title: Synthesis of (13)C-labeled gamma-hydroxybutyrates for EPR studies with 4-hydroxybutyryl-CoA dehydratase.
abstract: 4-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.
authors.affiliation: School of Natural Sciences--Chemistry, University of Newcastle upon Tyne, Bedson Building, Newcastle upon Tyne, NE1 7RU, United Kingdom.
authors.forename: Ulrike
authors.initials: U
authors.lastname: Näser
authors.forename: Antonio J
authors.initials: AJ
authors.lastname: Pierik
authors.forename: Richard
authors.initials: R
authors.lastname: Scott
authors.forename: Irfan
authors.initials: I
authors.lastname: Cinkaya
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Buckel
authors.forename: Bernard T
authors.initials: BT
authors.lastname: Golding
mesh_terms: D000214:Acyl Coenzyme A / Q000737:chemistry / Q000378:metabolism; D002247:Carbon Isotopes; D002384:Catalysis; D003013:Clostridium / Q000201:enzymology*; D003681:Dehydration; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D005182:Flavin-Adenine Dinucleotide / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D006836:Hydro-Lyases / Q000737:chemistry / Q000378:metabolism*; D008956:Models, Chemical; D012978:Sodium Oxybate / Q000138:chemical synthesis* / Q000494:pharmacology
chemical_list: D000214:Acyl Coenzyme A; D002247:Carbon Isotopes; D005182:Flavin-Adenine Dinucleotide; C029276:flavin semiquinone; D012978:Sodium Oxybate; C010701:crotonyl-coenzyme A; C066370:4-hydroxybutyryl-CoA dehydratase; D006836:Hydro-Lyases