(R)-lactyl-CoA dehydratase from Clostridium propionicum. Stereochemistry of the dehydration of (R)-2-hydroxybutyryl-CoA to crotonyl-CoA.
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| Abstract |
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.
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| Authors | A E Hofmeister, W Buckel |
| Journal | European journal of biochemistry (Eur J Biochem) Vol. 206 Issue 2 Pg. 547-52 (Jun 01 1992) ISSN: 0014-2956 [Print] England |
| PMID | 1597194 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't) |
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