(R)-lactyl-CoA dehydratase from Clostridium propionicum. Stereochemistry of the dehydration of (R)-2-hydroxybutyryl-CoA to crotonyl-CoA.
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.
Hofmeister
A E
AE
Laboratorium für Mikrobiologie, Fachbereichs Biologie, Philipps-Universität, Marburg, Federal Republic of Germany.
Buckel
W
W
D000214:Acyl Coenzyme A / Q000737:chemistry / Q000378:metabolism*; D003013:Clostridium / Q000201:enzymology*; D005486:Flavin Mononucleotide / Q000378:metabolism; D006836:Hydro-Lyases / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D012256:Riboflavin / Q000378:metabolism; D013237:Stereoisomerism; D013912:Threonine / Q000378:metabolism; D014867:Water / Q000737:chemistry
C074884:2-hydroxybutyryl-coenzyme A; D000214:Acyl Coenzyme A; D014867:Water; D013912:Threonine; D005486:Flavin Mononucleotide; C010701:crotonyl-coenzyme A; D006836:Hydro-Lyases; C045581:lactate dehydratase; D012256:Riboflavin