Lactate reduction in Clostridium propionicum. Purification and properties of lactyl-CoA dehydratase.
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.
D000179:Acrylates / Q000378:metabolism; D000214:Acyl Coenzyme A / Q000378:metabolism / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D003013:Clostridium / Q000201:enzymology*; D003903:Deuterium; D005486:Flavin Mononucleotide / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D006836:Hydro-Lyases / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D007773:Lactates / Q000378:metabolism*; D019344:Lactic Acid; D008274:Magnesium / Q000494:pharmacology; D009682:Magnetic Resonance Spectroscopy; D008970:Molecular Weight; D010084:Oxidation-Reduction; D010100:Oxygen / Q000494:pharmacology; D011422:Propionates / Q000378:metabolism; D013053:Spectrophotometry; D013329:Structure-Activity Relationship
D000179:Acrylates; D000214:Acyl Coenzyme A; D007773:Lactates; D011422:Propionates; D005182:Flavin-Adenine Dinucleotide; C047009:lactoyl-coenzyme A; D019344:Lactic Acid; C047010:3-pentynoyl-coenzyme A; C047011:acryloyl-coenzyme A; D005486:Flavin Mononucleotide; D000255:Adenosine Triphosphate; D003903:Deuterium; D006836:Hydro-Lyases; C045581:lactate dehydratase; D008274:Magnesium; C036658:acrylic acid; C029658:propionic acid; D010100:Oxygen