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Research report:

Microbiology AG Buckel

Wolfgang Buckel (born 22. November 1940)

Diplom (Chemistry), Universität München, 1965

Dr. rer. nat. (Biochemistry), Universität München, 1968

Akademischer Rat/Direktor, Universität Regensburg, 1969-1987

Postdoc (Microbiology), University of California, Berkeley, 1970-1971

Professor of Microbiology at the Philipps-Universität, since 1987

Main research

Unusual enzymes involved amino acid fermenting anaerobes

Members of the orders Clostridiales and Fusobacteriales have the unique ability to ferment amino acids for energy conserving purposes. Since there are twenty proteinogenous amino acids, each of which is fermented via at least one specific pathway, these microorganisms are among the biochemical most versatile Bacteria. The best-studied amino acid in this respect is glutamate, which is fermented by two different pathways to identical products: ammonia, CO2, acetate, butyrate and H2. Organisms living in the soil, as are Clostridium tetani, Clostridium tetanomorphum and Clostridium cochlearium, use the methylaspartate pathway, in which the linear carbon skeleton of (S)-glutamate is rearranged to the branched one of (2S,3S)-3-methylaspartate catalysed by the coenzyme B12-dependent glutamate mutase. On the other hand, organisms living in the gastrointestinal tract of animals and humans, as are Acidaminococcus fermentans, Clostridium symbiosum and Fusobacterium nucleatum, ferment glutamate via the FMN- and [4Fe-4S]-containing (R)-2-hydroxyglutaryl-CoA dehydratase, which has to be activated by ATP and reduced ferredoxin. The subsequent decarboxylation of the product glutaconyl-CoA to crotonyl-CoA catalysed by a membrane enzyme, which conserves energy via an electrochemical Na+-gradient, concludes the special part of this pathway. The hydrophilic carboxyl-transferase part of the biotin-containing glutaconyl-CoA decarboxylase has been produced in E. coli and crystallised. Its X-ray structure revealed that the enzyme belongs to the crotonase superfamily, in which the thiol ester carbonyl is hydrogen-bonded to two backbone amides. This type of binding facilitates decarboxylation, but the liberated CO2 is immediately transferred to biotin activated in a similar manner. In collaboration with the group of Lars-Oliver Essen (Fachbereich Chemie, Philipps-Universität) we have succeeded to get crystals of the whole decarboxylase from C. symbiosum.


Figure 1. Proposed mechanism of 2-hydroxyisocaproyl-CoA dehydratase from the pathogenic anaerobe Clostridium difficile.

2-Hydroxyacyl-CoA dehydratases

The difficult elimination of water from (R)-2-hydroxyglutaryl-CoA to glutaconyl-CoA in A. fermentans can be readily explained with a ketyl radical anion as intermediate, which is formed by injection of an electron into the thiolester carbonyl driven by ATP hydrolysis (Fig. 1). This ‘activation’ is catalysed by an extremely oxygen sensitive iron-sulfur protein, whose structure has been solved by X-ray crystallography. The structure of the homodimeric protein revealed a helix-cluster-helix motif forming an angle of 105°. Upon reduction of the cluster by one electron and binding of 2 ATP, the angle is probably opened to 180°, in order to facilitate the electron transfer to component D, the actual dehydratase. This conformational change is similar to that of an archer shooting an arrow like an electron driven by ATP hydrolysis in his muscles (Fig. 2). Using AlF4 and ATP, a complex between activator and dehydratase could be isolated. The complex is stabilised by ADP-AlF4, a transition state analogue of ATP hydrolysis. Recently we have purified (R)-2-hydroxyisocaproyl-CoA dehydratase and its activating protein from Clostridium difficile, whose genome has been sequenced by the Sanger Centre, U.K. Although the enzyme system is similar to that of A. fermentans, the comparative analysis revealed further insights into the mechanism. Especially the highly active dehydratase from C. difficile (specific activity 150 U/mg) revealed several features that could not be seen in the 2-hydroxyglutaryl-CoA dehydratases.

1. The enzyme contains definitely no molybdenum, but 5 Fe, which, according to Mössbauer spectroscopy, form a [4Fe-4S]-cluster with an additional iron that becomes reduced during catalysis.

2. The enzyme catalyses 10,000 turnover until the single electron ‘cofactor’ is lost and another activation by ATP and reduced activator is required.

3. The product-related ketyl radical anion, one of the three postulated radical intermediates, could be detected and characterised by EPR spectroscopy.

The activator has counterparts in the analogous nitrogenase system and in the homologous benzoyl-CoA reductase complex from Thauera aromatica as well as in unknown homologous enzymes from E. coli, Clostridium acetobutylicum and several methanogens. Ketyl mechanisms may also be involved in the coenzyme B12-dependent and independent diol dehydratase, ethanolamine ammonia lyase, glycerol dehydratase and ribonucleotide reductase.


Figure 2. The picture shows the helix-cluster-helix architecture of the activator, which we call Archerase. We propose that upon binding of ATP the angle of 105° opens to 180°, which enables docking of the activator at the dehydratase and the ATP-driven electron transfer. The same conformational change happens at the string of the archer's bow during shooting. The relief of the background depicts the Assyrian king Ashurbanibal hunting wild asses, ca. 650 BC, British Museum, London.

4-Hydroxybutyryl-CoA dehydratase

The formation of a ketyl radical anion in an oxidative process has been proposed for the FAD- and [4Fe-4S]-cluster-containing 4-hydroxybutyryl-CoA dehydratase from Clostridium aminobutyricum. The crystal structure of the FAD- and [4Fe-4S]-cluster-part of one of the four identical subunits is shown in Fig. 3. Notably the cluster is coordinated by three cysteine and one histidine residues with an unusual long N-Fe-bond (2.4 Å). In analogy to butyryl-CoA dehydrogenase, which has a fold related to that of 4-hydroxybutyryl-CoA dehydratase, we have shown that the stereochemistry of the α- and β-hydrogens to be abstracted by both enzymes are identical. We postulate that the hydroxyl group of 4-hydroxybutyryl-CoA coordinates to the Fe-His and releases the histidine, which acts as a base to remove the a-proton. Electron transfer to the flavin generates an enoy radical, which is deprotonated in the ß-position by the flavin semiquinone to yield the ketyl radical anion. Now the hydroxyl group is eliminated and the resulting dienoxy radical is reduced and protonated to the product crotonyl-CoA.


Figure 3. Active site of 4-hydroxybutyryl-CoA dehydratase from Clostridium aminbutyricum. The picture shows the unusual [4Fe-4S]cluster, in which Fe1 is coordinated to His292, and the adjacent FAD. The holes in the electron density of the aromatic isoalloxazine ring demonstrate the high resolution of 1.6 Å.


The activation of the 2-hydroxyacyl-CoA dehydratases requires the reduction of component A or activase by ferredoxin, which usually is reduced by pyruvate ferredoxin-oxidoreductase. In A. fermentans, however, only NADH is formed. Therefore we postulated an enzyme, which catalyses the thermodynamically uphill reduction of ferredoxin by NADH driven by an electrochemical Na+-gradient, which is generated by decarboxylation of glutaconyl-CoA (vide supra). We purified a membrane bound NADH-ferredoxin oxidoreductase from C. tetanomorphum, in which the reaction proceeds in the reverse direction and the generated electrochemical H+/Na+-gradient might be used for transport processes. The enzyme is related to the Rnf-enzyme complex (Rhodobacter capsulatus nitrogen fixation), which has been postulated to be involved in the electron transport to nitrogenase iron protein.

Comparative biochemistry reveals a hitherto unrecognised function of coenzyme B12

Coenzyme B12 initiates radical chemistry in two types of enzymatic reactions, the irreversible eliminases (e.g. diol dehydratases) and the reversible mutases (e.g. methylmalonyl-CoA mutase). Whereas eliminases are known, which use radical generators other than coenzyme B12, no alternative coenzyme B12-independent mutases have been detected for substrates in which a methyl group is reversibly converted to a methylene radical. We predict that such mutases do not exist. However, coenzyme B12-independent pathways have been detected that circumvent the need for glutamate, ß-lysine or methylmalonyl-CoA mutases by proceeding via different intermediates. In humans the methylcitrate cycle, which is ostensibly an alternative to the coenzyme B12-dependent methylmalonyl-CoA pathway for propionate oxidation, is not used because it would interfere with the Krebs cycle and thereby compromise the high energy requirement of the nervous system. In the diol dehydratases the 5'-deoxyadenosyl radical generated by homolysis of the carbon-cobalt-bond of coenzyme B12 moves ca. 10 Å away from the cobalt of cob(II)alamin. The substrate and product radicals are generated at a similar distance from cob(II)alamin, which acts solely as spectator of the catalysis. In glutamate and methylmalonyl-CoA mutases the 5'-deoxyadenosyl radical remains within 3-4 Å of the cobalt, with the substrate and product radicals ca. 3 Å further away. It is suggested that cob(II)alamin acts as a conductor by stabilising both the 5'-deoxyadenosyl radical and the product/substrate-related methylene radicals.

Nicotinate fermentation by Eubacterium barkeri (work performed by Dr. Antonio J. Pierik)

Eubacterium barkeri (formerly called Clostridium barkeri) ferments nicotinate to ammonia, CO2, acetate and propionate. The pathway involves 12 enzymes, 6 of which are specific for nicotinate fermentation. We are interested in the mechanism of the coenzyme B12-dependent 2-methyleneglutarate mutase. It is well established that binding of coenzyme and substrate leads to homolysis of the carbon-cobalt-bond of coenzyme B12 (adenosylcobalamin). The formed 5'-deoxyadenosyl radical abstracts the 4-Re-hydrogen of 2-methyleneglutarate to generate 2-methyleneglutar-4-yl radical (substrate derived radical), which rearranges to the 3-methyleneitaconate radical. Redonation of the abstracted hydrogen to the product related radical yields (R)-3-methylitaconate and regenerates the 5'-deoxyadenosyl radical. The radical rearrangement can occur in two ways: (i) Addition of the substrate-derived radical at the double bond of the methylene group yields a three-membered-ring, which can be reopened by elimination to the product-derived radical. (ii) Alternatively, the substrate-derived radical may fragment into acrylate and acryl-2-yl radical, which can recombine to the product-derived radical. Our experiments with isotope-labelled substrates and analogues thereof using UV/vis, NMR and EPR-spectroscopy as well as scintillation counting favour the fragmentation mechanism, whereas no evidence could be obtained for the addition/elimination pathway. In addition we study the function of MgmL, a protein postulated to be involved in repair or stabilisation of 2-methyleneglutarate mutase.

A 23 kbp-gene cluster encoding the specific enzymes of the nicotinate pathway has been cloned and sequenced. The first enzyme, nicotinate dehydrogenase, comprises four subunits: a regular FAD-subunit, NdhF, and an usual 2x[2Fe-2S] cluster containing subunit, NdhS. Contrary to all sequenced enzymes of the xanthine dehydrogenase family, the E. barkeri nicotinate dehydrogenase has two separate molybdopterin subunits and contains non-selenocysteinyl selenium. UV-Vis and EPR spectroscopy in conjunction with analysis of the primary sequence of the purified 6-hydroxynicotinate reductase showed that this enzyme has one covalently bound flavin, two [4Fe-4S] and one [2Fe-2S] clusters. Purification of two novel enzymes, enamidase and 2-(hydroxymethyl)glutarate dehydrogenase both from E. barkeri and after heterologous expression in E. coli, allowed the identification of 2-formyl- and 2-(hydroxymethyl)glutarate as chiral intermediates. Enamidase belongs to the amidohydrolase enzyme superfamily. It contains a binuclear (Fe-Zn) active site, which appears to catalyse two reactions: hydrolysis of the amide bond of 1,4,5,6-tetrahydro-6-oxo-nicotinate as well as tautomerization of the unstable enamine intermediate to chiral 2-formylglutarate. The 3-dimensional structure of enamidase has been solved to a resolution of 1.89 Å in collaboration with the group of Prof. L.-O. Essen (Fachbereich Chemie, Marburg). 2-(Hydroxymethyl)glutarate dehydrogenase belongs to the 3-hydroxyisobutyrate/ phosphogluconate dehydrogenase superfamily. The enzyme is NADH-specific and as inferred from activity with (S)-3-hydroxyisobutyrate is only active on the (S)-stereoisomer of 2-formyl- and 2-(hydroxymethyl)glutarate. Crystals diffracting up to a resolution of 2.3 Å have been obtained also in collaboration with the Essen group. Bioinformatic analysis identified that an enzyme similar to [4Fe-4S]-cluster containing labile serine dehydratases was encoded in the nicotinate gene cluster. Contrary to our expectations, the dehydration of (S)-2-(hydroxymethyl)glutarate does not occur at the CoA-ester level. This redefined mechanistic demands for dehydrations of -substituted 3-hydroxyacids. The second dehydratase/hydratase in the nicotinate fermentation pathway, (2R,3S)-2,3-dimethylmalate dehydratase, belongs to the aconitase superfamily, a much more common type of dehydratase. The gene encoding the last step of the nicotinate fermentation, 2,3-dimethylmalate lyase, was expressed in E. coli and a functional enzyme could be obtained catalysing the cleavage of (2R,3S)-2,3-dimethylmalate to propionate and pyruvate. Interestingly the enzyme is closely related to isocitrate and 2-methylisocitrate lyases.


Scott, R., Näser, U., Friedrich, P. Cinkaya, I., Buckel, W., & Golding, B. T. (2004) Stereospecific elimination of the 3-Si hydrogen of 4-hydroxybutyryl-CoA during catalysis of 4-hydroxybutyryl-CoA dehydratase from Clostridium aminobutyricum. Chem. Com. (Camb.) 2004, 1210-1211.

Brock, M. & Buckel, W. (2004) On the mechanism of action of the antifungal agent propionate: Propionyl-CoA inhibits glucose metabolism in Aspergillus nidulans. Eur. J. Biochem. 271, 3227-3241.

Speranza, G., Buckel, W. & Golding, B. T. (2004) Coenzyme B12-dependent enzymatic dehydration of 1,2-diols: simple reaction, complex mechanism. J. Porphyrins Phthalocyanines 8, 290-300.

Kim, J., Hetzel, M., Boiangiu, C. D. & Buckel, W. (2004) Dehydration of (R)-2-hydroxyacyl-CoA to enoyl-CoA in the fermentation of a-amino acids by anaerobic bacteria. FEMS Microbiol. Rev. 28, 455-468.

Buckel, W., Hetzel, M. & Kim, J. (2004) ATP-driven electron transfer in enzymatic radical reactions. Curr. Opin. Chem. Biol. 8, 462-467.

Martins, B. M., Dobbek, H., Çinkaya. I., Buckel, W. & Messerschmidt, A. (2004) Crystal structure of 4-hydroxybutyryl-CoA dehydratase: Radical catalysis involving a [4Fe-4S] cluster and flavin. Proc. Natl. Acad. Sci. USA 101, 15645-15649.

Verfürth, K., Pierik, A.J., Leutwein, C., Zorn, S. & Heider, J. (2004) Substrate specificities and electron paramagnetic resonance properties of benzylsuccinate synthases in anaerobic toluene and m-xylene metabolism. Arch. Microbiol. 181, 155-162.

Mander, G.J., Pierik, A.J., Huber, H. & Hedderich, R. (2004) Two distinct heterodisulfide reductase-like enzymes in the sulfate-reducing archaeon Archaeoglobus profundus. Eur. J. Biochem. 271, 1106-1116.

Balk, J., Pierik, A.J., Aguilar Netz, D.J., Mühlenhoff, U. & Lill, R. (2004) The essential yeast hydrogenase-like protein Nar1p is involved in maturation of cytosolic and nuclear iron-sulfur proteins. EMBO J. 23, 2105-2115.

Andrei, P., Pierik, A.J., Zauner, S., Andrei-Selmer, L. & Selmer, T. (2004) Subunit composition of the glycyl radical enzyme p-hydroxyphenylacetate decarboxylase: a small subunit, HpdC, is essential for catalytic activity. Eur. J. Biochem. 271, 2225-2230.

Martins, B. M., Macedo-Ribeiro, S., Bresser, J., Buckel, W. & Messerschmidt, A. (2005) Structural basis for stereo-specific catalysis in NAD+-dependent (R)-2-hydroxyglutarate dehydrogenase from Acidaminococcus fermentans. FEBS Journal 272, 269–281

Näser, U., Pierik, A. J., Scott, R., Çinkaya, I., Buckel, W. & Golding, B. T. (2005) Synthesis of 13C-labeled y-butyrolactones and EPR studies with 4-hydroxybutyryl-CoA dehydratase. Bioorganic Chemistry 33, 53-66.

Kim, J., Darley, D. & Buckel, W. (2005) 2-Hydroxyisocaproyl-CoA dehydratase and its activator from Clostridium difficile. FEBS Journal 272, 550-561.

Herrmann, G., Selmer, T., Jessen, H. J., Gokarn, R. R., Selifonova, O., Gort, S. J & Buckel, W. (2005) Two beta-alanyl-CoA:ammonia lyases in Clostridium propionicum. FEBS Journal 272, 813–821.

Buckel, W. (2005) Special clostridial enzymes and fermentation pathways. In Clostrida (P. Dürre, ed). CRC Press, Taylor & Francis Group, Atlanta, GA, USA, pp. 177-220.

Pierik, A. J., Ciceri, D., Lopez, R. F., Kroll, F., Bröker, G., Beatrix, B., Buckel, W. & Golding, B. T. (2005) Searching for intermediates in the carbon skeleton rearrangement of 2-methyleneglutarate to (R)-3-methylitaconate catalysed by coenzyme B12-dependent 2-methyleneglutarate mutase from Eubacterium barkeri, Biochemistry 44, 10541-10551.

Buckel, W. (2005) On the road to bioremediation of ‘dioxin’. Chem. Biol. 12, 723-724.

Buckel, W. (2005) Highlight: radicals in enzymatic catalysis. Biol. Chem. 386, 949-950.

Buckel, W., Martins, B. M., Messerschmidt, A. & Golding, B. T. (2005) Radical mediated dehydrations in anaerobic bacteria. Biol. Chem. 389, 951-959.

Balk, J, Pierik, A.J., Aguilar Netz, D.J., Mühlenhoff, U. & Lill, R. (2005) Nar1p, a conserved eukaryotic protein with similarity to Fe-only hydrogenases, functions in cytosolic iron-sulphur protein biogenesis. Biochem. Soc. Trans. 33, 86-89.

Hausmann, A., Aguilar Netz, D.J., Balk, J., Pierik, A.J., Mühlenhoff, U. & Lill. R. (2005) The eukaryotic P loop NTPase Nbp35: an essential component of the cytosolic and nuclear iron-sulfur protein assembly machinery. Proc. Natl. Acad. Sci. USA 102, 3266-3271.

Seedorf, H., Kahnt, J., Pierik, A.J. & Thauer, R.K. (2005) Si-face stereospecificity at C5 of coenzyme F420 for F420H2 oxidase from methanogenic Archaea as determined by mass spectrometry. FEBS J. 272, 5337-5342.

Selmer, T., Pierik, A.J. & Heider, J. (2005) New glycyl radical enzymes catalysing key metabolic steps in anaerobic bacteria. Biol. Chem. 386, 981-988.

Balk, J., Aguilar Netz, D.J., Tepper, K., Pierik, A.J. & Lill, R. (2005) The essential WD40 protein Cia1 is involved in a late step of cytosolic and nuclear iron-sulfur protein assembly. Mol. Cell. Biol. 25, 10833-10841.

Buckel, W., Kratky, C. & Golding, B. T. (2005) Stabilisation of methylene radicals by cob(II)alamin in coenzyme B12-dependent mutases, Chem. Eur. J. 12, 352-362.

Boiangiu, C. D., Jayamani, E., Brügel, D., Herrmann, G., Forzi, L., Hedderich, R., Vgenopoulou, I., Pierik, A. J., Steuber, J. & Buckel, W. (2006) Sodium ion pumps and hydrogen production in glutamate fermenting anaerobic bacteria, J. Mol. Microbiol. Biotechnol., in press.

Buckel, W. & Golding, B. T. (2006) Radical enzymes in anaerobes. Ann. Rev. Microbiol., in press.

Mack, M., Liesert, M., Zschocke, J., Peters, V., Linder, D. & Buckel, W. (2006) 3-Methylglutaconyl-CoA hydratase from Acinetobacter sp. Arch. Microbiol., in press.

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Gokarn, R. R., Selifonova, O. V., Jessen, H. J., Gort, S. J., Selmer, T. & Buckel, W. (2004) 3-Hydroxypropionic acid and other organic compounds. US Patent Application 20040076982, Cargill, Inc., USA.

Diploma theses

Sandra Plett (2005) Neue Inaktivatoren der Coenzym B12-abhängigen 2-Methylenglutarat-Mutase aus Eubacterium barkeri

Leslie Schlüter (2005) Zur Klonierung der Gene der 2-Hydroxyglutaryl-CoA-Dehydratase aus Clostridium symbiosum

Martin Schuster (2005) Biochemische und molekularbiologische Untersuchungen zur Nicotinatverwertung durch Proteobakterien

Doctoral theses

Marc Hetzel (2004) Zum Mechanismus der 2-Hydroxyglutaryl-CoA-Dehydratase aus Clostridium symbiosum

Jihoe Kim (2004) On the enzymatic mechanism of 2-hydroxyisocaproyl-CoA dehydratase from Clostridium difficile

Ashraf Al Hapel (2005) Nicotinatfermentation in Eubacterium barkeri: Klonierung des kompletten Nicotinat-Locus und Charakterisierung der 2-(Hydroxymethyl)glutarat-Dehydrogenase und Enamidase

Members of the group (12/2005)

Group leader:

Prof. Dr. Wolfgang Buckel

Postdoctoral fellows:

Dr. Ashraf Alhapel
Dr. Dan Darley
Dr. Jihoe Kim
Dr. Antonio J. Pierik

PhD/Diploma students:

Andrew Bozarth
Daniela Brügel
Peter Friedrich
Gloria Herrmann
Elamparithi Jayamani
Ying Lu
Anutthaman Parthasarathy
Jin Zhang

Technical assistants:

Elke Eckel
Iris Schall

Laboratory technician:

Uta Weber


Marco Hornung


Patricia Wagner


Deutsche Forschungsgemeinschaft: Support for 2 PhD students and 1 Post-doc

Europäische Kommission: Support für 2 Postdocs

Fonds der Chemischen Industrie: Support for Equipment and consumables

Graduiertenkolleg „Protein functions at the atomic level“: Support for 2 PhD students

Sonderforschungsbereich 395: Support for 2 PhD students


Prof. Dr. Wolfgang Buckel
Laboratorium für Mikrobiologie
Fachbereich Biologie
Philipps-Universität Marburg
D-35032 Marburg/Germany

Parcel address:
Karl-von-Frisch-Straße 8
D-35043 Marburg/Germany

Phone +49-6421-2821527
FAX +49-6421-2828979
E-mail: buckel@staff.uni-marburg.de

Zuletzt aktualisiert: 22.05.2006 · dohle

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