Microbiology AG Buckel
Wolfgang Buckel (born 22. November
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
- Unusual enzymes involved amino acid fermenting anaerobes
- 2-Hydroxyacyl-CoA dehydratases
- 4-Hydroxybutyryl-CoA dehydratase
- Comparative biochemistry reveals a hitherto unrecognised function of coenzyme B12
- Nicotinate fermentation by Eubacterium barkeri (work performed by Dr. Antonio J. Pierik)
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
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
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.
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.
Sandra Plett (2005) Neue Inaktivatoren der Coenzym
B12-abhängigen 2-Methylenglutarat-Mutase aus Eubacterium
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
Marc Hetzel (2004) Zum Mechanismus der
2-Hydroxyglutaryl-CoA-Dehydratase aus Clostridium
Jihoe Kim (2004) On the enzymatic mechanism of
2-hydroxyisocaproyl-CoA dehydratase from Clostridium
Ashraf Al Hapel (2005) Nicotinatfermentation in
Eubacterium barkeri: Klonierung des kompletten Nicotinat-Locus
und Charakterisierung der 2-(Hydroxymethyl)glutarat-Dehydrogenase und
Members of the group (12/2005)
Prof. Dr. Wolfgang Buckel
Dr. Ashraf Alhapel
Dr. Dan Darley
Dr. Jihoe Kim
Dr. Antonio J. Pierik
Deutsche Forschungsgemeinschaft: Support for 2 PhD students and 1
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