Mechanism of enzymatic Birch reduction: stereochemical course and exchange reactions of benzoyl-CoA reductase.
Dearomatizing benzoyl-coenzyme A reductases (BCR) from facultatively anaerobic bacteria are key enzymes in the anaerobic degradation of aromatic compounds. They catalyze the ATP-dependent reduction of benzoyl-CoA (BCoA) to cyclohexa-1,5-diene-1-carboxyl-CoA (dienoyl-CoA). A Birch reduction mechanism involving alternate electron transfer and protonation steps has been proposed for BCR. In this work we reacted BCoA in H2O and D2O, and d5-BCoA in H2O with BCR and the second enzyme of the pathway, dienoyl-CoA hydratase (DCH). The 1,4 hydration product formed from the dienoyl-CoA, 6-hydroxycyclohex-1-ene-1-carbonyl-CoA, was analyzed by several NMR techniques. The results obtained indicate that BCR stereoselectively forms the trans-dienoyl-CoA product, and DCH stereoselectively catalyzes a trans-1,4 water addition. Moreover, unexpected proton exchanges at C-2 and C-6 were observed. They indicate that a free radical intermediate with an unusual low pKa is formed during BCR catalysis. This finding provides evidence for the proposed Birch reduction mechanism of BCR and is in agreement with the established radical mechanism of homologous alpha-hydroxyacyl-CoA dehydratases.
Thiele
Bärbel
B
Institute of Biochemistry, University of Leipzig, 04103 Leipzig, Germany.
Rieder
Oliver
O
Golding
Bernard T
BT
Müller
Michael
M
Boll
Matthias
M
D000214:Acyl Coenzyme A / Q000737:chemistry*; D017666:Deuterium Oxide / Q000737:chemistry; D006836:Hydro-Lyases / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D015394:Molecular Structure; D010084:Oxidation-Reduction; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000737:chemistry*; D013237:Stereoisomerism; D014867:Water / Q000737:chemistry
D000214:Acyl Coenzyme A; D014867:Water; C037817:benzoyl-coenzyme A; D044925:Oxidoreductases Acting on CH-CH Group Donors; C109030:benzoyl-coenzyme A reductase (dearomatizing); D006836:Hydro-Lyases; C114397:cyclohexa-1,5-diene-1-carboxyl-CoA hydratase; D017666:Deuterium Oxide