programme 1319

Biological transformations of hydrocarbons without oxygen:

from the molecular to the global scale

08 – 10 March, 2010 First Meeting

Schloss Machern/Leipzig

SPP 1319


DFG – Priority Programme 1319

Biological transformations of hydrocarbons without

oxygen: from the molecular to the global scale

1. Meeting 08 – 10 March, 2010

Schloss Machern / Leipzig

ORGANISATION: Prof. Dr. Matthias Boll Institute of Biochemistry Brüderstr. 34 University of Leipzig 04103 Leipzig phone: +49‐341‐9736996 e‐mail: boll@uni‐leipzig.de Prof. Dr. Rainer U. Meckenstock Helmholtz Zentrum München Institute of Groundwater Ecology Ingolstädter Landstr. 1 85764 Neuherberg phone: +49‐89‐3187‐2561 e‐mail: rainer.meckenstock@helmholtz‐muenchen.de


VENUE: Schloss Machern Schlossplatz 1 04827 Machern Tel. 03 42 92 / 7 20 79 Fax 03 42 92 / 7 28 30 [email protected] http://www.schlossmachern.de/


Monday, 08 March, 2010 .

13.00 Lunch

14.00 Welcome Matthias Boll

Anaerobic metabolism of aromatic compounds

Chair: Matthias Boll

14.10‐14.40

Markus Hilberg

AG Heider, Marburg

Anaerobic toluene metabolism: Biochemistry of benzylsuccinate synthase and its activating enzyme

14.40‐15.10

Ralf Rabus

Oldenburg

Anaerobic degradation of p‐alkylbenzoates and –toluenes by the denitrifying strains pMbN1 and pCyN1

15.10‐15.40

Ramon Diesveld

AG Meckenstock, Munich

The initial step of anaerobic benzene degradation in the iron reducing culture BF is a carboxylation to benzoate

15.40‐16.10

Martin Taubert

AG von Bergen, Leipzig Application of protein based stable isotope probing (Protein‐SIP) to unravel anoxic benzene degradation

16.10 – 16.40

Coffee Break

16.40‐17.10

Johannes Kung

AG Boll, Leipzig

The tungsten‐containing class of benzoyl‐CoA reductases: characterization and distribution in obligately anaerobic bacteria

17.10‐17.40

Tobias Weinert

AG Ermler, Frankfurt Crystallization of benzoyl‐CoA reductase and acetophenone carboxylase both involved in the anaerobic degradation of hydrocarbons

17.40‐18.10

Berta Martins

Berlin Crystal structure of 4‐hydroxyphenylacetate decarboxylase from Clostridium scatologenes, a 2[4Fe‐4S] cluster‐containing glycyl radical dependent enzyme

18.15‐19.30

Dinner

Evening Lectures I Chair: Wolfgang Buckel

19.30‐20.30

Rudolf K. Thauer

Marburg

Reverse methanogenesis: the key nickel enzyme MCR catalyses anaerobic oxidation of methane.

20.30‐21.30

Marc Fontecave

Grenoble

Iron‐sulfur clusters and radical biocatalysis: applications in the modification of macromolecules


Tuesday, 09 March, 2010 .

7.30 Breakfast

Anaerobic metabolism of aliphatic compounds

Chair: Hans Heider

8.30‐ 9.00

Marc Staßen

AG Rother, Frankfurt

Genetic and biochemical analysis of methyltransferase function and mechanism in Methanosarcina acetivorans C2A

9.00‐ 9.30

Marie Kim

Ivana Djurdjevic

AG Buckel, Marburg

Early Steps of Benzoate Synthesis in Syntrophus aciditrophicus

9.30‐10.00

Felix tenBrink

AG Kroneck, Konstanz

Acetylene Hydratase: a closer look at its active site

10.00‐ 10.30

Carlos Dullius

AG Schink, Konstanz

Acetone carboxylation in facultatively and obligately anaerobic bacteria

10.30‐11.00

Coffee Break

12.00‐12.30

Heinz Wilkes

Potsdam

Mechanistic investigations on the pathway of n‐alkane oxidation in anaerobic bacteria

11.30‐ 12.00

Marta Drozdowska

Masih Sadeghi

AG Golding, Newcastle

Synthesis of Putative Intermediates in Anaerobic Hexane Metabolism

12.00‐ 12.30

Daniel Knack

AG Heider, Marburg

Anaerobic alkene metabolism and biological alkene hydration

12.30‐ 15.00

Lunch + Free time


Biophysical, computational approaches

Chair: Bernard Golding

15.00‐15.30

Alistair J. Fielding

AG Bennati, Göttingen

Multifrequency ENDOR spectroscopy identifies a unique iron site on the iron‐sulphur cluster involved in substrate reduction of heterodisulfide reductase

15.30‐16.00

Mikolaj Feliks

AG Ullmann, Bayreuth

Molecular Modelling Study of Mechanisms of Enzymes Involved in the Anaerobic Degradation of Hydrocarbons – Acetylene Hydratase and 4‐Hydroxyphenylacetate Decarboxylase

16.00‐16.30

Mario Kampa

AG Neese, Bonn

Electronic structure calculations of HDR, FTR and Acetylene Reductase

16.30‐ 16.45

Coffee Break

Anaerobic metabolism of isoprenoids

Chair: Bernhard Schink

16.45‐ 17.15

Juri Dermer

AG Fuchs, Freiburg

Anaerobic bacterial metabolism of cholesterol

17.15‐17.45

Jens Harder

Bremen

A potential pathway for anaerobic monoterpene metabolism: enzymes transforming myrcene to geranic acid

18.00‐19.00

Dinner

Evening Lectures II

Chair: Rainer Meckenstock

19.00‐20.00

Alfons J.M. Stams

Wageningen

Microbial degradation of aromatic and aliphatic hydrocarbons with chlorate as electron acceptor

20.00‐21.00

Wilfred F.M. Röling

Amsterdam

Anaerobic degradation of low concentrations of BTEX in landfill leachate.


Wednesday, 10 March, 2010 .

7.30 Breakfast

Ecophysiology/in situ techniques

Chair: Ralf Rabus

8.30‐ 9.00

Friederike Gründger

AG Krüger, Hannover

New life in old reservoirs – the microbial conversion of oil to methane

9.00‐ 9.30

Ulrike Jaeckel

AG Musat, Bremen

Phylogenetic characterization and two‐dimensional stable isotope fractionation analysis of sulphate‐reducing enrichment cultures degrading short‐chain alkanes

9.30‐10.00

Sara Kleindienst

AG Knittel, Bremen

Global distribution of hydrocarbon‐degrading SRB at marine gas and oil seeps

10.00‐

10.30

Coffee Break

10.30‐ 11.00

Conrad Dorer

AG Richnow/Vogt, Leipzig

Characterization of the initial reaction of anaerobic hydrocarbon degradation pathways by two‐dimensional compound specific isotope analysis (2D‐CSIA)

11.00‐11.30

Frederick von Netzer

AG Lüders, Munich

BSS and beyond ‐ unravelling the diversity and structure of anaerobic hydrocarbon degrader communities in natural systems

11.30‐ 12.00

Kevin Kuntze

AG Boll, Leipzig

The BamA targeting molecular tool for the detection of aromatic compound degrading anaerobes

12.15 Lunch and Departure


AG Bennati: Multifrequency ENDOR spectroscopy identifies a unique iron site on the iron-sulphur cluster involved in substrate reduction of heterodisulfide reductase Alistair J. Fielding1, Kristian Parey2, Ulrich Ermler2, Bernhard Jaun3, Silvan Scheller3, Marina Bennati1 1Max-Plank Institute for Biophysical Chemistry, Göttingen 2Max-Plank Institute for Biophysics, Frankfurt 3Laboratory of Organic Chemistry, ETHZ Heterodisulfide reductase (HDR) is a key enzyme in the energy metabolism of methanogenic archaea. The enzyme catalyzes the reversible reduction of the heterodisulfide (CoM-S-S-CoB) to the thiol coenzymes, coenzyme M (CoM-SH) and coenzyme B (CoB-SH) in the final step of methanogenesis. It employs an unusual [4Fe4S] cluster to carry out substrate chemistry. Previous studies have identified a mechanistic-based paramagnetic intermediate generated upon half-reaction of the oxidized enzyme with CoM-SH in the absence of CoB-SH (1). The unusual [4Fe4S]-cluster is bound in subunit HdrB of the Methanothermobacter marburgenis HdrABC holoenzyme within the C-terminal domain of two cysteine-rich sequence motifs (Cx31-

39CCx35-36CxxC). EPR studies on the isolated subunit HdrB heterologously produced in E. coli have shown that the [4Fe4S] cluster can be observed after oxidation in the absence of substrate (2). Our previous electron nuclear double resonance (ENDOR) investigations on the cluster within the Hdr-CoM-SH complex have lead to the assignment of four distinct iron sites and to the determination of the full hyperfine tensor elements. However, we were unable to provide conclusive evidence of a unique iron site and it was postulated that the cluster could interact with the substrate at multiple sites (1). Recently, Mössbauer measurements on the CCG-domain-containing subunit SdhE of succinate:quinone oxidreductase from Sulfolobus sulfataricus P2 has unambiguously indicated the presence of a unique iron site in the reduced cluster (3). We report multifrequency 57Fe ENDOR spectroscopic measurements on the iron sulphur cluster in HdrABC, HdrB and SdhE. Specifically, we report on the first results at 34 GHz, which show enhanced resolution compared to previous measurements at 9 and 94 GHz arising from the absence of proton resonances and polarization effects. The combined results at different frequencies allow assignment of all four 57Fe resonances and provide evidence of a unique iron site. We also report first 13C ENDOR results of 13C labelled CoM-SH bound to the cluster. (1) Hedderich R., Hamann N. and Bennati, M. (2005) Biological Chemistry 386: 961-970 (2) Hamann N., Mander G.J., Shokes J.E., Scott R.A, Bennati M. and Hedderich R. (2007) Biochemistry 46:12875-12885 (3) Hamann N., Bill E., Shokes J.E., Scott R.A., Bennati M. and Hedderich R. (2009), J. Biol. Inorg. Chem. 14: 457-470


AG von Bergen: Application of protein based stable isotope probing (Protein-SIP) to unravel anoxic benzene degradation Martin Taubert1, Jana Seifert1, Martin von Bergen1, Carsten Vogt2, Hans-Hermann Richnow2 1Department of Proteomics, Helmholtz-Centre for Environmental Research - UFZ 1Department of Isotope Biogeochemistry, Helmholtz-Centre for Environmental Research - UFZ In order to detect functional relationships within a microbial community, recently stable isotope probing (SIP) of proteins was developed (1). Metabolically active species assimilate the labeled substrate and incorporate stable isotopes (13C or 15N) into their biomass by synthesizing biopolymers like DNA, RNA and proteins. For Protein-SIP, mass spectrometry offers a tool for simultaneously identification and sensitive determination of stable isotope incorporation (down to ~2%). In contrast to SIP of nucleic acids, this allows direct linkage between physiology and taxonomy.

In this study, we report on the use of Protein-SIP on a benzene degrading, sulfate reducing microbial community isolated from a contaminated aquifer near Zeitz, Saxony-Anhalt. Benzene is a widespread environmental contaminant, especially problematic in soil or ground water, and knowledge about the initial steps of its degradation under anoxic conditions is still lacking. First clues on taxonomic composition of the microbial community have been acquired by DNA-SIP experiments (2). Our approach will give further insights into the function and trophic structure of the community by identifying the key players of anaerobic benzene degradation on proteomic level. Furthermore, in combination with other proteomic methods, the identification of key enzymes involved in the degradation pathway is possible. (1) Jehmlich N., Schmidt F., von Bergen M., Richnow H.H., Vogt C. (2008), ISME J. 2:1122-33 (2) Herrmann S., Kleinsteuber S., Chatzinotas A., Kuppardt S., Lueders T., Richnow H.H., Vogt C. (2009), Environ Microbiol. doi10.1111/j.1462-2920.2009.02077.

1220 1240 1260 1220 1240 1260800 820 840 860m/z

800 820 840 860

Inte

nsity

• „light“ substrate • „heavy“ substrate

•

•

•

•

•

•

e.g [12C6]-benzene e.g [13C6]-benzene

normal situation labeled situation

mixed culture (A+B)

A AB B

mass spectra


AG Boll: The tungsten-containing class of benzoyl-CoA reductases: characterization and distribution in obligately anaerobic bacteria Johannes Kung1, Claudia Löffler1, Thorsten Friedrich2, Martin von Bergen3, Sven Baumann3, Matthias Boll1 1Institute of Biochemistry, University of Leipzig 2Institute of Organic Chemistry and Biochemistry, University of Freiburg 3Dep. of Proteomics, Helmholtz Centre for Environmental Research Leipzig Benzoyl-CoA reductases (BCR) are key enzymes in the anaerobic degradation of aromatic compounds and catalyze the two-electron reduction of the aromatic ring to a cyclic dienoyl-CoA. The are two different classes of BCR enzymes: facultative anaerobes use a 3x[4Fe-4S] clusters containing ATP-dependent BCR, whereas in obligately anaerobic bacteria an ATP-independent BCR complex, encoded by the benzoate-induced bamBCDEFGHI genes is involved (1). We report on the first isolation and characterization of BamBC, the prototype of the active site containing components of the ATP-independent class of BCRs from Geobacter metallireducens (2). The enzyme has an a2b2 composition and contains 0.9 W, 15 Fe, 2.2 Ca and 1.0 Zn per ab module. EPR and cofactor analysis suggested the presence of a W-bis-MGD-pterin, three [4Fe-4S], and one [3Fe-4S] cluster per ab-unit. The cofactors could only be reduced by dienoyl-CoA but not with dithionite or Ti(III)-citrate. Low potential viologens served as electron acceptors in the backward reaction suggesting that BamBC transfers electrons on an extremely negative redox potential. A number of unusual kinetic properties of BamBC are presented. The distribution of BamBC-like enzymes was investigated in a number of obligately anaerobic bacteria that use aromatic growth substrates including Fe(III)-respiring, sulphate reducing and fermenting bacteria by (i) bamBC gene identification and regulation, (ii) in vitro enzyme activity assays, and (iii) Western Blot analysis using antibodies against BamBC from Geobacter metallireducens. (1) Wischgoll S., Heintz D., Peters F., Erxleben A., Sarnighausen E., Reski R., van Dorsselaer A. and Boll M.. (2005), Mol Microbiol. 58:1238-52 (2) Kung J.W., Löffler C., Dörner K., Heintz D., Gallien S., van Dorsselaer A., Friedrich T. and Boll M. (2009), PNAS 106:17687-92

C O S CoA COSCoA

BcrABCD

2 ATP Fdred

2 ADP 2 Pi + Fdox

BamBCDEFGHI2 [H]

Facultative Anaerobes

Obligate Anaerobes


AG Boll: The BamA targeting molecular tool for the detection of aromatic compound degrading anaerobes Kevin Kuntze1, Carsten Vogt2, Hans-Hermann Richnow2, Matthias Boll1 1Institute of Biochemistry, University of Leipzig, 2Departement of Isotope Biochemistry, Helmholtz Centre for Environmental Research Leipzig In anaerobic bacteria, most aromatic growth substrates (e.g. toluene, phenol, cresols, xylenes, ethylbenzenes, benzoate analogues, etc.) are channelled to the central intermediate benzoyl-CoA, which is then dearomatized to a cyclic dienoyl-CoA (1). After a series of ß-oxidation-like reactions 6-oxocyclohexenoyl-CoA (6-OCH) is formed. This intermediate serves as substrate for a ring opening hydrolase, referred to as BamA (bam = benzoic acid metabolism), yielding the aliphatic 6-OH-pimelyl-CoA. We established an assay with degenerated primers targeting the bamA gene from all known anaerobic aromatic compound degrading bacteria (2) including Gram-positive bacteria. Two benzene contaminated aquifers were analyzed using in situ microcosms (BACTRAP®), loaded with benzene and incubated for five months at two different sites. The combined application of both, bamA and 16S-RNA analysis identified a Geobacter species as the only dominating species at one site, whereas at another site a species belonging to the Rhodocyclaceae dominated, most possibly representing a novel species or genus.

The bamA-targeting PCR assay provides a new and widely applicable tool for the detection of all types of anaerobic bacteria capable of degrading a wide variety of aromatic compounds and for monitoring anaerobic biodegradation processes. (1) Boll (2005) J. Mol. Microbiol. Biotechnol. 10:132-142 (2) Kuntze et al. (2008) Environ Microbiol. 10(6):1547-56

Magnetospirillum AMB1

Magnetospirillum MS1

Magnetospirillum CC26

Magnetospirillum TS6

Thauera sp. MZ1T

Thauera aromatica

Thauera chlorobenzoica strain 3CB-1

Geobacter FRC-32

Geobacter metallireducens

Geobacter bemidjiensis

Geobacter sp. M21

Azoarcus CIB

Azoarcus evansii

Aromatoleum aromaticum EbN1

Hansemann-clone bamA bF25sA11

Hansemann-clone bamA bF25sG12

Hansemann-clone bamA bF25sE11

Hansemann-clone bamA bF25sG07



Desulfobacterium anilini

Syntrophus aciditrophicus SB

Desulfosarcina ovata

Desulfosarcina cetonica

Desulfococcus multivorans

D. gibsoniae

D. thermobenzoicum

100

83100

100

91

88

97

99

45

97

100

56100

99

98

94

79

60

99

96

84

64

51

78

0.05

Phylogenetic tree of bamA-sequences from aromatic compound degrading anaerobic bacteria. D. gibsoniae and D. thermobenzoicum are Gram-positive sulfate reders


AG Buckel: Early Steps of Benzoate Synthesis in Syntrophus aciditrophicus Marie Kim1, Ivana Djurdjevic1, Wolfgang Buckel1,2 1Laboratorium für Mikrobiologie, Fachbereich Biologie, Philipps-Universität, 35032 Marburg 2Max-Planck-Institut für terrestrische Mikrobiologie, Karl-von-Frisch-Str. 4, 35043 Marburg The anaerobic Deltaproteobacterium Syntrophus aciditrophicus thrives syntrophically on benzoate and axenically on crotonate, which is oxidized to acetate and reduced to cyclohexane carboxylate and some benzoate (1). Hence, the degradation of benzoate is reversible whereby glutaconyl-CoA may serve as central intermediate. S. aciditrophicus contains three genes coding for the energy conserving glutaconyl-CoA decarboxylase (Gcd), which could catalyse the endergonic carboxylation of crotonyl-CoA driven by an electrochemical Na+-gradient (2). For the subsequent reduction of glutaconyl-CoA to glutaryl-CoA by NAD(P)H, a non-decarboxylating glutaryl-CoA dehydrogenase/electron transferring flavoprotein complex (Gdh/Etf) could be responsible. Similar to butyryl-CoA dehydrogenase/Etf from Clostridium kluyveri (3), Gcd/Etf could bifurcate electrons to ferredoxin (see Scheme). Alternatively, the reductant could be menaquinol also present in S. aciditrophicus. In addition, glutaconyl-CoA could serve as biosynthetic precursor of glutamate via the reverse 2-hydroxyglutarate pathway of glutamate fermentation.

CoAS

OCOO-CoAS

OCOO-

CO2

Na+

Membrane

Ferredoxin2-Ferredoxin

2 NADHGlutaconyl-CoA Glutaryl-CoA

CoAS

OCrotonyl-CoA out

2 NAD+

Gdh/EtfGcd

Results 1. The genes coding for GcdA, B and C were expressed in E. coli. Currently we check whether GcdA catalyses the transfer of CO2 from glutaconyl-CoA to biotin. 2. Gdh and Etf were produced separately in E. coli. GdH was characterized as a non-decarboxylating glutaryl-CoA dehydrogenase using ferricenium as electron acceptor (4). Etf catalyses the reduction of iodonitrosotetrazolium by NADH and NADPH. 3. The genome of S. aciditrophicus revealed no genes for a functional 2-hydroxyglutaryl-CoA dehydratase/glutaconyl-CoA hydratase but for a Re-citrate synthase (5). The expression of the latter gene in an IBA vector together with groEL in E. coli yielded a protein complex, from which the chaperon could be removed with ATP, K+, and Mg++. The resulting pure enzyme was characterized as Re-citrate synthase. The aerotolerant enzyme required Mn++ or preferentially Co++ for activity. At this time, we raise polyclonal antibodies against Re-citrate synthase, in order to show whether the low citrate synthase activity in cell-free extracts of S. aciditrophicus stems from this enzyme. 1. Mouttaki, H., Nanny, M. A., and McInerney, M. J. (2007). Appl Environ Microbiol 73, 930-938. 2. McInerney, M. J., Rohlin, L., Mouttaki, H., Kim, U., Krupp, R. S., Rios-Hernandez, L., Sieber, J.,

Struchtemeyer, C. G., Bhattacharyya, A., Campbell, J. W., and Gunsalus, R. P. (2007). Proc Natl Acad Sci U S A 104, 7600-7605.

3. Herrmann, G., Jayamani, E., Mai, G., and Buckel, W. (2008). J Bacteriol 190, 784-791. 4. Wischgoll, S., Taubert, M., Peters, F., Jehmlich, N., von Bergen, M., and Boll, M. (2009). J Bacteriol

191, 4401-4409. 5. Li, F., Hagemeier, C. H., Seedorf, H., Gottschalk, G., and Thauer, R. K. (2007). J Bacteriol 189,

4299-4304.


AG Ermler: Crystallization of benzoyl-CoA reductase and acetophenone carboxylase both involved in the anaerobic degradation of hydrocarbons Tobias Weinert1, Diana Kathrey, Johannes Kung2, Karola Schühle3, Matthias Boll2, Johann Heider3, Ulrich Ermler1 1Max-Planck-Institute of Biophysics, Frankfurt/Main 2Institute of Biochemistry, University of Leipzig 3Laboratorium für Mikrobiologie Fachbereich Biologie Philipps-Universität

Crystallization attempts were initiated with fragment BamBC of a benzoyl-CoA reductase from Geobacter metallireducens and of acetophenone/acetone carboxylase from Azoarcus EbN1. BamBC is the catalytic unit of benzoyl-CoA reductase, the key enzyme of the anaerobic degradation of aromatic compounds (1). The heterotetrameric α2β2 complex with a molecular mass of 185 kDa was crystallized with the hanging drop method under anaerobic conditions using PEG 4000 as precipitant. Crystals normally grew as needles but in combination with microseeding a few crystals became more compact and a data set at 2.9 Å resolution could be collected. Initial phases could be calculated using the distantly related aldehyde ferredoxin oxidoreductase and formaldehyde ferredoxin oxidoreductase from Pyrococcus furiosus as models. Although the bis-MGD-pterin-cofactor and the function of the small subunit β for αβ tetramerization could be identified the quality of the phases was not sufficient for complete model building and refinement. Acetophenone carboxylase (APC) catalyzes the second step in ethylbenzene degradation to benzoyl-CoA by carboxylating acetophenone. The 485 kDa protein complex (αβγδ)2ε consists of an (αβγδ)2 core and a loosely attached ε subunit. Crystals of the acetophenone carboxylase core grew with pentaerythritol ethoxylate as precipitant and diffracted to 3.7 Å resolution. The space group was P61/522 and the cell axis 241.2 Å and 337.3 Å. Crystals were only obtained when APC was purified from cells cultivated with ethylbenzene which is an extremely non-reproducible process. Crystallization of APC purified from an organism grown on acetophenone as sole energy source failed. Preliminary crystallization attempts were initiated with acetone carboxylase of Azoacrus EbN1 which is also a member of the same group of C-C forming ligases. (1) Kung J.W., Löffler C., Dörner K., Heintz D., Gallien S., van Dorsselaer A., Friedrich T. and Boll M. (2009), PNAS 106:17687-92


Invited Speaker: M. Fontecave Iron-sulfur clusters and radical biocatalysis: applications in the modification of macromolecules Marc Fontecave Laboratoire de Chimie et Biologie des Métaux, UMR CNRS-CEA-Université Joseph Fourier 5249, CEA Grenoble, 17 Avenue des Martyrs, 38054 Grenoble Cedex 9 – France Collège de France, 11 Place Marcelin Berthelot, 75231 Paris Cedex 05-France Radical-SAM enzymes constitute a very large family of iron sulphur proteins involved in a great variety of metabolic pathways and biosynthetic reactions. The active site, a complex between a [4Fe-4S] cluster and S-adenosylmethionine (SAM), serves to generate free radicals for reaction initiation. This unique and complex radical chemistry has been recently shown to be used in Nature during the specific modification of biological macromolecules: DNA, transfer RNA and proteins. This will be specifically illustrated through examples of enzymes studied in our laboratory. First, mechanistic studies of the sporephotoproduct lyase, an enzyme essential for repairing DNA in pathogenic sporulating bacteria exposed to UV radiation, will be discussed (1,2). Second, enzymes participating in the biosynthesis of sulfur-containing compounds and catalyzing C-H to C-S bond formation, will be presented with special emphasis on methylthio-transferases present in both prokaryotes and eukaryotes involved in the specific methylthiolation of tRNAs (3,4), as well as on a methylthio-transferase involved in the methylthiolation of a specific aspartate residue of a ribosomal protein (5,6). . (1) A. Chandor-Proust, O. Berteau, T. Douki, D. Gasparutto, S. Ollagnier-de-Choudens, M. Atta, M. Fontecave (2008), J. Biol. Chem. 283 : 36361-68 (2) C. Mantel, A. Chandor, D. Gasparutto, T. Douki, M. Atta, M. Fontecave, P-A. Bayle, J-M. Mouesca, M. Bardet (2008), J. Am. Chem. Soc. 130: 16978-84 (3) M. Fontecave, S. Ollagnier-de Choudens, E. Mulliez (2003), Chem. Rev. 103: 2149-66 (4) H. L. Hernández, F. Pierrel, E. Elleingand, R. García-Serres, Boi Hanh Huynh, M. K. Johnson, M. Fontecave, M. Atta (2007), Biochemistry 46 : 5140-47 (5) M. Fontecave, E. Mulliez, M. Atta (2008), Chemistry and Biology 15, 209-10. (6) S. Arragain, R. Garcia-Serres, G. Blondin, T. Douki, M. Clemancey, J.-M.Latour, F. Forouhar, H. Neely, G.T. Montelione, J.F. Hunt, E. Mulliez, M. Fontecave, M. Atta (2010) J. Biol. Chem. (in press)


AG Fuchs:

Anaerobic bacterial metabolism of cholesterol Juri Dermer & Georg Fuchs, Mikrobiologie, Fak. Biologie, Universität Freiburg

The anoxic metabolism of the ubiquitous triterpene cholesterol is challenging

because of its complex chemical structure, low solubility in water, low number of

active functional groups, and the presence of four alicyclic rings and two quaternary

carbon atoms. Consequently, the aerobic metabolism depends on oxygenase

catalyzed reactions requiring molecular oxygen as co-substrate. Sterolibacterium

denitrificans was shown to metabolize cholesterol completely to CO2 under anoxic

(denitrifying) conditions. The pathway proceeds via the oxidation of ring A, followed

by an oxygen-independent hydroxylation of the subterminal tertiary C-25 of the side

chain. The anaerobic hydroxylation of a tertiary carbon using water as oxygen donor

is unprecedented and may be catalyzed by a novel molybdenum containing enzyme.

The initial two enzymes of the pathway have been isolated and studied before (1-3).

To identify the postulated cholesterol C25 hydroxylase, we applied various

chromatographic techniques and obtained a yet impure enzyme fraction that showed

major bands at 110, 60, and 30 kDa. The discontinuous enzyme assay was based on

HPLC analysis of products using ferricyanide as electron acceptor. The substrate

needs to be added together with Tween 20, which results in turbidity and hampers a

spectrophotometric assay. We have sequenced the genome of the bacterium (28

contigs, ~ 3.2 Mbp) and have identified the putative genes for the hydroxylase based

on mass spectrometric characterization of the protein bands on SDS-Gels. The

enzyme subunits are indeed related to subunits of Mo dependent anaerobic

hydroxylases, notably DMSO dehydrogenase or ethylbenzene dehydrogenase.

1. Chiang Y-R, Ismail W, Heintz D, Schaeffer C, Van Dorsselaer A, Fuchs G. 2008. Study of anoxic and oxic cholesterol metabolism by Sterolibacterium denitrificans. J. Bacteriol. 190:905-914.

2. Chiang Y.-R, Ismail W, Gallien S, Heintz D, Van Dorsselaer A, Fuchs G. 2008. Cholest-4-en-3-one-Δ1-dehydrogenase: A flavoprotein catalyzing the second step in anoxic cholesterol metabolism. Appl. Environm. Microbiol. 74:107-113.

3. Chiang YR, Ismail W, Muller M, Fuchs G. 2007. Initial steps in the anoxic metabolism of cholesterol by the denitrifying Sterolibacterium denitrificans. J Biol Chem. 282:13240-13249.


AG Golding:

Synthesis of Putative Intermediates in Anaerobic Hexane Metabolism Marta Drozdowska, Masih Sadeghi, and Bernard T Golding School of Chemistry, Newcastle University, Newcastle upon Tyne, NE1 7RU, UK Radical enzymes catalyse reactions in which ‘free’ radicals are protein-bound intermediates (e.g. the human enzyme methylmalonyl-CoA mutase; CoA = coenzyme A). A similar rearrangement (1 → 2) has been discovered in the anaerobic oxidation of alkanes that occurs e.g. in deep sediments (1, 2).

RCH2

OACoS

HO2C

HO

SCoA

H

HO2CH

Re

from propionate, a toxic degradation product of

certain amino acids

enters Krebs cycle

(R)-methylmalonyl-CoA succinyl-CoA

CO2H

COSCoA

COSCoA

HO2C

COSCoA

fumaratethioester migration - CO2

1 2 3 To define the mechanism of this rearrangement we are synthesising intermediates 1 and 2, as well as the end product 4-methyloctanoic acid (3, CoA ester) and stereospecifically deuterated hexanes. A representative scheme is (PG = protecting group, e.g. Ph2

tBuSi) starting with the commercially available methyl (S)-3-hydroxy-2-methylpropanoate: MeO2C Me

OH

Me

OPG

OHS

Me

OPG

Br

MeOH

Me

=

steps

MeO2C Me

OPG

Me

OH

OPG

Me

OPG

Me

CO2H

Me

Me

O

SR

R

Me

Me

CO2H

target compounds

e. g. R = CoA

alternative steps

i ii iii iv

v

Reagents and conditions: i) Ph2

tBuSiCl, imidazole, DMF, r.t., 3 h; ii) iBu2AlH, toluene, -78 oC, 2 h; iii) CBr4, PPh3, THF, r.t., 2 h; iv) C3H7MgCl (2.0 M solution in ether), isoprene, NiCl2, THF, -78 oC then 0 oC, 1 h; v) Bu4NF (1.0 M solution in THF), THF, 23 oC. The synthetic approaches make use of chiral pool starting materials and enantioselective reductions. The above scheme is flexible and allows the synthesis of both enantiomers of 4-methyloctanoic acid. In addition, by using alternative Grignard reagents in step iv, other branched fatty acids can be prepared. (1) H Wilkes, R Rabus, T Fischer, A Armstroff, A Behrends, F Widdel, Arch Microbiol, 2002, 177, 235-243. (2) H Wilkes, S Kühner, C Bolm, T Fischer, A Classon, F Widdel, R Rabus, Org Geochem, 2003, 34, 1313-1323.


AG Harder: A potential pathway for anaerobic monoterpene metabolism: enzymes transforming myrcene to geranic acid Jens Harder, Danny Brodkorb, Frauke Lüddeke, Annika Wülfing, Aytac Dikfidan, Frauke Germer, Markus Timke, Matthias Gottschall, Robert Marmulla and Christina Probian Department of Microbiology, Max Planck Institute for Marine Microbiology, Celsiusstr. 1, D-28359 Bremen, contact: [email protected] Isoprenoids are characterized by tertiary and quaternary carbon atoms and carbon-carbon double bonds. To demonstrate the anaerobic mineralization of these compounds for the first time, we enriched and isolated denitrifying bacteria on individual monoterpenes (1,2) and essential oils (3), the monoterpenoids eucalyptol and linalool (4), cholesterol (5) and the quaternary compounds dimethylmalonate (6) and dimethylpropionate (7). Initial studies demonstrated a cometabolic allylic hydrogen shift with isoterpinolene as dead-end metabolite from isolimonene (8). The transformation of myrcene into geranic acid as first ionic intermediate was catalyzed with cell-free soluble fractions (9).

A simple hypothesis is the hydration of myrcene to linalool, followed

by an allylic rearrangement to geraniol and an oxidation via geranial to geranic acid. Castellaniella (ex Alcaligenes) defragrans strain 65Phen contained a geraniol dehydrogenase activity induced by monoterpenes. The protein was purified and the gene was cloned. Genomic knowledge was extended to a 50 kb genomic fragment and we found a non-energy conserving aldehyde dehydrogenase gene provisionally considered as geranial dehydrogenase. The corresponding protein is induced on monoterpenes. We developed a genetic system for C. defragrans. The geraniol dehydrogenase deletion mutant is impaired in the monoterpene utilisation. The initial reactions are catalyzed by a novel enzyme, a bifunctional linalool dehydratase-isomerase. The protein was purified to homogeneity as linalool dehydratase and catalyzes also the geraniol isomerisation to linalool and the thermodynamically less favourable reverse reactions of myrcene to linalool and of linalool to geraniol. The gene was also detected on the 50 kb genomic fragment and codes for a preprotein, suggesting a periplasmic location of the linalool dehydratase-isomerase. The novel enzyme differs from the previously detected linalool isomerase in Thauera linaloolentis (10). A third enzyme activity, a geraniol dehydratase forming myrcene without linalool formation was detected in Thauera terpenica. 1. Appl. Environ. Microbiol. 61, 3804 (1995) 2. Syst. Appl. Microbiol. 21, 237 (1998) 3. Biodegradation 11, 55 (2000) 4. Syst. Appl. Microbiol. 21, 365 (1998) 5. Arch. Microbiol. 167, 269 (1997) 6. Appl. Environ. Microbiol. 65, 3319 (1999) 7. Appl. Environ. Microbiol. 69, 1866 (2003) 8. FEMS Microbiol. Lett. 169, 67 (1998) 9. Appl. Env. Microbiol. 66, 3004 (2000) 10. FEMS Microbiol. Lett. 149, 71 (1997)


AG Heider: Anaerobic toluene metabolism: Biochemistry of benzylsuccinate synthase and its activating enzyme Markus Hilberg, Johann Heider Fachbereich Biologie, Laboratorium für Mikrobiologie, Philipps-Universität Marburg Anaerobic degradation of toluene in Thauera aromatica is initiated by an unusual addition reaction of the toluene methyl group to the double bond of a fumarate cosubstrate to form the first intermediate (R)-benzylsuccinate. This reaction is catalyzed by the glycyl radical enzyme benzylsuccinate synthase (BSS).

All enzymes required for the degradation of toluene to (R)-benzylsuccinate are encoded in the toluene-inducible bss-operon. The bssDCAB genes code for the subunits of benzylsuccinate synthase (BssA, B and C) and an additional enzyme (BssD) showing strong similarity with activating enzymes needed for radical formation in other glycyl radical enzymes. The three subunits of benzylsuccinate synthase are arranged in a α2β2γ2 composition, where the large α-subunits contain the essential glycine and cysteine residues that are conserved in all glycyl radical enzymes. Metal analysis and EPR studies of the non-reconstituted benzylsuccinate synthase indicated the presence of two [Fe3S4]-clusters per holoenzyme. We are currently trying to reconstitute the enzyme to potential [Fe4S4]-clusters to elucidate the nature of the iron-sulfur clusters in further detail and we want to investigate the reaction mechanism of benzylsuccinate synthase. The activating enzyme (BSSAE) was recombinantly produced in E.coli and purified by His-Tag affinity-chromatography. All purification steps and assays were performed in an anoxic glovebox and BSSAE was characterized by UV/vis and EPR spectroscopy. Activation and crystallization assays with the purified BSSAE and the recombinantly produced BSS are planed.

CH3CH2

COO-

-OOC

COO-

COO-COO-

COO-

BSS BSS- H

Toluol Benzylradikal

(R)-Benzylsuccinylradikal

Fumarat

(R)-Benzylsuccinat

toluene benzylradical

fumarate

(R)-benzylsuccinylradical (R)-benzylsuccinate


AG Heider: Anaerobic alkene metabolism and biological alkene hydration Daniel H. Knack und Johann Heider Laboratorium für Mikrobiologie, Philipps-Universität Marburg

In nature the degradation of alkenes is managed by microorganisms under aerobic and anaerobic conditions. In contrast to the mechanisms of aerobic biological alkene degradation, the mechanisms of anaerobic alkene degradation are virtually unknown. Among a few known anaerobic alkene degrading microorganisms, the sulfate-reducing bacterium Desulfococcus oleovorans is characterised. The genome of D. oleovorans is sequenced making it suitable for studying mechanisms of anaerobic alkene degradation. It is known that this organism can covert 1-alkenes to their corresponding alcohols and fatty acids, respectively (1). For this reason, an alkene hydration mechanism is proposed as initial reaction of alkene degradation in this organism (2).

A model enzyme capable of water-addition to alkenes is ethylbenzene dehydrogenase (EbDH) the initial enzyme of the anaerobic degradation of ethylbenzene in the denitrifying bacterium Aromatoleum aromaticum EbN1. During studies of the EbDH substrate spectrum, a side activity of EbDH was detected that catalyzes the addition of water to the double bond of the alkene ethylidenecyclohexane (3; Fig. 1).

Fig. 1: top: EbDH catalysed addition of water to ethylidenecyclohexane resulting in the product 1-ethylcyclohexanol. bottom: EbDH catalyzed oxidation of 2-ethyl-1H-indene to the intermediate 2-ethylidene-2H-indene and EbDH catalyzed subsequent addition of water resulting in the product 2-ethyl-2H-indene-2-ol.

During my PhD work I was able to identify a new alkene substrate which seems also to be oxidized and subsequently hydrated to the corresponding alcohol (Fig.1). The catalytical mechanism of alkene hydration by this enzyme will be investigated further.

Significantly, a gene cluster was identified in the genome of D. oleovorans that encodes gene products which show a high similarity to the subunits of ethylbenzene dehydrogenase in A. aromaticum. Because D. oleovorans cannot use ethylbenzene as carbon source, the EbDH-like enzyme may catalyse another reaction and may be involved in the hydration of alkenes. In my PhD work I will clone and overexpress this operon and characterize the gene products by biochemical and biophysical methods.

A third part of my PhD work involves cultivation of D. oleovorans on alkene substrates to identify induced proteins by 2D-PAGE and MALDI-TOF-MS/MS, as well as substrate depletion and product formation analysis by HPLC- or GC-MS. 1. Aeckersberg, F.; Bak, F.; Widdel, F. (1991) Anaerobic oxidation of saturated hydrocarbons to CO2 by a new type of sulphate‐reducing bacterium. Arch Microbiol 156:5‐14. 2. Aeckersberg, F.; Rainey, F. A.; Widdel, F. (1998) Growth, natural relationships, cellular fatty acids and metabolic adaptation of sulfate‐reducing bacteria that utilize long chain alkanes under anoxic conditions. Arch Microbiol 170:361‐369. 3. Szaleniec, M.; Hagel, C.; Menke, M.; Nowak, P.; Witko, M.; Heider, J. (2007) Kinetics and mechanism of oxygen‐independent hydrocarbon‐hydroxylation by ethylbenzene dehydrogenase. Biochemistry 46:7637‐7647.

OHEbDH

EbDH

H20

H20

EbDH

2 [H]


AG Knittel:

Global distribution of hydrocarbon-degrading SRB at marine gas and oil seeps

Sara Kleindienst & Katrin Knittel

Hot spots of sulfate-reduction (SR) occur in anoxic marine sediments, where methane, short-chain

alkanes or oil components dominate. In methane seeps, SR is tightly coupled to the anaerobic

oxidation of methane (AOM). However, in sediments with natural oil seepage methane-dependent SR

drops to less than 10% of total SR rates as an indicator for hydrocarbon degradation by sulfate-

reducing bacteria (SRB). Up to now several SRB have been described to be capable of hydrocarbon

degradation but it is still unknown which SRB dominate in situ.

We have investigated diverse oil and gas seep sediments by CARD-FISH to study the global

distribution and abundance of specific sulfate reducers. Of particular interest were known

hydrocarbon-degrading SRB groups as well as the so-called “SEEP-SRB” groups 1 to 4, which

include only uncultivated members.

Maximal relative abundances of free-living Deltaproteobacteria within different habitats ranged from

4 % to 23 % of total single cells. Steepest gradients within the cores were found at hydrothermal

sediments of the Guaymas Basin, where abundances correlated negatively with depth resulting from

high temperature gradients. The main portion of detected Deltaproteobacteria could be further

assigned to a specific SRB group. Members of the Desulfosarcina/Desulfococcus (DSS) branch

strongly dominated gassy as well as oily seep sediments with up to 17 % of total single cells. Of these,

members of SEEP-SRB1, also known as bacterial partners of methanotrophic archaea of the ANME-2

clade, accounted for up to 2 %. The remaining part of DSS cells seemed to be highly diverse. Results

suggest rather the presence of numerous diverse and specialized but low abundant SRB than the

presence of one dominant subgroup or species. Members of the family Desulfobulbaceae (including

groups SEEP-SRB3 and -4) were mainly detected at Mediterranean and Arctic mud volcanoes, with

highest abundances of up to 8 %. Desulfobacterium anilini and related species, known to degrade

aromatic hydrocarbons, showed relative abundances up to 5 % of total cells. Results will be discussed

with respect to available hydrocarbons and correlated with environmental and biogeochemical data.


AG Kroneck:

Acetylene Hydratase: a closer look at its active site Felix tenBrink1, Kinga Gerber1, Oliver Einsle2, Bernhard Schink1, Peter MH Kroneck1 1Department of Biology, University of Konstanz 2Institute of Organic Chemistry and Biochemistry, University of Freiburg Today, acetylene (C2H2) is only a minor trace gas on Earth. Interestingly, it is quite abundant on Titan which is considered to represent a cold model of Earth’s early atmosphere around four billion years ago. The Cassini/Huygens mission to Titan detected several large lakes consisting of hydrocarbons (eg CH4, HCN, or C2H2). Similar processes on Earth including volcanic eruptions may have provided C2H2 as a readily available source of carbon and energy (1,2). Recently, we reported the first crystal structure of the enzyme acetylene hydratase (AH), a tungsten enzyme from Pelobacter acetylenicus (3).

A crucial question about the reaction mechanism of AH is related to the oxygen ligand (OH- or H2O) and the Asp 13 residue bound at the W center. A hydrophobic pocket to accommodate the substrate is located directly above this ligand (Figure). To tackle this problem, we began to replace several relevant amino acids at the active site of AH and investigated the catalytic properties of these variants. Furthermore we will report on experiments, where crystals of AH had been exposed to C2H2 (or CO) in a cell at elevated pressures to identify the substrate (or inhibitor) binding site. (1) Tokano T. (2009), Astrobiology 9, 147-64; Cordier D., Mousis O., Lunine J. I., Lavvas P. and Vuitton V. (2009), ApJL eprint arXiv0911.186 (2) Oremland R. S. and Voytek M. A. (2008), Astrobiology 8: 45-58 (3) Seiffert G. B., Ullmann G. M., Messerschmidt A., Schink B., Kroneck P. M.H., and Einsle O. (2007), Proc. Natl. Acad. Sci. (U S A) 104, 3073-3077.


AG Krüger:

New life in old reservoirs – the microbial conversion of oil to methane F. Gründger1, S. Feisthauer2, H. H. Richnow2, M. Siegert1, M. Krüger1 1 Bundesanstalt für Geowissenschaften und Rohstoffe, Abteilung Geomikrobiologie,

Stilleweg 2, D-305655 Hannover, Germany 2 Helmholtz-Zentrum für Umweltforschung GmbH, Department Isotopenbiogeochemie,

Permoserstr. 15, D-04318 Leipzig, Germany Since almost 20 years it is known from stable isotope studies that large amounts of biogenic methane are formed in oil reservoirs. The investigation of this degradation process and of the underlying biogeochemical controls are of economical and social importance, since even under optimal conditions, not more than 30-40 % of the oil in a reservoir is actually recovered. The conversion of parts of this non-recoverable oil via an appropriate biotechnological treatment into easily recoverable methane would provide an extensive and ecologically sound energy resource. Laboratory mesocosm as well as high pressure autoclave experiments with samples from different geosystems showed high methane production rates after the addition of oils, single hydrocarbons or coals. The variation of parameters, like temperature, pressure or salinity, showed a broad tolerance to environmental conditions. The fingerprinting of the microbial enrichments with DGGE showed a large bacterial diversity while that of Archaea was limited to three to four dominant species. The Q-PCR results showed the presence of high numbers of Archaea and Bacteria. To analyse their function, we measured the abundances of genes indicative of metal reduction (16S rRNA gene for Geobacteraceae), sulphate reduction (sulphate reductase, dsr), and methanogenesis (methyl coenzyme M-reductase, mcrA). The methanogenic consortia will be further characterised to determine enzymatic pathways and the individual role of each partner. Degradation pathways for different compounds will be studied using 13C-labelled substrates and molecular techniques. Our stable isotope data from both, methane produced in our incubations with samples from various ecosystems and field studies, implies a common methanogenic biodegradation mechanism, resulting in consistent patterns of hydrocarbon alteration.


AG Lueders:

BSS and beyond - unravelling the diversity and structure of anaerobic hydrocarbon degrader communities in natural systems. Frederick von Netzer, Tillmann Lueders Institute of Groundwater Ecology, Helmholtz Zentrum München - German Research Centre for Environmental Health Anaerobic microbes responsible for natural attenuation in contaminated aquifers can be monitored via respective catabolic marker genes. Recently, we have established an approach based on the detection of the α-subunit of the benzylsuccinate synthase (bssA) (1). This enzyme and its homologues are key to the breakdown of hydrocarbons such as toluene, xylenes, cresols, but also aliphatic compounds and methylated naphtalenes via fumarate addition (2-5). In “real world scenarios” such as contaminated aquifers, the diversity of bssA and its homologues can be extremely valuable to characterize intrinsic degrader populations, and to predict and monitor natural attenuation. However, the true identity and diversity of anaerobic degraders present at such sites is still an enigma. Several lineages of bssA and homologues with unclear phylogenetic affiliation have been detected:

Here, we have comparatively screened intrinsic degrader populations in samples from different hydrocarbon contaminated habitats as well as selected pure cultures and enrichments for intrinsic bssA-like sequences using novel primer permutations. This new set of bssA detection assays was tested for its specificity to recover different gene lineages of fumarate adding enzymes. We provide evidence for a previously unrecognized clostridial bssA branch and their importance in situ in contaminated aquifers. Next, this project aims to verify whether general concepts of ecological theory (6) can help to understand principles of degrader community assembly and functioning. Using laboratory flow-through columns and field degrader communities as inocula, we will test how degrader diversity and functional redundancy within communities affects the stability and performance of degradation processes under stable or changing environmental conditions. 1. C. Winderl, S. Schaefer, T. Lueders, Environ. Microbiol. 9, 1035 (2007). 2. D. Selesi et al., J. Bacteriol., (2010, in press). 3. F. Musat et al., Environ. Microbiol. 11, 209 (2009). 4. A. V. Callaghan et al., Biochem. Biophys. Res. Commun. 366, 142 (2008). 5. O. Grundmann et al., Environ. Microbiol. 10, 376 (2008). 6. J. I. Prosser et al., Nat Rev Micro 5, 384 (2007).


OO

OH

SHCH3

OH

S

SOO

OH

H

H+

SOO

OH

-

SH

OH O

H+

H+

p-cresolphenoxymethyl radicalphenoxyacetyl radical

(b)

(a)

(c) (d)-

CO2

AG Martins: Crystal structure of 4-hydroxyphenylacetate decarboxylase from Clostridium scatologenes, a 2[4Fe-4S] cluster-containing glycyl radical dependent enzyme Lisa Schilder1, Lihua Yu2, Mikolaj Feliks3, G. Matthias Ullmann3, Thorsten Selmer2, Berta M Martins1 1Research Center for Bio-Macromolecules, University of Bayreuth 2Biotechnology/Enzyme technology, Fachhochschule Aachen-Jülich 3Structural Biology/Bioinformatics, University of Bayreuth Glycyl radical-dependent enzymes (GREs) are key players in anoxic biological processes and require post-translational activation by a specific activating S-adenosylmethionine (SAM)-dependent Fe/S protein. The 4-hydroxyphenylacetate decarboxylase enzyme system (4-HPAD) found in Clostridia represents a new class of GREs acting on aromatic compounds. The decarboxylase component has, in addition to the glycyl radical containing subunit an extra subunit that binds two [4Fe-4S] clusters. The respective activating enzyme has one Fe/S cluster in addition to the SAM-binding [4Fe-4S] cluster. 4-HPAD is proposed to use a glycyl-thiyl radical dyad to catalyze the last step of tyrosine fermentation by Clostridium difficile and C. scatologenes. The decarboxylation product p-cresol is a virulence factor of C. difficile.

Scheme 1 - Putative decarboxylation mechanism of 4-HPAD We report on the crystal structures at 1.8 Å resolution of substrate-free and substrate-bound forms of 4-HPAD from Clostridium scatologenes. These are the first structures of a Fe/S cluster-containing glycyl radical enzyme and provide insights into its mechanism. The subtrate-free state shows a tetramer of heterodimers ((βγ)4) where each heterodimer is composed of a catalytic β-subunit harboring the putative Gly-873/Cys-503 radical dyad, and a distinct small γ-subunit with two [4Fe-4S] clusters. The γ-subunit comprises a N-terminal domain that coordinates one cluster with 1 histidine and 3 cysteines and a C-terminal domain that binds the second cluster with 4 cysteines. The two domains display pseudo-twofold symmetry and are structurally related to the [4Fe-4S] cluster-binding scaffold of high-potential iron-sulfur proteins. Our results support a redox active N-terminal cluster potentially involved in radical dissipation, and a structural role for the C-terminal cluster within the catalytic competent (βγ)4 complex. In contrast to expectation, the substrate-bound structure shows a direct interaction between the substrate’s carboxylate group and the putative active site Cys-503. This form captures a possible catalytic competent complex and suggests a Kolbe-type decarboxylation as alternative mechanism for p-cresol formation (scheme 1). (1) Lihua Y., Blaser M., Andrei P.I., Pierik A.J. and Selmer T. (2006), Biochemistry. 45:9584-92 (2) Martins B.M. et al Crystal structure of 4-hydroxyphenylacetate decarboxylase, a 2[4Fe-4S] clusters-containing glycyl radical enzyme (submitted)


AG Meckenstock:

The initial step of anaerobic benzene degradation in the iron reducing culture BF is a carboxylation to benzoate Ramon Diesveld1, Nidal Abu Laban1, Draženka Selesi1, Thomas Rattei2, Patrick Tischler2, Rainer U. Meckenstock1 1 Institute of Groundwater Ecology, Helmholtz Zentrum München 2 Genome-oriented Bioinformatics, Technische Universität München Three possible routes are proposed for the initial step in anaerobic benzene degradation (figure 1). Recent studies on metabolite analysis with stable isotope-labelled benzene, water or bicarbonate buffer [1] suggested either hydroxylation of benzene to phenol (a), direct carboxylation to benzoate (b), or methylation to toluene (c).

CH3

OH

COOH

COO-

-OOC

COO-

P O-

OH

O

COSCoA

C-

O

ATP AMP+Pi OH- Pi CO2

PpsABC PpcABCD PpcABCD

Ohb1

BssABCD

BadA, BclA, BzdABamY

COSCoA

OH

COO-

OH

CoA+ATP AMP+PPi

2H+ +

2FdredH2O +2Fdox

CoA+ATP AMP+PPi

CO2

HbaAHcrL

HbaBCDHcrCABPcmRST

CO2

Benzene

Toluene Benzylsuccinate

Benzoate

Phenol

Phenylphosphate4-hydroxybenzoate 4-hydroxybenzoyl-CoA

Benzoyl-CoAa

b

c

COO-

In order to identify enzymes involved in the initial activation reaction of anaerobic benzene degradation by our iron-reducing culture BF, we compared the proteomes of cells grown with benzene, phenol, or benzoate as carbon source. SDS-PAGE revealed specific bands of benzene-grown cells which could not be observed during growth on phenol or benzoate. The N-terminal sequence of a strongly expressed 60 kDa protein was determined by Edman-sequencing. Alignment of the obtained amino acid sequence of the 60 kDa band to the shotgun metagenome of culture BF revealed that it is encoded by ORF 138. Both ORF 138 as well as the adjacent ORF 137 showed 43% and 37% sequence identity to phenylphosphate carboxylase subunit PpcA and PpcD of Aromatoleum aromaticum strain EbN1, respectively. In total proteome analysis with LC/MS/MS analysis of peptides from the SDS gels these two genes and further ORFs in direct genetic neighbourhood were shown to be expressed specifically with benzene as carbon source. We propose these ORFs as constituents of a putative benzene degradation gene cluster (~17 Kb). Due to the similarity of these genes with known carboxylases we propose that the initial reaction in anaerobic benzene degradation is a direct carboxylation to benzoate. In order to characterize the putative benzene degradation gene cluster both genes ORF 137 and ORF 138 as well as the adjacent ORF 139 encoding for a predicted benzoate-CoA ligase were cloned into specific expression vectors and expressed in different E. coli strains under anaerobic conditions. [1] Identification of enzymes involved in anaerobic benzene degradation by a strictly anaerobic iron-reducing enrichment culture. N. Abu Laban, D. Selesi, T. Rattei, P. Tischler, and R. U. Meckenstock. (Environ. Microbiol. submitted)


AG Musat:

Phylogenetic characterization and two-dimensional stable isotope fractionation analysis of sulphate-reducing enrichment cultures degrading short-chain alkanes Ulrike Jaekel 1, Carsten Vogt 2, Niculina Musat 1, Marcel Kuypers 1, Hans-Hermann Richnow 2, Friedrich Widdel 1 and Florin Musat 1

1 Max Planck Institute for Marine Microbiology, Celsiusstr. 1, 28359 Bremen, Germany 2 UFZ-Helmholtz Centre for Environmental Research, Permoserstr. 15, 4318 Leipzig, Germany

The short-chain, gaseous alkanes ethane, propane and butane are important constituents of natural gas, and as such they enter the biosphere, for example through natural gas seeps in the marine environment. While the aerobic degradation of short-chain alkanes is known from many years, their anaerobic degradation was only recently reported with a pure culture, strain BuS5, and several enrichment cultures obtained from diverse marine habitats1. Strain BuS5, a sulphate-reducing bacterium affiliated to the Desulfosarcina – Desulfococcus cluster of the Deltaproteobacteria, is able to utilize only propane and butane as growth substrates. In the present study, the propane- and butane-degrading enrichment cultures were analyzed regarding the microbial community structure. Whole-cell hybridization with developed sequence-specific oligonucleotide probes showed that the enrichment cultures were dominated by phylotypes closely related to strain BuS5. Application of Halogen In Situ Hybridization-Secondary Ion Mass Spectroscopy2 showed that the dominant phylotypes were the actual alkane degraders, as revealed by incorporation of 13C from supplied 13C-labeled butane (figure).

In addition, the degradation of propane and butane was investigated in more detail using two-dimensional compound specific stable isotope fractionation analysis (2D-CSIA)3. The slope of the linear regression for hydrogen (Δδ2H) vs. carbon (Δδ13C) discrimination (λ= Δδ 2H/ Δδ 13C ≈ εH/εC) was determined to be higher for propane degradation (6.3 to 8.6 for propane vs. 4.6 to 6.0 for butane). The hydrogen fractionation of all cultures (εH between −5.2 and −27.7) was low in comparison to that observed for the anaerobic degradation of aromatic compounds. This findings suggests that in the present cultures the rate-limiting step of the activation via addition to fumarate is the formation of the C-C bond and not the cleavage of the C-H bond, as postulated for the similar anaerobic activation of aromatic hydrocarbons. References

1. Kniemeyer, O., Musat, F., Sievert, S. M., Knittel, K., Wilkes, H., Blumenberg, M., Michaelis, W., Classen, A., Bolm, C., Joye, S.B., and Widdel, F. (2007). Nature 449, 898-810.

2. Musat, N. , Halm, N., Winterholler, B., Hoppe, P., Peduzzi S. , Hillion, F., Horreard, F., Amann, R., Jørgensen, B., and Kuypers, M. (2008). PNAS 105, 17861–17866.

3. Elsner, M., Zwank, L., Hunkeler, D. and Schwarzenbach, R. (2005). Environ Sci Technol, 39, 6896-6916.


AG Neese: Electronic structure calculations of HDR, FTR and Acetylene Reductase Mario Kampa1,2, Anoop Ayyappan1, Frank Neese1,2 1 Institute for Physical and Theoretical Chemistry, University of Bonn 2 Max Planck Institiute for Bioinorganic Chemistry Mülheim a.d. Ruhr Heterodisulfide reductase (HDR) of methanogenic archaea catalyze the reduction of the heterodisulfide (CoM-S-S-CoB) to the thiols coenzyme M (CoM-SH) and coenzyme B (CoB-SH) which play a key role in methane metabolism (1). The [4Fe-4S] cluster in the active site mediates the reduction via a paramagnetic intermediate which has been characterized by EPR spectroscopy. Our contribution to SPP 1319 is to do electronic structure calculations of HDR using the ORCA program package (3) to come up with a consistent reaction mechanism. However, no crystal structure is available yet. Ferredoxin:thioredoxin reductase (FTR) for which an X-ray structure has been determined, also contains a catalytically active [4Fe-4S] cluster. We have created a cluster model of the active site of FTR (Figure 1). The presence of several open-shell electrons interacting magnetically makes the electronic structure calculations more challenging requiring broken-symmetry DFT methods. In addition, we are working on the enzyme acetylene hydratase. The reaction mechanism for acetylene hydration, as deduced form the X-ray structure at 1.26 Å (2) may proceed either via a nucleophilic attack of a hydroxo ligand at the acetylene triple bond or via a Markovnikov-type addition including a vinyl cation intermediate. The reaction surface of the first mechanistic alternative has been studied by the Ullmann group using DFT revealing that the energy barriers are too high for the reaction to proceed. We have carried out single point calculations with LPNO-CCSD and DFT using higher quality basis sets confirming the DFT results of the Ullmann group. A nucleophilic attack of a hydroxylate is therefore unlikely and other mechanisms have to be taken into account. (1) Walters, E. and Johnson, M. (2004), Photosynthesis Research 79: 249-264 (2) Seiffert, G., Ullmann, M., Messerschmidt, A., Schink, B., Kroneck, Pa and Einsle, O. ( 2007), PNAS 104: 3073-3077. (3) http://www.thch.uni-bonn.de/tc/orca/

Figure 1: Cluster model for FTR


AG Rabus:

Anaerobic degradation of p-alkylbenzoates and –toluenes by the denitrifying strains pMbN1 and pCyN1 Ralf Rabus Institute for Chemistry and Biology of the Marine Environment, University of Oldenburg Slow utilization of para-alkylated aromatic hydrocarbons (e.g. p-xylene or p-cymene) is attributed to mechanistically challenging degradation of the p-alkylated benzoate intermediates, since the latter cannot be transformed by benzoyl-CoA reductase, the central enzyme of anaerobic aromatic compound degradation. We have isolated a new nitrate-reducing bacterium, strain pMbN1, which grows anaerobically with p-methylbenzoate as sole source of carbon and energy. Preliminary proteomic and metabolic comparison of p-methylbenzoate versus benzoate adapted cells provide first hints to a special anaerobic degradation pathway for p-methylbenzoate, which should be distinct from the generally used benzoyl-CoA reductase route.The planned project pursues the following main objectives: (i) extraction and mass-spectrometric identification of intermediates specifically formed during anaerobic growth of strain pMbN1 with p-methylbenzoate, (ii) further proteomic analysis including identification of subtrate-specific proteins in strain pMbN1, (iii) determine the corresponding catabolic gene cluster(s) in strain pMbN1, (iv) testing for an analogous pathway for p-ethyl- and p-isopropylbenzoate degradation in denitrifying strain pCyN1, und (v) elucidation of the initial reaction in anaerobic p-ethyl- and p-isopropyltoluene (p-cymene) degradation in strain pCyN1.


AG Richnow/Vogt:

Characterization of the initial reaction of anaerobic hydrocarbon degradation pathways by two-dimensional compound specific isotope analysis (2D-CSIA) Conrad Dorer1, Anko Fischer2, Hans-Hermann Richnow1, Carsten Vogt1

1Helmholtz Centre for Environmental Research, Department of Isotope Biogeochemistry, Permoserstraße 15, 04318 Leipzig, Germany 2Isodetect – Company for Isotope Monitoring (Branch Leipzig), Permoserstraße 15, 04318 Leipzig, Germany

In the last decade, several studies have demonstrated that stable isotope tools are highly applicable for monitoring anaerobic biodegradation processes. Compound specific isotope analysis (CSIA) makes use of isotope fractionation processes occurring in biochemical reactions. H-C-bond cleavages are often accompanied by strong kinetic isotope effects for carbon and hydrogen and thus provide clues to characterize distinct biochemical degradation pathways and to quantify the extent of biodegradation. Enrichment factors (εbulk) needed for a CSIA field site approach have to be determined in laboratory reference experiments. Recent research results from different laboratories have shown that single εbulk values for similar biochemical reactions can be highly variable; thus, the use of two-dimensional compound specific isotope analysis (2D-CSIA) has been encouraged for characterizing biodegradation pathways more precisely. 2D-CSIA for hydrocarbons can be expressed by the slope of the linear regression for hydrogen (Δδ2H) versus carbon (Δδ13C) discrimination known as lambda (Λbulk) = Δδ2H/Δδ13C ≈ εHbulk/εCbulk. We determined the carbon and hydrogen isotope fractionation for the biodegradation of benzene, toluene and xylenes by various reference cultures. Specific enzymatic reactions initiating different biodegradation pathways could be distinguished by 2D-CSIA. For the aerobic di- and monohydroxylation of the benzene ring, Λ-values always lower than 9 were observed. Enrichment cultures degrading benzene anaerobically produced significant different values: Λ-values between 8-19 were oberved for nitrate-reducing consortia, whereas sulfate-reducing and methanogenic consortia showed always Λ-values greater than 20 [1,2]. The observed variations suggest that (i) aerobic benzene biodegradation can be distinguished from anaerobic biodegradation, and (ii) that more than a single mechanism seems to exist for the activation of benzene under anoxic conditions. Λ-values for anaerobic toluene degradation initiated by the enzyme benzylsuccinate synthase (BSS) ranged from 4 to 41, tested with strains using nitrate, sulfate or ferric iron as electron acceptor or using light as energy source [3,4,5]. Significantly different Λ values were also observed for the anaerobic degradation of xylenes initiated by the BSS [5]. The different Λ-values obtained for the anaerobic degradation of toluene and xylenes might be caused by slightly different reaction mechanisms of BSS isoenzymes. In comparison, Λ and/or εbulk values for the methyl monohydroxylation of toluene with oxygen as co-substrate were significantly different for two tested strains each containing a different toluene attacking enzyme, indicating that specific enzymes for aerobic methyl group oxidation reactions can be detected by CSIA and 2D-CSIA. Our results show that the combined carbon and hydrogen isotope fractionation approach has great potential to elucidate biodegradation pathways of monoaromatic hydrocarbons in microcosm and field studies. Current work focus on (i) 2D-CSIA of aromatic and aliphatic hydrocarbons in degradation experiments using whole cells, and (ii) 2D-CSIA of aromatic hydrocarbons in in vitro experiments using cell extracts. [1] Fischer et al. (2008) Environ. Sci. Technol. 42, 4356-4363 [2] Mancini et al. (2008) Environ. Sci. Technol. 42, 8290-8296 [3] Vogt et al. (2008) Environ. Sci. Technol. 42, 7793-7800 [4] Tobler et al. (2008) Environ. Sci. Technol. 42, 7786-7792 [5] Herrmann et al. (2009) Environ. Microbiol. Reports 1, 535-544


Invited Speaker W.F.M Röling:

Anaerobic degradation of low concentrations of BTEX in landfill leachate. Wilfred F.M. Röling1, Boris M. van Breukelen2, Martin Braster1, Martijn Staats1 1 Molecular Cell Physiology, VU University Amsterdam, the Netherlands 2 Geo-environmental Sciences and Hydrology and, VU University Amsterdam, the Netherlands Leachate of old landfills in the Netherlands poses a serious treat to the environment. We examined the microbiology and biogeochemistry of the aquifer polluted by the Banisveld landfill, Boxtel, the Netherlands, in order to understand natural attenuation of landfill leachate better. Concentrations of BTEX were low (<180 microgram/l) and comprised a small part of the Dissolved Organic Carbon (around 100 mg/l). BTEX disappeared by microbial degradation. Microbial communities in polluted areas of the landfill were found to be dominated by anaerobic, iron-reducing Geobacteraceae, a family that also contains members able to degrade mono-aromatics. Our expectation was that also at Banisveld mono-aromate degradation would be performed by Geobacteraceae. However, a combination of culturing and culture-independent analysis indicated otherwise. Georgfuchsia toluolica, a new iron-reducing microorganism that belongs to the Betaproteobacteria was isolated from the aquifer. It is only capable of mono-aromate degradation. A culture-independent survey revealed that benzylsuccinate synthase genes (bssA) sequences closest related to this species, were dominantly present throughout the aquifer. We suggest that due to its specialization on mono-aromatics, Georgfuchsia competes successfully with Geobacter for mono-aromatics. Geobacter species are more generalists and might thrive on the other carbon sources in landfill leachate. bamA genes were found at about tenfold higher copy numbers than bssA genes, and only a small fraction was closest related to Georgfuchsia. This suggests the presence of a considerable pool of microorganisms that possess the benzoyl-coA pathway and degrade other aromatics or are involved in syntrophic interactions with BTEX degraders. While most research on anaerobic BTEX degradation focuses on the bacteria that actually consume the BTEX, it may well be that the control over the rate of biodegradation is determined for a large part by other microbial network members. An example relating to predation by protozoa will be briefly discussed.


AG Rother:

Genetic and biochemical analysis of methyltransferase function and mechanism in Methanosarcina acetivorans C2A Marc Staßen1, Ellen Oelgeschläger1, Michael Rother1

1Institute of Molecular Biosciences, University of Frankfurt Methanosarcina acetivorans encodes a remarkable number of methyltransferases, which are predicted to be involved in methylotrophic energy metabolism. However, for all methylotrophic substrates known for M. acetivorans the methyltransferases involved are also known, leaving 7 “orphan” methyltransferases encoded in the chromosome, which indicates that the methylotrophic capabilities of this organism exceeds our knowledge. To address this issue a number of naturally and non- naturally occurring methylated and methoxylated compounds are being tested for their ability to support growth of M. acetivorans. Also, we are in the process of systematically deleting the respective genes to unravel the physiological function of the respective proteins. Additionally, we are addressing the catalytic mechanism of MtsF, a “fused” methyltransferase required for methylsulfide metabolism in this organism. MtsF consists of a N-terminal corrinoid and a C-terminal methyltransferase 2 domain and it is possible that this protein alone can meditate methylation of coenzyme M with dimethylsulfide (DMS) as the methyl donor. To this end we tried to overproduce MtsF heterologously in E. coli but found it to be insoluble. We therefore established a homologous overexpression system in M. acetivorans to generate sufficient amounts of MtsF for biochemical analysis. The results of these efforts, together with initial characteristics of MtsF, are presented.


AG Schink:

Acetone carboxylation in facultatively and obligately anaerobic bacteria Carlos Dullius, Ching-Yuan Chen, Olga Gutierrez, Bernhard Schink Microbial Ecology, Dept. of Biology, University of Konstanz, [email protected] Both in aerobic and anaerobic bacteria, acetone is activated by carboxylation to an acetoacetate derivative. Earlier work in the group of Scott Ensign, Utah, has shown that acetone carboxylation by aerobic chemotrophic bacteria proceeds according to the equation CH3COCH3 + CO2 + ATP → Acetoacetate- + H+ + AMP + 2 Pi The reaction proceeds in a two-step process, in which acetone is first converted to a reactive enol structure, and in the second step the carboxylic group is added. Thus, the carboxylation which theoretically would need only an energy expenditure of about 25 kJ per mol costs the cell two ATP equivalents. We studied acetone carboxylation with nitrate-reducing bacteria in the past, but for a long time we could not measure an ATP-dependent carboxylation reaction. In cell-free extracts of Paracoccus pantotrophus, we could recently demonstrate that the energy supply for acetone carboxylation comes from UTP rather than ATP, and that the overall stoichiometry of UTP hydrolysis resembles that described above for aerobic bacteria. Other nitrate-reducing bacteria appear to use a similar activation system. On the contrary, in strictly anaerobic sulfate-reducing bacteria, acetoacetate is not a free intermediate in acetone activation. According to physiological experiments with intact cells, we could show that the reaction is sodium-dependent, and that also CO can act as a carboxylic group donor. Further work in our group will concentrate on the acetone activation mechanism in sulfate-reducing and syntrophically fermenting bacteria.


Invited Speaker A.J.M. Stams:

Microbial degradation of aromatic and aliphatic hydrocarbons with chlorate as electron acceptor Alfons J.M. Stams1, Farrakh Mehboob1, Sander A.B. Weelink1, Teun Veuskens1, Marjet J. Oosterkamp1, Alette A.M. Langenhoff2, Jan Gerritse2, Gosse Schraa1, Caroline Plugge1, Peter J. Schaap1 1Laboratory of Microbiology, Wageningen University 2Deltares, Utrecht Bacteria that respire with chlorate are able to produce molecular oxygen, which can be used as terminal electron acceptor and to degrade organic compounds by means of mono- and dioxygenases. Chlorate-reducing bacteria may thus have the ability to employ aerobic pathways for the degradation of hydrocarbons under seemingly anaerobic conditions. Key conversions are: Chlorate reduction: ClO3

- + 2[H] ClO2- + H2O

ClO2- Cl- + O2

Hydrocarbon oxygenation: R-CH + 2[H] + O2 R-COH + H2O We have studied the degradation of benzene with chlorate by a newly isolated Alicycliphilus denitrificans strain (1) and decane degradation with chlorate by Pseudomonas dechloritidismutans (2). These strains are able to denitrify, but with nitrate as electron acceptor they cannot grow with benzene and decane, respectively. This shows the unique physiological feature of respiration with chlorate. The genomes of the two bacteria were sequenced. All key genes required for presumed oxygenase-dependent pathways for benzene degradation in A. denitrificans and decane degradation in P. chloritidismutans were present in the genomes. Differential proteome analysis revealed insight into regulatory aspects in chlorate-reducing bacteria. (1) Weelink SAB, Tan NCG, ten Broeke H, van den Kieboom C, van Doesburg W, Langenhoff AAM, Gerritse J, Junca H and Stams AJM (2008) Isolation and characterization of Alicycliphilus denitrificans strain BC that grows on benzene with chlorate as electron acceptor. Appl Environ Microbiol 74: 6672-6681. (2) Mehboob F, Junca H, Schraa G and Stams AJM (2009) Oxidation of alkanes coupled to chlorate reduction by Pseudomonas chloritidismutans AW-1T. Appl Microbiol Biotechnol 83:739-747


Invited Speaker R.K. Thauer:

Reverse methanogenesis: the key nickel enzyme MCR catalyses anaerobic oxidation of methane.

Silvan Scheller1, Meike Goenrich2, Reinhard Boecher2, Rudolf K. Thauer2 & Bernhard Jaun1 1Laboratory of Organic Chemistry, ETH Zurich, 8093 Zurich, Switzerland

2Max-Planck-Institute for Terrestrial Microbiology, 35403 Marburg, Germany

Large amounts (estimates range from 70 Tg/y to 300 Tg/y) of the potent green house gas methane are oxidised to CO2 in marine sediments by consortia of methanotrophic archaea and sulphate-reducing bacteria and thus are prevented from escaping into the atmosphere. Indirect evidence suggests that the anaerobic oxidation of methane (AOM) might proceed as the reverse of archaeal methanogenesis from CO2 with the nickel-containing methyl-coenzyme M reductase (MCR) as the methane-activating enzyme. However, experiments showing that MCR can catalyse the endergonic back reaction have been lacking. Here we report that purified MCR from Methanothermobacter marburgensis converts methane into methyl-coenzyme M under equilibrium conditions with apparent Vmax and KM values consistent with the observed in vivo kinetics of AOM with sulphate. This result supports the hypothesis of “reverse methanogenesis” and is paramount to understanding the still unknown mechanism of the last step of methanogenesis. The ability of MCR to cleave the particularly strong C-H bond of methane without involvement of highly reactive oxygen-derived intermediates is directly relevant to catalytic C-H activation, currently a hot topic in chemistry.


AG Ullmann:

Molecular Modelling Study of Mechanisms of Enzymes Involved in the Anaerobic Degradation of Hydrocarbons – Acetylene Hydratase and 4-Hydroxyphenylacetate Decarboxylase Mikolaj Feliks1, G. Matthias Ullmann1

1) Structural Biology/Bioinformatics Group, Bayreuth University Over the last decades computer-aided modelling has become an important tool for studying molecular details of chemical reactions. The rapid growth of computing power and the intense development of theoretical methods have provided an insight into systems of thousands of atoms, such as enzymes. In the following work we present the computational analysis of two distinct enzymes that facilitate chemically difficult proton and electron transfer reactions. Acetylene Hydratase1,2 is a tungsten-iron-sulfur protein which catalyzes the conversion of acetylene to acetaldehyde. Tungsten ion is coordinated by two pterin cofactors and a cysteine. Aspartatic acid at the active site also seems to be involved in the reaction. Although the enzyme has been already investigated both experimentally3 and theoretically4, the molecular basis of its catalytic activity still remain undiscovered. The key questions are how the protein environment contributes to the catalysis and why the substitution of tungsten by chemically similar molybdenum leads to decrease of the activity.

Active site model of Acetylene Hydratase – rate-limiting step of the reaction

4-Hydroxyphenylacetate Decarboxylase5 is a glycyl radical enzyme that utilizes glycyl-thiyl radical dyad to catalyze the conversion of 4-hydroxyphenylacetate to p-cresol. The unexpected binding mode and preliminary docking studies suggest a variety of possible decarboxylation mechanisms. In our calculations, we established a hierarchy of models of increasing complexity. The most simple gas-phase models provide high-accuracy structural and electronic information that serves as a guideline for the study of larger systems, such as cluster or full-protein QM/MM models. Our ultimate goal is to identify all intermediates and transition states along the catalytic cycles of both enzymes and to understand the role of protein environment in the catalysis. Finally, we present further plans for the development of methodology and computational tools for studying enzymes theoretically.

(1) Rosner B. M., Schink B., Journal of Bacteriology, 1995, 5767-5772 (2) Seiffert G. B., Ullmann G. M. et al., PNAS vol. 104 no. 9, 2007, 3073-3077 (3) Yadav J. et al., J. Am. Chem. Soc., 1997, 119, 4315-4316 (4) Antony S., Bayse CA, Organometallics 2009, 28, 4938-4944 (5) Martins B. M., Yu L., Blaser M., Selmer T., Dobbek H., 2009, in preparation


AG Wilkes: Mechanistic investigations on the pathway of n-alkane oxidation in anaerobic bacteria Heinz Wilkes1, Eline Basilio Janke1, Ralf Rabus2 and Friedrich Widdel3 1Helmholtz Centre Potsdam GFZ German Research Centre for Geosciences 2Institute for Chemistry and Biology of the Marine Environment, University of Oldenburg 3Max Planck Institute for Marine Microbiology, Bremen Alkanes are the main constituents of crude oil and various petroleum products. Their biodegradation in anoxic natural environments such as contaminated aquifers or petroleum reservoirs plays an important role in the global carbon cycle. During the last 15 years several denitrifying and sulphate-reducing bacteria capable of utilising alkanes as sole source of carbon and energy have been isolated and characterised. Based on the identification of metabolites and studies with isotope labeled substrates a pathway for the complete oxidation of n-hexane to carbon dioxide in the denitrifying bacterium strain HxN1 has been proposed (Fig. 1; 1-2). In the initial activation reaction the n-alkane is added to the double bond of fumarate yielding (1-methylpentyl)succinate (MPS). MPS is further transformed via a sequence of enzyme reactions to a branched fatty acid (4-methyloctanoyl-CoA) which then is degraded by β-oxidation. Main goal of this project will be to obtain new insights into important mechanistic aspects of the proposed degradation pathway, and to contribute to a better understanding of possible fundamental differences in the biochemical mechanisms involved in n-alkane versus alkylbenzene oxidation. In particular, the stereochemistry of key metabolites will be established by stereoselective synthesis of authentic standards and rigid structure assignment of isolated metabolites. The substrate range of anaerobic hydrocarbon oxidising bacteria will be characterised through co-transformation experiments with model substrates and crude oil. Fig. 1. Key steps of n-hexane oxidation in the denitrifying bacterium HxN1. (1) Rabus R., Wilkes H., Behrends A., Armstroff A., Fischer T., Pierik A.J. and Widdel F. (2001), J. Bacteriol. 183:1707-15. (2) Wilkes H., Rabus R., Fischer T., Armstroff A., Behrends A. and Widdel F. (2002), Arch Microbiol 177:235-43.

COO-

COO-

COO-

COO-

GlycylRadicalEnzyme

CO-SCoA

CH3CO-SCoA+

β-Oxidation

Regeneration of Fumarate

CO-SCoA+

2 CH3CO-SCoA

2 β-Oxidation Cycles

6 CO2Reduction ofElectron Acceptor

[H] [H]TCA-Cycle

CO-SCoA

ActivationRearrangementDecarboxylation

- CO2+


Addresses of participating groups/speakers

Dr. Marina Bennati Max‐Planck‐Institut für biophysikalische Chemie Am Faßberg 11 D‐37077 Göttingen Phone: +49‐551‐2011911 [email protected] PD Dr. Martin von Bergen Helmholtz‐Zentrum für Umweltforschung (UFZ) Permoserstrasse 15. D 04318 Leipzig. Phone +49‐341‐2351265 [email protected] Prof. Matthias Boll Institut für Biochemie Universität Leipzig Brüderstr. 34 D 04103 Leipzig Phone +49‐341‐9736996 boll@uni‐leipzig.de Prof. Dr. Wolfgang Buckel Philipps‐Universit Marburg Fb. 17 ‐ Biologie, Laboratory for Microbial Biochemistry Karl‐von‐Frisch‐Straße 8 D 35043 Marburg Phone +49‐6421‐28 21527 [email protected]‐marburg.de Prof. Dr. Bernhard Golding Newcastle University School of Natural Sciences Research in Chemistry Newcastle upon Tyne NE1 7RU, United Kingdom Großbritannien Phone: +44‐0191‐2226647 [email protected]


PD Dr. Ulrich Ermler Max‐Planck‐Institut für Biophysik Abteilung Molekulare Membranbiologie Max‐von‐Laue‐Straße 3 D 60438 Frankfurt/Main Phone +49‐69‐6303 1054 ermler@mpibp‐frankfurt.mpg.de Prof. Dr. Marc Fontecave Laboratoire de Chimie et Biologie des Métaux CEA‐Grenoble iRTSV/LCBM. Bat. K' 17 av. des Martyrs. 38054 Grenoble, France Phone +33 04 3878 9106 [email protected] Prof. Dr. Georg Fuchs Institut für Biologie II (Mikrobiologie) Albert‐Ludwigs‐Universität Freiburg Schänzlestr. 1 D 79104 Freiburg Phone +49‐ 761‐2032649 [email protected]‐freiburg.de PD Dr. Jens Harder Max‐Planck‐Institut für marine Mikrobiologie Celsiusstr. 1 D‐28359 Bremen Phone: +49‐421‐2028‐750 jharder@mpi‐bremen.de Prof. Dr. Johann Heider Laboratorium für Mikrobiologie Fachbereich Biologie Philipps‐Universität Marburg Karl‐von‐Frisch Strasse 8 D‐35043 Marburg/Germany Phone: +49‐6421‐28 21527 [email protected]‐marburg.de Dr. Katrin Knittel Max‐Planck‐lnstitut für Marine Mikrobiologie Celsiusstr. 1 D 28359 Bremen Phone +49‐421‐2028935 kknittel@mpi‐bremen.de


Prof. Dr. Peter M. H. Kroneck Universität Konstanz Mathematisch‐Naturwissenschaftliche Sektion Fachbereich Biologie D 78457 Konstanz Phone +49‐7531‐88‐2103 Peter.Kroneck@uni‐konstanz.de Dr. Martin Krüger Bundesanstalt für Geowissenschaften und Rohstoffe (BGR) Stilleweg 2 D 30655 Hannover Phone +49‐511‐ 6433102 [email protected] Dr. Tilmann Lüders Forschungszentrum für Umwelt und Gesundheit (GSF) GSF ‐ Forschungszentrum für Umwelt und Gesundheit Institut für Grundwasserökologie Ingolstädter Landstraße 1 D 85764 Oberschleißheim Phone +49‐89‐3187 3687 [email protected] Dr. Berta M.D.P. Martins Humboldt Universität zu Berlin Institut für Biologie Philippstr. 13, Haus 18 D 10115 Berlin Phone +49‐921‐55‐4358 Berta.Martins@uni‐bayreuth.de Dr. Rainer U. Meckenstock GSF‐Forschungszentrum für Umwelt und Gesundheit GmbH Institut für Grundwasserökologie Ingolstädter Landstr. 1 85764 Neuherberg Phone +49‐89‐3187‐2561 [email protected] rainer.meckenstock@helmholtz‐muenchen.de Dr. Florin Musat Max‐Planck‐Institut für Marine Mikrobiologie Abteilung Mikrobiologie Celsiusstr. 1 D 28359 Bremen Phone +49‐421‐2028740 fmusat@mpi‐bremen.de


Prof. Dr. Frank Neese Rheinische Friedrich‐Wilhelms‐Universität Bonn Institut für Physikalische und Theoretische Chemie Wegelerstraße 12 D – 53115 Bonn Phone +49‐ 228‐732351 [email protected]‐bonn.de Prof. Dr. Ralf Rabus Carl von Ossietzky Universität Oldenburg Institut für Chemie und Biologie des Meeres (ICBM) Carl von Ossietzky Universität Oldenburg Ammerländer Heerstraße 114‐118 D 26129 Oldenburg Phone +49‐441‐798‐3884 [email protected] PD Dr. Hans Hermann Richnow Department für Isotopenbiogeochemie Helmholtz‐Zentrum für Umweltforschung (UFZ) Permoserstrasse 15 D 04318 Leipzig Phone +49‐341‐235 1212 [email protected] Prof. Dr. Wilfred A. M. Röling Vrije Universiteit Amsterdam Department Molecular Cell Physiology 1081 HV Amsterdam, The Netherlands Phone +31 317 483102 [email protected] Dr. Michael Rother Johann Wolfgang Goethe‐Universität Frankfurt am Main Institut für Molekulare Biowissenschaften Max‐von‐Laue‐Str. 9 60439 Frankfurt a. M. Phone +49‐69‐79829320 [email protected]‐frankfurt.de Prof. Dr. Bernhard Schink Universität Konstanz Mathematisch‐Naturwissenschaftliche Sektion Fachbereich Biologie, Lehrstuhl für Mikrobielle Ökologie Postfach 55 60 <M654> D 78457 Konstanz Phone +49‐7531‐882140 bernhard.schink@uni‐konstanz.de


Prof. Dr. Alfons J.M. Stams Department of Microbiology, Wageningen Agricultural University, Hesselink van Suchtelenweg 4 6703 CT Wageningen, The Netherlands Phone +31‐8370‐83101 [email protected] Prof. Dr. Dr. h. c. Rudolf K. Thauer Max‐Planck‐Institut für terrestrische Mikrobiologie Karl‐von‐Frisch‐Straße D‐35043 Marburg Phone: +49‐6421‐178101 thauer@mpi‐marburg.mpg.de Prof. Dr. G. Matthias Ullmann Universität Bayreuth Fakultät für Biologie, Chemie und Geowissenschaften Universitätstr. 30, BGI D 95447 Bayreuth Phone +49‐921‐553545 Matthias.Ullmann@uni‐bayreuth.de Dr. Carsten Vogt Helmholtz‐Zentrum für Umweltforschung (UFZ) Permoserstraße 15 D 04318 Leipzig Phone + 49‐341‐235 1317 [email protected] Dr. Heinz Wilkes GeoForschungsZentrum Potsdam (GFZ) Section 4.3 Telegrafenberg B228 D 14473 Potsdam, Germany Phone +49‐331‐2881784 wilkes@gfz‐potsdam.de

Documents

Biological transformations of hydrocarbons without oxygen: from the