Publications

• Purification and properties of an iron-sulfur and FAD-containing 4-hydroxybutyryl-CoA dehydratase/vinylacetyl-CoA delta 3-delta 2-isomerase from Clostridium aminobutyricum.

  Scherf U., Buckel W.

  4-Hydroxybutyryl-CoA dehydratase, the key enzyme in the metabolism of gamma-aminobutyrate in Clostridium aminobutyricum, represents approximately 15-25% of the soluble protein. The enzyme was purified to homogeneity under anaerobic conditions to a specific activity of 209 nkat mg-1. The dehydratas ... >> More

  4-Hydroxybutyryl-CoA dehydratase, the key enzyme in the metabolism of gamma-aminobutyrate in Clostridium aminobutyricum, represents approximately 15-25% of the soluble protein. The enzyme was purified to homogeneity under anaerobic conditions to a specific activity of 209 nkat mg-1. The dehydratase catalyses the reversible conversion of 4-hydroxybutyryl-CoA (Km = 50 microM) to crotonyl-CoA and possesses a probably intrinsic vinylacetyl-CoA delta 3-delta 2-isomerase with a specific activity of 223 nkat mg-1. The equilibrium of the reversible dehydration was determined from both sides as K = [crotonyl-CoA]/[4-hydroxybutyryl-CoA] = 4.2 +/-0.3. Cyclopropylcarboxyl-CoA was not converted to crotonyl-CoA. The native enzyme has an apparent molecular mass of 232 kDa and is composed of four apparently identical subunits (molecular mass = 56 kDa), indicating a homotetrameric structure. Under anaerobic conditions the active enzyme revealed a brown colour and contained 2 +/- 0.2 mol FAD (64 +/- 5% oxidized), 16 +/-0.8 mol Fe and 14.4 +/-1.2 mol inorganic sulfur, which probably form iron-sulfur clusters. Exposure to air resulted initially in a slight activation followed by irreversible inactivation. Concomitantly the vinylacetyl-CoA delta-isomerase activity was lost and the colour of the enzyme changed to yellow. Reduction by sodium dithionite yielded inactive enzyme which could be completely reactivated by oxidation with potassium hexacyanoferrate(III). The data indicate that the active enzyme contains oxidized FAD despite its sensitivity towards oxygen. During the dehydration a non activated C-H bond at C-3 of 4-hydroxybutyryl-CoA has to be cleaved. A putative mechanism for 4-hydroxybutyryl-CoA dehydratase is proposed in which this cleavage is achieved by a FAD-dependent oxidation of 4-hydroxybutyryl-CoA to 4-hydroxycrotonyl-CoA. In a second step the hydroxyl group is substituted by a hydride derived from the now reduced FAD in an SN2' reaction leading to vinylacetyl-CoA. Finally isomerisation yields crotonyl-CoA. 4-Hydroxybutyryl-CoA dehydratase is quite distinct from 3-hydroxyacyl-CoA dehydratase (crotonase) and 2-hydroxyacyl-CoA dehydratases. Contrary to the latter enzyme [e.g. (R)-lactyl-CoA dehydratase and (R)-2-hydroxyglutaryl-CoA dehydratase] which are composed of three different subunits and similarly catalyse the cleavage of a non activated C-H bond at C-3, 4-hydroxybutyryl-CoA dehydratase does not require ATP, MgCl2 and Ti(III)citrate for activity. Furthermore 4-hydroxybutyryl-CoA dehydratase is not inactivated by oxidants such as 5 mM 4-nitrophenol, 5 mM chloramphenicol and 5 mM hydroxylamine. << Less

  Eur. J. Biochem. 215:421-429(1993) [PubMed] [EuropePMC]

• 4-Hydroxybutyryl-CoA dehydratase from Clostridium aminobutyricum: characterization of FAD and iron-sulfur clusters involved in an overall non-redox reaction.

  Muh U., Cinkaya I., Albracht S.P., Buckel W.

  4-Hydroxybutyryl-CoA dehydratase catalyzes the reversible dehydration of 4-hydroxybutyryl-CoA to crotonyl-CoA, which involves cleavage of an unactivated beta-C-H bond. The enzyme also catalyzes the apparently irreversible isomerization of vinylacetyl-CoA to crotonyl-CoA. Addition of crotonyl-CoA t ... >> More

  4-Hydroxybutyryl-CoA dehydratase catalyzes the reversible dehydration of 4-hydroxybutyryl-CoA to crotonyl-CoA, which involves cleavage of an unactivated beta-C-H bond. The enzyme also catalyzes the apparently irreversible isomerization of vinylacetyl-CoA to crotonyl-CoA. Addition of crotonyl-CoA to the dehydratase, which contains FAD as well as non-heme iron and acid labile sulfur, led to a decrease of the flavin absorbance at 438 nm and an increase in the region from 500 to 800 nm. The protein-bound FAD was easily reduced to the semiquinone (redox equilibration within seconds) and only slowly to the hydroquinone (redox equilibration minutes to hours): the redox potentials were not unusual for flavoproteins (Eox/sq = -140 +/- 15 mV and Esq/red = -240 +/-15 mV; pH 7.0, 25 degrees C). There was no equilibration of electrons between the flavin and the Fe-S cluster, which was difficult to reduce. After extensive photoreduction, an EPR signal indicative of a [4Fe-4S]+ cluster was detected (g-values: 2.037, 1.895, 1.844). Upon exposure to air at 0 degrees C, the enzyme lost dehydration activity completely within 40 min, but isomerase activity dropped to about 40% of the initial value and persisted for more than a day. The properties of the protein-bound FAD are consistent with a mechanism involving transient one-electron oxidation of the substrate to activate the the beta-C-H bond. The putative [4Fe-4S]2+ cluster could serve a structural role and/or as Lewis acid facilitating the leaving of the hydroxyl group. << Less

  Biochemistry 35:11710-11718(1996) [PubMed] [EuropePMC]

• Assay of 4-hydroxybutyryl-CoA dehydratase from Clostridium aminobutyricum.

  Willadsen P., Buckel W.

  It has been proposed that Clostridium aminobutyricum contains an enzyme catalyzing an unusual reaction: the dehydration of 4-hydroxybutyryl-CoA to vinylacetyl-CoA. 4-Hydroxy-[3-3H]butyric acid has been prepared which allows the activity of this enzyme to be assayed in the presence of acetyl-CoA un ... >> More

  It has been proposed that Clostridium aminobutyricum contains an enzyme catalyzing an unusual reaction: the dehydration of 4-hydroxybutyryl-CoA to vinylacetyl-CoA. 4-Hydroxy-[3-3H]butyric acid has been prepared which allows the activity of this enzyme to be assayed in the presence of acetyl-CoA under anaerobic conditions by the release of tritiated water. Initial characterization of the enzyme from C. aminobutyricum has shown it to be largely membrane or particle bound in the crude lysates. It can be solubilized in detergent. It is inactivated by oxygen, but stable under anaerobic conditions. Only 49 +/-2% of the label is removed after enzyme-catalyzed equilibration with water. This stereospecific release is consistent with the formation of vinylacetyl-CoA and excludes a vitamin B12 coenzyme-dependent rearrangement to 3-hydroxybutyryl-CoA followed by dehydration to crotonyl-CoA. << Less

  FEMS Microbiol Lett 58:187-191(1990) [PubMed] [EuropePMC]

• A 3-hydroxypropionate/4-hydroxybutyrate autotrophic carbon dioxide assimilation pathway in Archaea.

  Berg I.A., Kockelkorn D., Buckel W., Fuchs G.

  The assimilation of carbon dioxide (CO2) into organic material is quantitatively the most important biosynthetic process. We discovered that an autotrophic member of the archaeal order Sulfolobales, Metallosphaera sedula, fixed CO2 with acetyl-coenzyme A (acetyl-CoA)/propionyl-CoA carboxylase as t ... >> More

  The assimilation of carbon dioxide (CO2) into organic material is quantitatively the most important biosynthetic process. We discovered that an autotrophic member of the archaeal order Sulfolobales, Metallosphaera sedula, fixed CO2 with acetyl-coenzyme A (acetyl-CoA)/propionyl-CoA carboxylase as the key carboxylating enzyme. In this system, one acetyl-CoA and two bicarbonate molecules were reductively converted via 3-hydroxypropionate to succinyl-CoA. This intermediate was reduced to 4-hydroxybutyrate and converted into two acetyl-CoA molecules via 4-hydroxybutyryl-CoA dehydratase. The key genes of this pathway were found not only in Metallosphaera but also in Sulfolobus, Archaeoglobus, and Cenarchaeum species. Moreover, the Global Ocean Sampling database contains half as many 4-hydroxybutyryl-CoA dehydratase sequences as compared with those found for another key photosynthetic CO2-fixing enzyme, ribulose-1,5-bisphosphate carboxylase-oxygenase. This indicates the importance of this enzyme in global carbon cycling. << Less

  Science 318:1782-1786(2007) [PubMed] [EuropePMC]

  This publication is cited by 6 other entries.

• Crystal structure of 4-hydroxybutyryl-CoA dehydratase: radical catalysis involving a [4Fe-4S] cluster and flavin.

  Martins B.M., Dobbek H., Cinkaya I., Buckel W., Messerschmidt A.

  Dehydratases catalyze the breakage of a carbon-oxygen bond leading to unsaturated products via the elimination of water. The 1.6-A resolution crystal structure of 4-hydroxybutyryl-CoA dehydratase from the gamma-aminobutyrate-fermenting Clostridium aminobutyricum represents a new class of dehydrata ... >> More

  Dehydratases catalyze the breakage of a carbon-oxygen bond leading to unsaturated products via the elimination of water. The 1.6-A resolution crystal structure of 4-hydroxybutyryl-CoA dehydratase from the gamma-aminobutyrate-fermenting Clostridium aminobutyricum represents a new class of dehydratases with an unprecedented active site architecture. A [4Fe-4S](2+) cluster, coordinated by three cysteine and one histidine residues, is located 7 A from the Re-side of a flavin adenine dinucleotide (FAD) moiety. The structure provides insight into the function of these ubiquitous prosthetic groups in the chemically nonfacile, radical-mediated dehydration of 4-hydroxybutyryl-CoA. The substrate can be bound between the [4Fe-4S](2+) cluster and the FAD with both cofactors contributing to its radical activation and catalytic conversion. Our results raise interesting questions regarding the mechanism of acyl-CoA dehydrogenases, which are involved in fatty acid oxidation, and address the divergent evolution of the ancestral common gene. << Less

  Proc. Natl. Acad. Sci. U.S.A. 101:15645-15649(2004) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.