Publications

• (R)-lactyl-CoA dehydratase from Clostridium propionicum. Stereochemistry of the dehydration of (R)-2-hydroxybutyryl-CoA to crotonyl-CoA.

  Hofmeister A.E., Buckel W.

  1. A new two-step method for purifying component E II of lactyl-CoA dehydratase was developed. The source of the enzyme was Clostridium propionicum grown on either D,L-alanine or L-threonine. No difference in these preparations was observed whether during purification or by SDS/PAGE of the pure en ... >> More

  1. A new two-step method for purifying component E II of lactyl-CoA dehydratase was developed. The source of the enzyme was Clostridium propionicum grown on either D,L-alanine or L-threonine. No difference in these preparations was observed whether during purification or by SDS/PAGE of the pure enzymes. Both preparations exhibited similar activities towards (R)-lactyl-CoA as well as towards (R)-2-hydroxybutyryl-CoA, the latter being the superior substrate. 2. Three species of (2R)-2-hydroxybutyrate labelled with 3H at C3 were prepared containing 96%, 37% and 63% of the 3H in the 3S-position. By incubation of these species with acetyl-CoA, propionate CoA-transferase and lactyl-CoA dehydratase 104%, 32% and 70% of the 3H, respectively, was release as 3HOH. The data indicate that stereospecific abstraction of the 3Si hydrogen of (2R)-2-hydroxybutyryl-CoA during the dehydration. 3. The identity of the product of the dehydration as crotonyl-CoA was established by the combined action of the enzymes crotonase and (S)-3-hydroxyacyl-CoA dehydrogenase. The results indicate that the elimination of water from (R)-2-hydroxybutyryl-CoA occurs in a syn mode. 4. All enzyme activities necessary for the conversion of L-threonine via (R)-2-hydroxybutyryl-CoA to butyrate were detected in cell-free extracts of C. propionicum. 5. A new mechanism for the dehydration of lactyl-CoA is proposed. << Less

  Eur. J. Biochem. 206:547-552(1992) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• On the dehydration of (R)-lactate in the fermentation of alanine to propionate by Clostridium propionicum.

  Schweiger G., Buckel W.

  All the enzymes of the pathway of (S)-alanine fermentation to acetate and propionate were detected in cell-free extracts of Clostridium propionicum . Among these (S)-glutamate dehydrogenase (NAD), (R)-lactate dehydrogenase (NAD) and propionate CoA-transferase were purified to apparent homogeneity. ... >> More

  All the enzymes of the pathway of (S)-alanine fermentation to acetate and propionate were detected in cell-free extracts of Clostridium propionicum . Among these (S)-glutamate dehydrogenase (NAD), (R)-lactate dehydrogenase (NAD) and propionate CoA-transferase were purified to apparent homogeneity. Their structures were presumably alpha 6, alpha 2 and alpha 4, respectively. The latter enzyme was specific for short-chain monocarboxylic acids with a pronounced preference for (R)-lactate over the (S)-enantiomer. The key step of the pathway, the dehydration of (R)-lactate required acetyl phosphate and CoASH under anaerobic conditions. It was inhibited by hydroxylamine, arsenate, azide (1 mM each) or by 0.1 mM 2,4-dinitrophenol. Thus it closely resembled the dehydration of (R)-2-hydroxyglutarate in Acidaminococcus fermentans , although an activation was not necessary. << Less

  FEBS Lett 171:79-84(1984) [PubMed] [EuropePMC]

• Fe-S centers in lactyl-CoA dehydratase.

  Kuchta R.D., Hanson G.R., Holmquist B., Abeles R.H.

  Lactyl-CoA dehydratase consists of two enzymes, E1 and E2, and requires catalytic quantities of ATP for activity [Kuchta, R. D., & Abeles, R. H. (1985) J. Biol. Chem. 260, 13181-13189]. In contrast to E1, which contains no Fe, E2 contains 8.20 +/-0.04 mol of Fe/mol of E2, one of which can be remov ... >> More

  Lactyl-CoA dehydratase consists of two enzymes, E1 and E2, and requires catalytic quantities of ATP for activity [Kuchta, R. D., & Abeles, R. H. (1985) J. Biol. Chem. 260, 13181-13189]. In contrast to E1, which contains no Fe, E2 contains 8.20 +/-0.04 mol of Fe/mol of E2, one of which can be removed by 1,10-phenanthroline. E2 also contains 7.33 +/-0.68 mol of inorganic sulfur/mol of E2, indicating that at least seven of the Fe atoms are present as Fe-S clusters. E1 and E2 contain less than 0.14 mol of Cu, Co, Zn, Mn, and Ni/mol of E1 or E2. Both reduced and oxidized E1 are EPR silent over a 10,000-G scan range at 4 K, while two signals in E2 are observable at 4 K. Identical spectra were obtained with E2 containing either seven or eight Fe atoms, and both signals were only observable at T less than 30 K. Signal 1 has axial symmetry with g = 2.0232 and g = 2.0006. Signal 2 is orthorhombic with g1 = 1.982, g2 = 1.995, and g3 = 2.019. Computer simulation of these spectra with a S = 1/2 spin Hamiltonian was used to extract the g matrices. The intensity of both signals decreases when E2 is reduced with Na2S2O4. We propose that signal 1 is due to an unusual [4Fe-4S] cluster and signal 2 to a [3Fe-3/4S] cluster. Addition of either acrylyl-CoA or lactyl-CoA dramatically alters signal 2.(ABSTRACT TRUNCATED AT 250 WORDS) << Less

  Biochemistry 25:7301-7307(1986) [PubMed] [EuropePMC]

• Lactate reduction in Clostridium propionicum. Purification and properties of lactyl-CoA dehydratase.

  Kuchta R.D., Abeles R.H.

  Clostridium propionicum converts lactate to propionate (Cardon, B.P., and Barker, H.A. (1947) Arch. Biochem. Biophys. 12, 165-171). We have obtained a soluble system that carries out this conversion as well as the hydration of acrylate to lactate and the reduction of acrylate to propionate. 3-Pent ... >> More

  Clostridium propionicum converts lactate to propionate (Cardon, B.P., and Barker, H.A. (1947) Arch. Biochem. Biophys. 12, 165-171). We have obtained a soluble system that carries out this conversion as well as the hydration of acrylate to lactate and the reduction of acrylate to propionate. 3-Pentynyl-CoA inhibits reduction of acrylate and lactate to propionate, but not hydration of acrylate to lactate by cell extracts. The conversion probably involves CoA esters. When [beta-2H3] lactate is used as a substrate, the rate of propionate formation is reduced 1.8-fold, and the methyl group of the resulting propionate has lost 1.4 deuterium atoms. These results are consistent with the intermediate formation of acrylate (acrylyl-CoA) in the conversion of D-lactate to propionate. Two proteins, which we designate E I and E II, were purified to greater than 90% homogeneity. Together, they catalyze the hydration of acrylyl-CoA to lactyl-CoA. E I has an apparent molecular mass of 27,000 daltons and is rapidly and irreversibly inactivated by O2. E II consists of two subunits of molecular mass 41,000 and 48,000 daltons and contains equal amounts of riboflavin and flavin mononucleotide. Hydration of acrylyl-CoA to lactyl-CoA requires Mg2+ and catalytic quantities of ATP. GTP can replace ATP, but ADP and adenylyl imidodiphosphate cannot. We were unable to detect any stable intermediate during acrylyl-CoA hydration. Finally, we proposed a mechanism for this reaction. << Less

  J. Biol. Chem. 260:13181-13189(1985) [PubMed] [EuropePMC]