Differential induction of enzymes involved in anaerobic metabolism of aromatic compounds in the denitrifying bacterium Thauera aromatica

Heider, Johann; Boll, Matthias; Breese, Klaus; Breinig, Sabine; Ebenau-Jehle, Christa; Feil, Ulrich; Gad’on, Nasser; Laempe, Diana; Leuthner, Birgitta; Mohamed, Magdy; Schneider, Sabine; Burchhardt, Gerhard; Fuchs, Georg

doi:10.1007/s002030050623

Cited by 81 publications

(77 citation statements)

References 29 publications

(45 reference statements)

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“…In extracts of cells grown on acetate, this labeled band was only Ϸ5% of the one observed in benzoategrown cells. This finding fits perfectly with the differential expression of BCR when grown on different substrates (23).…”

Section: Resultssupporting

confidence: 86%

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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Section: Resultssupporting

confidence: 86%

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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“…For immunoblot analysis of benzoyl-CoA reductase (Heider et al 1998), cell-free extracts were separated on SDS gels and subsequently transferred on nitrocellulose filters (Schleicher & Schüll, Dassel, Germany) with a Multiphor system (Pharmacia, Freiburg, Germany). Benzoyl-CoA reductase was detected immunologically by luminescence using the ECL system (Amersham).…”

Section: Sds-page and Immunoblottingmentioning

confidence: 99%

Two distinct pathways for anaerobic degradation of aromatic compounds in the denitrifying bacterium Thauera aromatica strain AR-1

Philipp

Schink

1999

Arch Microbiol

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Denitrifying bacteria degrade many different aromatic compounds anaerobically via the well-described benzoyl-CoA pathway. We have shown recently that the denitrifiers Azoarcus anaerobius and Thauera aromatica strain AR-1 use a different pathway for anaerobic degradation of resorcinol (1,3-dihydroxybenzene) and 3,5-dihydroxybenzoate, respectively. Both substrates are converted to hydroxyhydroquinone (1,2,4-trihydroxybenzene).In the membrane fraction of T. aromatica strain AR-1 cells grown with 3,5-dihydroxybenzoate, a hydroxyhydroquinone-dehydrogenating activity of 74 nmol min -1 (mg protein) -1 was found. This activity was significantly lower in benzoate-grown cells. Benzoate-grown cells were not induced for degradation of 3,5-dihydroxybenzoate, and cells grown with 3,5-dihydroxybenzoate degraded benzoate only at a very low rate. With a substrate mixture of benzoate plus 3,5-dihydroxybenzoate, the cells showed diauxic growth. Benzoate was degraded first, while complete degradation of 3,5-dihydroxybenzoate occurred only after a long lag phase. The 3,5-dihydroxybenzoate-oxidizing and the hydroxyhydroquinone-dehydrogenating activities were fully induced only during 3,5-dihydroxybenzoate degradation. Synthesis of benzoyl-CoA reductase appeared to be significantly lower in 3,5-dihydroxybenzoate-grown cells as shown by immunoblotting. These results confirm that T. aromatica strain AR-1 harbors, in addition to the benzoyl-CoA pathway, a second, mechanistically distinct pathway for anaerobic degradation of aromatic compounds. This pathway is inducible and subject to catabolite repression by benzoate.

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“…aromatic compounds is well documented (Collier et al, 1996;Ohtsubo et al, 2006;Prieto et al, 2004;Rojo & Dinamarca, 2004), only a couple of examples of this phenomenon have been reported for the anaerobic degradation of aromatic compounds in T. aromatica (Heider et al, 1998) and Azoarcus sp. CIB (Ló pez Barragán et al, 2004), and there are no studies on the molecular mechanisms that control such catabolite repression.…”

Section: Introductionmentioning

confidence: 99%

New insights into the BzdR-mediated transcriptional regulation of the anaerobic catabolism of benzoate in Azoarcus sp. CIB

et al. 2008

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Differential induction of enzymes involved in anaerobic metabolism of aromatic compounds in the denitrifying bacterium Thauera aromatica

Cited by 81 publications

References 29 publications

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

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

Two distinct pathways for anaerobic degradation of aromatic compounds in the denitrifying bacterium Thauera aromatica strain AR-1

New insights into the BzdR-mediated transcriptional regulation of the anaerobic catabolism of benzoate in Azoarcus sp. CIB

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