Ulrike Näser, Antonio J Pierik, Richard Scott, Irfan Cinkaya, Wolfgang Buckel, Bernard T Golding
School of Natural Sciences--Chemistry, University of Newcastle upon Tyne, Bedson Building, Newcastle upon Tyne, NE1 7RU, United Kingdom.
Bioorganic chemistry 2005 Feb4-Hydroxybutyryl-CoA dehydratase from Clostridium aminobutyricum catalyses the reversible dehydration of its substrate 4-hydroxybutyryl-CoA (4-HB-CoA) to crotonyl CoA. The enzyme contains one [4Fe-4S](2+) cluster and one flavin adenine dinucleotide (FAD) molecule per homotetramer. Incubation of the enzyme with its substrate under equilibrium conditions followed by freezing at 77K induced the EPR-spectrum of a neutral flavin semiquinone (g=2.005, linewidth 2.1 mT), while at 10K additional signals were detected. In an attempt to characterize these signals, 4-HB-CoA molecules specifically labeled with (13)C have been synthesized. This was achieved via (13)C-labeled gamma-butyrolactones, which were obtained from (13)C-labeled bromoacetic acids by efficient synthetic routes. Incubation of the (13)C-labeled 4-hydroxybutyrate-CoA molecules with 4-hydroxybutyryl-CoA dehydratase did not lead to marked broadening of the signals.
Ulrike Näser, Antonio J Pierik, Richard Scott, Irfan Cinkaya, Wolfgang Buckel, Bernard T Golding. Synthesis of (13)C-labeled gamma-hydroxybutyrates for EPR studies with 4-hydroxybutyryl-CoA dehydratase. Bioorganic chemistry. 2005 Feb;33(1):53-66
PMID: 15668183
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