Search

Research has been conducted on the Acidaminococcus fermentans 2-hydroxyglutaryl-CoA dehydratase. The isolation of flavodoxin from A. fermentans cell-free extracts has been carried out and the results demonstrate the postulated one-electron transfer from flavodoxin to the homodimeric activator.

Radio-labeled glutamates were utilized in the investigation of the dynamics of coenzyme B12-dependent glutamate mutase-catalyzed reaction forming methylaspartate from glutamate. Electron paramagnetic resonance spectroscopy was used to follow the reaction. Simulations were used in the interpretation of the data generated which indicate that 4-glutamyl radical formation is an intermediate in the reaction catalyzed by coenzyme B12-dependent glutamate mutase.

The catalytic mechanism of 4-hydroxybutyryl-CoA dehydratase from Clostridium aminobutyricum was determined by characterization of the FAD and iron-sulfur clusters involved in the reaction. Electron paramagnetic resonance measurements indicate transient one-electron oxidation that initiates cleavage of the beta-C-H bond. This suggests that redox reactions involving FAD and the Fe-S cluster are involved in catalysis by 4-hydroxybutyryl-CoA dehydratase.

The different enzymes which isomerase's on a group of carbon skeleton mutases have important functions in catabolic processes in human liver and bacterial fermentations. It is indicated that the involvement of cob(II) alaminorganic radical partnership with 2-methylenegluterate mutase is similar to that in glutamate mutase and thus would be useful in conjugating high frequency and pulsed electron paramagnetic resonance techniques.

Two-methyleneglutarate mutase from the anaerobe eubacterium barkeri is an adenosylcobalamin (coenzyme B(sub 12))-dependent enzyme that catalyzes the equilibrium of 2-methylene-glutarate with (R)-3-methylitaconate. It is concluded that the fragmentation-recombination mechanism can provide an explanation for results in terms of an additional barrier, despite the higher calculated activation energy for this pathway.

EXAFS spectroscopy was used to study the axial Co-N bond length in two adenosylcobalamin-depednent enzymes glutamate mutase and 2-methyleneglutarate mutase.

Members of the 2-hydroxyacyl-CoA dehydratase enzyme family catalyze the β,α-dehydration of various CoA-esters in the fermentation of amino acids by clostridia. Abstraction of the nonacidic β-proton of the 2-hydroxyacyl-CoA compounds is achieved by the reductive generation of ketyl radicals on the substrate, which is initiated by the transfer of an electron at low redox potentials. The highly energetic electron needed on the dehydratase is donated by a [4Fe-4S] cluster containing ATPase, termed activator. We investigated the activator of the 2-hydroxyisocaproyl-CoA dehydratase from Clostridium difficile. The activator is a homodimeric protein structurally related to acetate and sugar kinases, Hsc70 and actin, and has a [4Fe-4S] cluster bound in the dimer interface. The crystal structures of the Mg-ADP, Mg-ADPNP, and nucleotide-free states of the reduced activator have been solved at 1.6-3.0 Å resolution, allowing us to define the position of Mg2+ and water molecules in the vicinity of the nucleotides and the [4Fe-4S] cluster. The structures reveal redox- and nucleotide dependent changes agreeing with the modulation of the reduction potential of the [4Fe-4S] cluster by conformational changes. We also investigated the propensity of the activator to form a complex with its cognate dehydratase in the presence of Mg-ADP and Mg-ADPNP and together with the structural data present a refined mechanistic scheme for the ATP-dependent electron transfer between activator and dehydratase. [ABSTRACT FROM AUTHOR]

The radical enzyme (R)-2-hydroxyisocaproyl-CoA dehydratase catalyzes the dehydration of (R)-2-hydroxyisocaproyl-CoA in the fermentation of l-leucine by the human pathogenic bacterium Clostridium difficile. In contrast to other radical enzymes, such as bacterial class II ribonucleotide reductase or biotin synthase, the Fe/S cluster containing (R)-2-hydroxyisocaproyl-CoA dehydratase requires no special cofactors such as coenzyme B12 or S-adenosylmethionine for radical generation. Instead it uses a single high-energy electron that is recycled after each turnover. The catalyzed reaction, an atypical α/β-dehydration, depends on the reductive formation of ketyl radicals on the substrate generated by injection of a single electron from the ATP-dependent activator protein. So far, it is unknown how the active electron is recycled and how unwanted side reactions are prevented, allowing for up to 10000 turnovers. The crystal structure reveals that the heterodimeric protein contains two [4Fe-4S] clusters at a distance of 12 Å, each coordinated by three cysteines and one terminal ligand. The cluster in the α-subunit is part of the active site. In the absence of substrate, a water/hydroxide ion acts as the fourth ligand. The substrate replaces this ligand and coordinates the cluster via the carbonyl-oxygen of the thioester group. The cluster in the β-subunit has a terminal sulfhydryl/sulfido ligand and can act as a reservoir to protect the electron from unwanted side reactions via a recycling mechanism. The crystal structure of (R)-2-hydroxyisocaproyl-CoA dehydratase serves as a model for the reductively radical-generating metalloenzymes of the (R)-2-hydroxyacyl-CoA dehydratase and benzoyl-CoA reductase families. [ABSTRACT FROM AUTHOR]

2-Methyleneglutarate mutase from the anaerobe Eubacterium (Clostridium) barkeri is an adenosylcobalamin (coenzyme B(12))-dependent enzyme that catalyzes the equilibration of 2-methyleneglutarate with (R)-3-methylitaconate. Two possibilities for the mechanism of the carbon skeleton rearrangement of the substrate-derived radical to the product-related radical are considered. In both mechanisms an acrylate group migrates from C-3 of 2-methyleneglutarate to C-4. In the "addition-elimination" mechanism this 1,2-shift occurs via an intermediate, a 1-methylenecyclopropane-1,2-dicarboxylate radical, in which the migrating acrylate is simultaneously attached to both C-3 and C-4. In the "fragmentation-recombination" mechanism the migrating group, a 2-acrylyl radical, becomes detached from C-3 before it starts bonding to C-4. In an attempt to distinguish between these two possibilities we have investigated the action of 2-methyleneglutarate mutase on the stereospecifically deuterated substrates (Z)-3-methyl[2'-(2)H(1)]itaconate and (Z)-3-[2'-(2)H(1),methyl-(2)H(3)]methylitaconate. The enzyme catalyzes the equilibration of both compounds with their corresponding E-isomers and with a 1:1 mixture of the corresponding (E)- and (Z)-2-methylene[2'-(2)H(1)]glutarates, as shown by monitoring of the reactions with (1)H and (2)H NMR. In the initial phase of the enzyme-catalyzed equilibration a significant excess (8-11%) of (E)-3-methyl[2'-(2)H(1)]itaconate over its equilibrium value was observed ("E-overshoot"). The E-overshoot was only 3-4% with (Z)-3-[2'-(2)H(1),methyl-(2)H(3)]methylitaconate because the presence of the deuterated methyl group raises the energy barrier from 3-methylitaconate to the corresponding radical. The overshoot is explained by postulating that the migrating acrylate group has to overcome an additional energy barrier from the state leading back to the substrate-derived radical to the state leading forward to the product-related radical. It is concluded that the fragmentation-recombination mechanism can provide an explanation for the results in terms of an additional energy barrier, despite the higher calculated activation energy for this pathway.