Dehydratases catalyze the breakage of a carbonOoxygen bond leading to unsaturated products via the elimination of water. The 1.6-Å resolution crystal structure of 4-hydroxybutyryl-CoA dehydratase from the ␥-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 Å 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. Dehydration is a very common reaction in biochemical pathways. More than 100 dehydratases (carbon-oxygen lyases, EC 4.2.-.-) are known [Expert Protein Analysis System (ExPASy), www.expasy.org]. Most of these enzymes catalyze the ␣,␤-elimination of water, during which the ␣-hydrogen (C2 position) to be removed as a proton is activated by an adjacent electron-withdrawing carboxylate, carbonyl, or CoA-thiol ester group, and the hydroxyl group leaves from the ␤-position (C3 position) (1). In anaerobic microorganisms, the absence of the biradical dioxygen allows a rich chemistry of radical reactions involved in the removal of hydrogen atoms from nonactivated positions such as the ␤-or ␥-carbons of 2-, 4-, or 5-hydroxyacylCoA derivatives. These atypical dehydratases contain one or more prosthetic groups such as Fe-S clusters or flavins (2). An example is 4-hydroxybutyryl-CoA dehydratase (4-BUDH) from Clostridium aminobutyricum, which catalyzes both the reversible oxygen-sensitive dehydration of 4-hydroxybutyryl-CoA (Scheme 1a) and the oxygen-insensitive isomerization of vinylacetyl-CoA (Scheme 1c) to crotonyl-CoA (Scheme 1b) (3). The dehydration reaction requires the removal of a hydrogen atom from the least activated C3 position of the butyryl chain (pK a Ϸ 40) and is the mechanistically most demanding step in the fermentation of ␥-aminobutyrate (GABA) to ammonia, acetate, and butyrate by C. aminobutyricum (4). 4-BUDH is active as a homotetramer with up to one [4Fe-4S] 2ϩ cluster and one noncovalently bound flavin adenine dinucleotide (FAD) moiety per 54-kDa subunit. Despite the presence of a [4Fe-4S] 2ϩ cluster and the need of an oxidized FAD for catalysis, the overall reaction occurs with no net redox change (4). These observations have led to the proposal of a radical-based mechanism for the dehydration, where the one-electron oxidation of the enolate of 4-hydroxybutyryl-CoA to the enoxy radical makes the C3-proS-hydrogen (5) acidic enough [pK a ϭ 14 (6)] for deprotonation to a ketyl radical anion (F...

Clostridium aminobutyricum ferments 4-aminobutyrate via succinic semialdehyde, 4-hydroxybutyrate, 4-hydroxybutyryl-CoA and crotonyl-CoA to acetate and butyrate. The genes coding for the enzymes that catalyse the interconversion of these intermediates are arranged in the order abfD (4-hydroxybutyryl-CoA dehydratase), abfT (4-hydroxybutyrate CoA-transferase), and abfH (NAD-dependent 4-hydroxybutyrate dehydrogenase). The genes abfD and abfT were cloned, sequenced and expressed as active enzymes in Escherichia coli. Hence the insertion of the [4Fe-4S]clusters and FAD into the dehydratase required no additional specific protein from C. aminobutyricum. The amino acid sequences of the dehydratase and the CoA-transferase revealed close relationships to proteins deduced from the genomes of Clostridium difficile, Porphyromonas gingivalis and Archaeoglobus fulgidus. In addition the N-terminal part of the dehydratase is related to those of a family of FAD-containing mono-oxygenases from bacteria. The putative assignment in the databank of Cat2 (OrfZ) from Clostridium kluyveri as 4-hydroxybutyrate CoA-transferase, which is thought to be involved in the reductive pathway from succinate to butyrate, was confirmed by sequence comparison with AbfT (57% identity). Furthermore, an acetyl-CoA:4-hydroxybutyrate CoA-transferase activity could be detected in cell-free extracts of C. kluyveri. In contrast to glutaconate CoA-transferase from Acidaminococcus fermentans, mutation studies suggested that the glutamate residue of the motive EXG, which is conserved in many homologues of AbfT, does not form a CoA-ester during catalysis.

A FAD and [4Fe-4S]cluster-containing enzyme from Clostridium aminobutyricum catalyses the reversible dehydration of 4-hydroxybutyryl-CoA to crotonyl-CoA which involves the cleavage of an unactivated C-H bond at the beta-carbon. Transient oxidation of the substrate to an enoxy radical by FAD might facilitate the removal of this beta-proton, whereas no function could be attributed to the [4Fe-4S]cluster. In this paper the organic radical, which is formed by partial reduction of the enzyme with dithionite, was characterised as the neutral flavin semiquinone by EPR spectroscopy in H2O and D2O. The rapid electron-spin relaxation of the flavin semiquinone suggested a magnetic interaction with the [4Fe-4S]cluster. In order to obtain highly resolved information about nuclear spins in the vicinity of this paramagnetic centre, ENDOR spectroscopy was applied. The spectra were compared with those of the neutral semiquinone radicals of ferredoxin-NADP reductase and flavodoxin as well as with that of the anionic semiquinone radical of cholesterol oxidase. All ENDOR spectra showed strong couplings to the 8-methyl protons and to H-6 of the flavin. On addition of the substrates to the corresponding enzymes, the electron density changed significantly only at the 8-position. It decreased in the case of cholesterol oxidase and ferredoxin-NADP reductase, whereas an increase was observed with 4-hydroxybutyryl-CoA dehydratase. The results indicate an interaction of 4-hydroxybutyryl-CoA with the flavin as required by the proposed mechanism. Furthermore, the shift of electron density towards the benzoid ring of FAD in the dehydratase might be due to the location of the [4Fe-4S]cluster next to the 8-position as known from structurally characterised iron-sulfur flavoproteins.

4-Hydroxybutyryl-CoA dehydratase catalyzes the reversible dehydration of 4-hydroxybutyryl-CoA to crotonyl-CoA, which involves cleavage of an unactivated β-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 (E ox/sq = −140 ± 15 mV and E sq/red = −240 ± 15 mV; pH 7.0, 25 °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 °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 β-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.