Nonaromatic Products from Anoxic Conversion of Benzoyl-CoA with Benzoyl-CoA Reductase and Cyclohexa-1,5-diene-1-carbonyl-CoA Hydratase

Boll, Matthias; Laempe, Diana; Eisenreich, Wolfgang; Bacher, Adelbert; Mittelberger, Thomas; Heınze, Jürgen; Fuchs, Georg

doi:10.1074/jbc.m001833200

Cited by 62 publications

(72 citation statements)

References 28 publications

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“…BCoA was synthesized from benzoic acid anhydride and CoA (29). Dienoyl-CoA was enzymatically synthesized from BCoA by using enriched BCR from T. aromatica; the product was purified by preparative HPLC as described (7). Other CoA-esters were synthesized as described earlier (20).…”

Section: Methodsmentioning

confidence: 99%

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Identification and characterization of the tungsten-containing class of benzoyl-coenzyme A reductases

Kung

Löffler

Dörner

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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Section: Methodsmentioning

confidence: 99%

“…BCoA serves as the substrate for BCoA reductases (BCR), which dearomatize the aromatic ring by reduction yielding cyclohexa-1,5-diene-1-carboxyl-CoA (dienoyl-CoA; Fig. 1) (5)(6)(7)(8). A BCR enzyme has so far only been isolated and studied in the denitrifying, facultative anaerobe Thauera aromatica (9).…”

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confidence: 99%

Identification and characterization of the tungsten-containing class of benzoyl-coenzyme A reductases

Kung

Löffler

Dörner

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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115

139

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“…BCoA was synthesized from CoA and benzoic acid anhydride (19). Purification and purity control of the thioester was carried out by HPLC as described (3). The N-terminal amino acid sequence of the phosphorylated protein band was determined according to an automatized Edman degradation by H. Schägger (University of Frankfurt, Germany).…”

Section: Methodsmentioning

confidence: 99%

Mechanism of ATP-driven electron transfer catalyzed by the benzene ring-reducing enzyme benzoyl- CoA reductase

Unciuleac

Boll

2001

Proc. Natl. Acad. Sci. U.S.A.

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Benzoyl-CoA reductase (BCR) from the bacterium Thauera aromatica catalyzes the two-electron reduction of benzoyl-CoA (BCoA) to a nonaromatic cyclic diene. In a process analogous to enzymatic nitrogen reduction, BCR couples the electron transfer to the aromatic ring to a stoichiometric hydrolysis of 2 ATP͞2e ؊ . Reduced but not oxidized BCR hydrolyzes ATP to ADP. In this work, purified BCR was shown to catalyze an isotope exchange from [ 14 C]ADP to [ 14 C]ATP, which was Ϸ15% of the ATPase activity in the presence of equimolar amounts of ADP and ATP. In accordance, BCR (␣␤␥␦-composition) autophosphorylated its ␥-subunit when incubated with [␥-32 P]ATP. Formation of the enzyme-phosphate was independent of the redox state, whereas only dithionitereduced BCR catalyzed a dephosphorylation associated with the ATPase activity. This finding suggests that the ATPase-and autophosphatase-partial activities of BCR exhibit identical redox dependencies. BCoA or the nonphysiological electron-accepting substrate hydroxylamine stimulated the redox-dependent effects; the rates of both the overall ATPase and the autophosphatase activities of reduced BCR were increased 6-fold. In contrast, BCoA and hydroxylamine had no effect on oxidized and phosphorylated BCR. The reactivity of the phosphoamino acid fits best with a phosphoamidate or acylphosphate linkage. The results obtained suggest a mechanism of ATP hydrolysis-driven electron transfer, which differs from that of nitrogenase by the transient formation of a phosphorylated enzyme.B enzoyl-CoA (BCoA) is a central intermediate in the metabolism of many aromatic compounds in anaerobic bacteria. This compound becomes reduced by benzoyl-CoA reductase (BCR), a key enzyme in anaerobic aromatic metabolism (1, 2). It catalyzes the reductive dearomatization of BCoA to cyclohexa-1,5-diene-1-carbonyl-CoA ( Fig. 1; ref. 3). In this reaction, the hydrolysis of two ATP to ADP and Pi is stoichiometrically coupled to the transfer of two single electrons from reduced ferredoxin to the aromatic substrate. Notably, BCR also catalyzes the ATP-dependent reduction of the nonphysiological substrate hydroxylamine (2). The stoichiometric coupling of ATP hydrolysis to an electron transfer process has long been considered as a unique feature of nitrogenases (4, 5). However, there are no amino acid sequence similarities between BCR and nitrogenases.The reduction of the aromatic ring is a mechanistically and energetically difficult process that in organic synthesis requires solvated electrons, which are generated by dissolving alkali metals in liquid ammonia. In a so-called Birch mechanism, the reduction of the aromatic ring proceeds in single electron and proton transfer steps by means of radical intermediates (6). A similar mechanism has been proposed for enzymatic benzene ring reduction (7). The crucial step is the first electron transfer to the aromatic ring yielding a nonaromatic radical anion, which requires a redox potential of Ϫ1.9 V (3). BCR overcomes this high redox barrier by coupling a stoichiomet...

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“…Cyclohex-1-ene-1-carbonyl-CoA (1-monoenoyl-CoA) and 3-monoenoyl-CoA were synthesized from the free acids and CoA via succinimidyl ester synthesis as described (25). 2-Monoenoyl-CoA was enzymatically synthesized by a BCR/1,5-dienoyl-CoA hydratase-containing enzyme fraction using dithionite as electron donor as described earlier (26). 1,3-, 1,4-, and 2,5-dienoyl-CoA were synthesized from the free acids and CoA via modified succinimidyl ester synthesis.…”

Section: Growth Of Bacterial Cells and Preparation Of Cellmentioning

confidence: 99%

“…For activity determination in crude extracts, a discontinuous HPLC assay was used as described earlier (26).…”

Section: Growth Of Bacterial Cells and Preparation Of Cellmentioning

confidence: 99%

Aromatizing Cyclohexa-1,5-diene-1-carbonyl-Coenzyme A Oxidase

Thiele

Rieder

Jehmlich

et al. 2008

Journal of Biological Chemistry

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Benzoyl-CoA reductases (BCRs) are key enzymes of anaerobic aromatic metabolism in facultatively anaerobic bacteria. The highly oxygen-sensitive enzymes catalyze the ATP-dependent reductive dearomatization of the substrate, yielding cyclohexa-1,5-diene-1-carbonyl-CoA (1,5-dienoyl-CoA). In extracts from anaerobically grown denitrifying Thauera aromatica, we detected a benzoate-induced, benzoyl-CoA-forming, 1,5-dienoyl-CoA:acceptor oxidoreductase activity. This activity co-purified with BCR but could be partially separated from it by hydroxyapatite chromatography. After activity staining on native gels, a monomeric protein with a subunit molecular weight of M r 76,000 was identified. Mass spectrometric analysis of tryptic digests identified peptides from NADH oxidases/2,4-dienoyl-CoA reductases/"old yellow" enzymes. The UV-visible spectrum of the enriched enzyme suggested the presence of flavin and Fe/S-cofactors, and it was bleached upon the addition of 1,5-dienoyl-CoA. The enzyme had a high affinity for dioxygen as electron acceptor (K m ‫؍‬ 10 M) and therefore is referred to as 1,5-dienoyl-CoA oxidase (DCO). The likely product formed from dioxygen reduction was H 2 O. DCO was highly specific for 1,5-dienoyl-CoA (K m ‫؍‬ 27 M). The initial rate of DCO followed a Nernst curve with half-maximal activity at ؉10 mV. We propose that DCO provides protection for the extremely oxygensensitive BCR enzyme when the bacterium degrades aromatic compounds at the edge of steep oxygen gradients. The redox-dependent switch in DCO guarantees that DCO is only active during oxidative stress and circumvents futile dearomatization/ rearomatization reactions catalyzed by BCR and DCO.The central intermediate generated by most anaerobic bacteria capable of using aromatic compounds as growth substrates is benzoyl-CoA, which then serves as the substrate for dearomatizing benzoyl-CoA reductases (BCRs) 2 (1-5). With the exception of the enzyme from Rhodopseudomonas palustris, all BCRs appear to catalyze the two-electron reduction of benzoyl-CoA, yielding cyclohexa-1,5-diene-1-carbonyl-CoA (1,5-dienoyl-CoA; Fig. 1). The catalytic mechanism of BCR is considered to proceed in analogy to the Birch reduction in chemical synthesis and involves electron transfer steps at extremely low redox potentials (6). Two completely different classes of dearomatizing benzoyl-CoA reductases have been described. In BCR from facultative anaerobes, the electron transfer from reduced ferredoxin to benzoyl-CoA is coupled to a stoichiometric ATP-hydrolysis (1 ATP/electron transferred) (7,8). The biochemistry of ATP-dependent BCR enzymes has so far only been studied with the enzyme from the denitrifying bacterium Thauera aromatica. It has an ␣␤␥␦ subunit composition with two functionally different modules: (i) the electron activation module is composed of the ␣␦ subunits (49 and 29 kDa, respectively) and harbors two ATP-binding sites and the so-called electron entry 1ϩ/2ϩ cluster; (ii) the benzoylCoA reduction module is formed by the ␤␥ subunits (48 and 44 kDa, respe...

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Nonaromatic Products from Anoxic Conversion of Benzoyl-CoA with Benzoyl-CoA Reductase and Cyclohexa-1,5-diene-1-carbonyl-CoA Hydratase

Cited by 62 publications

References 28 publications

Identification and characterization of the tungsten-containing class of benzoyl-coenzyme A reductases

Identification and characterization of the tungsten-containing class of benzoyl-coenzyme A reductases

Mechanism of ATP-driven electron transfer catalyzed by the benzene ring-reducing enzyme benzoyl- CoA reductase

Aromatizing Cyclohexa-1,5-diene-1-carbonyl-Coenzyme A Oxidase

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