148471-94-7Relevant articles and documents
A catalytically versatile benzoyl-CoA reductase, key enzyme in the degradation of methyl- and halobenzoates in denitrifying bacteria
Tiedt, Oliver,Fuchs, Jonathan,Eisenreich, Wolfgang,Boll, Matthias
, p. 10264 - 10274 (2018)
Class I benzoyl-CoA (BzCoA) reductases (BCRs) are key enzymes in the anaerobic degradation of aromatic compounds. They catalyze the ATP-dependent reduction of the central BzCoA intermediate and analogues of it to conjugated cyclic 1,5-dienoyl-CoAs probably by a radical-based, Birch-like reduction mechanism. Discovered in 1995, the enzyme from the denitrifying bacterium Thauera aromatica (BCRTar) has so far remained the only isolated and biochemically accessible BCR, mainly because BCRs are extremely labile, and their heterologous production has largely failed so far. Here, we describe a platform for the heterologous expression of the four structural genes encoding a designated 3-methylbenzoyl-CoA reductase from the related denitrifying species Thauera chlorobenzoica (MBRTcl) in Escherichia coli. This reductase represents the prototype of a distinct subclass of ATP-dependent BCRs that were proposed to be involved in the degradation of methyl-substituted BzCoA analogues. The recombinant MBRTcl had an αβγδ-subunit architecture, contained three low-potential [4Fe- 4S] clusters, and was highly oxygen-labile. It catalyzed the ATPdependent reductive dearomatization of BzCoA with 2.3-2.8 ATPs hydrolyzed per two electrons transferred and preferentially dearomatized methyl- and chloro-substituted analogues in meta- and para-positions. NMR analyses revealed that 3-methylbenzoyl-CoA is regioselectively reduced to 3-methyl- 1,5-dienoyl-CoA. The unprecedented reductive dechlorination of 4-chloro-BzCoA toBzCoAprobably via HCl elimination from a reduced intermediate allowed for the previously unreported growth of T. chlorobenzoica on 4-chlorobenzoate. The heterologous expression platform established in this work enables the production, isolation, and characterization of bacterial and archaeal BCR and BCR-like radical enzymes, for many of which the function has remained unknown.
Mechanism of enzymatic Birch reduction: Stereochemical course and exchange reactions of benzoyl-CoA reductase
Thiele, Baerbel,Rieder, Oliver,Golding, Bernard T.,Mueller, Michael,Boll, Matthias
body text, p. 14050 - 14051 (2009/03/11)
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 establishedradical mechanism of homologous α-hydroxyacyl-CoA dehydratases. Copyright
