Anaerobic metabolism of catechol by the denitrifying bacterium Thauera aromatica--a result of promiscuous enzymes and regulators?

Abstract

The anaerobic metabolism of catechol (1,2-dihydroxybenzene) was studied in the betaproteobacterium Thauera aromatica that was grown with CO2 as a cosubstrate and nitrate as an electron acceptor. Based on different lines of evidence and on our knowledge of enzymes and genes involved in the anaerobic metabolism of other aromatic substrates, the following pathway is proposed. Catechol is converted to catechylphosphate by phenylphosphate synthase, which is followed by carboxylation by phenylphosphate carboxylase at the para position to the phosphorylated phenolic hydroxyl group. The product, protocatechuate (3,4-dihydroxybenzoate), is converted to its coenzyme A (CoA) thioester by 3-hydroxybenzoate-CoA ligase. Protocatechuyl-CoA is reductively dehydroxylated to 3-hydroxybenzoyl-CoA, possibly by 4-hydroxybenzoyl-CoA reductase. 3-Hydroxybenzoyl-CoA is further metabolized by reduction of the aromatic ring catalyzed by an ATP-driven benzoyl-CoA reductase. Hence, the promiscuity of several enzymes and regulatory proteins may be sufficient to create the catechol pathway that is made up of elements of phenol, 3-hydroxybenzoate, 4-hydroxybenzoate, and benzoate metabolism.

FIG. 1.

Anaerobic metabolism of representative aromatic compounds containing one or two phenolic hydroxyl groups. (A) Phenolic compounds metabolized via benzoyl-CoA or 3-hydroxybenzoyl-CoA. (B) Phenolic compounds and m-xylene metabolized via 3-methylbenzoyl-CoA. (C) Metabolism of resorcinol. (D) Metabolism of hydroquinone. The pathway of catechol was studied here, and the proposed scheme for the initial reactions is indicated by shading. The central intermediates that undergo aromatic ring reduction are enclosed in boxes. Note that several isomeric cresols are metabolized via different routes, depending on the organism and the redox potential of the terminal electron acceptor.

FIG. 2.

Anaerobic growth of T. aromatica on catechol with NaNO₃ as the electron acceptor in a 200-liter continuous fed-batch fermentor. The ratio of the catechol concentration supplied to the nitrate concentration supplied was 1:3.6.

FIG. 3.

Simultaneous adaptation experiments: substrate consumption by dense suspensions of cells (OD₅₇₈, 7) that were grown anaerobically with nitrate on different aromatic substrates. (A) Cells grown on benzoate. (B) Cells grown on protocatechuate. (C) Cells grown on phenol. (D) Cells grown on catechol. •, benzoate; ○, 3-hydroxybenzoate; ▾, 4-hydroxybenzoate; ▿, protocatechuate; ▪, catechol; □, phenol.

FIG. 4.

SDS-PAGE (13.5%) of the soluble protein fraction (20 to 30 μg protein) of cells grown on different aromatic substrates. Lane a, benzoate-grown cells; lane b, 4-hydroxybenzoate-grown cells; lane c, 3-hydroxybenzoate-grown cells; lane d, protocatechuate-grown cells; lane e, catechol-grown cells; lane f, phenol-grown cells.

FIG. 5.

Detection of (A) benzoyl-CoA reductase and (B) 3-hydroxybenzoate-CoA ligase by Western blotting, using polyclonal antibodies raised against the purified enzymes. Extracts (20 to 30 μg protein) of cells grown on different substrates were separated by SDS-PAGE and then hybridized. Lane a, benzoate-grown cells; lane b, 4-hydroxybenzoate-grown cells; lane c, 3-hydroxybenzoate-grown cells; lane d, protocatechuate-grown cells; lane e, catechol-grown cells; lane f, phenol-grown cells; lane g, purified enzyme (2 μg) used as a positive control for (A) benzoyl-CoA reductase with four subunits (Bcr A, Bcr B, Bcr C, and Bcr D) and for (B) 3-hydroxybenzoate-CoA ligase.

FIG. 6.

Transformation of [U-¹⁴C]catechol to [¹⁴C]protocatechuate and other unknown products in 15 min by extracts of cells grown on (A) catechol and (B) phenol. The assays were conducted in the absence or in the presence of Mg-ATP. An autoradiogram for samples separated by thin-layer chromatography is shown. For details see Materials and Methods.

FIG. 7.

UV spectra of (A) 75 μM protocatechuate and 33 μM protocatechuyl-CoA in 50 mM potassium phosphate buffer (pH 6.8) and (B) 4 μM protocatechuyl-CoA at different pH values. The maximum absorption of protocatechuyl-CoA at an alkaline pH in the near-UV region is at 345 nm. The enzyme activity was measured photometrically at pH 8.5 and 365 nm using a filter photometer with a strong 365-nm light source (Hg spectral line).

FIG. 8.

SDS-PAGE (13.5%) of purified 3-hydroxybenzoate-CoA ligase (3 μg, C-terminally His₆-tagged protein). The protein band is at the expected molecular mass, 63 kDa. The purification protocol started with 30 g of E. coli BL21(DE3)/plysS/pET5C/TOPO-3OHBCL cells and yielded 36 mg of enzyme.

FIG. 9.

Metabolic reactions and clusters of genes involved in (A) the catabolism of phenol to 4-hydroxybenzoate, (B) reductive dehydroxylation of 4-hydroxybenzoyl-CoA to benzoyl-CoA, and (C) metabolism of 3-hydroxybenzoate by T. aromatica, “A. aromaticum” (Azoarcus sp. strain EbN1), G. metallireducens, and M. magneticum. In panel A, orfs 1, 2, and 3 encode proteins 1, 2, and 3, the components of phenylphosphate synthase, which catalyzes the phosphorylation of phenol to phenylphosphate. orfs 4, 5, 6, and 12 encode subunits α, δ, β, and γ of phenylphosphate carboxylase, which catalyzes the carboxylation of phenylphosphate to 4-hydroxybenzoate and releases phosphate. In panel B, HcrA, HcrB, and HcrC are three subunits of 4-hydroxybenzoyl-CoA reductase, and the open arrows indicate a regulatory protein belonging to the MarR family. In panel C, the genes for 3-hydroxybenzoate-CoA ligase (3HBCL) and other open reading frames are shown. The annotation of 3HBCL in “A. aromaticum” and M. magneticum is tentative but is supported by the similar flanking genes and by the high e value compared with the Thauera enzyme. orfs 1 and 3 encode putative alcohol dehydrogenases. orf 4 is similar to the acyl-CoA dehydrogenase gene, and the orf 6 translated product is a putative hydrolase. The function of orf 2 is not known. The gray arrows indicate the open reading frames that were not found in the T. aromatica gene cluster.