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Benzoate-coenzyme A ligase from Thauera aromatica: an enzyme acting in anaerobic and aerobic pathways.

ABSTRACT: In the denitrifying member of the beta-Proteobacteria Thauera aromatica, the anaerobic metabolism of aromatic acids such as benzoate or 2-aminobenzoate is initiated by the formation of the coenzyme A (CoA) thioester, benzoyl-CoA and 2-aminobenzoyl-CoA, respectively. Both aromatic substrates were transformed to the acyl-CoA intermediate by a single CoA ligase (AMP forming) that preferentially acted on benzoate. This benzoate-CoA ligase was purified and characterized as a 57-kDa monomeric protein. Based on V(max)/K(m), the specificity constant for 2-aminobenzoate was 15 times lower than that for benzoate; this may be the reason for the slower growth on 2-aminobenzoate. The benzoate-CoA ligase gene was cloned and sequenced and was found not to be part of the gene cluster encoding the general benzoyl-CoA pathway of anaerobic aromatic metabolism. Rather, it was located in a cluster of genes coding for a novel aerobic benzoate oxidation pathway. In line with this finding, the same CoA ligase was induced during aerobic growth with benzoate. A deletion mutant not only was unable to grow anaerobically on benzoate or 2-aminobenzoate, but also aerobic growth on benzoate was affected. This suggests that benzoate induces a single benzoate-CoA ligase. The product of benzoate activation, benzoyl-CoA, then acts as inducer of separate anaerobic or aerobic pathways of benzoyl-CoA, depending on whether oxygen is lacking or present.

SUBMITTER: Schuhle K

PROVIDER: S-EPMC166471 | biostudies-literature | 2003 Aug

REPOSITORIES: biostudies-literature

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Benzoate-coenzyme A ligase from Thauera aromatica: an enzyme acting in anaerobic and aerobic pathways.

Schühle Karola K Gescher Johannes J Feil Ulrich U Paul Michael M Jahn Martina M Schägger Hermann H Fuchs Georg G

Journal of bacteriology 20030801 16

In the denitrifying member of the beta-Proteobacteria Thauera aromatica, the anaerobic metabolism of aromatic acids such as benzoate or 2-aminobenzoate is initiated by the formation of the coenzyme A (CoA) thioester, benzoyl-CoA and 2-aminobenzoyl-CoA, respectively. Both aromatic substrates were transformed to the acyl-CoA intermediate by a single CoA ligase (AMP forming) that preferentially acted on benzoate. This benzoate-CoA ligase was purified and characterized as a 57-kDa monomeric protein. ...[more]

PMID: 12897012

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Project description:The anaerobic metabolism of 3-hydroxybenzoate was studied in the denitrifying bacterium Thauera aromatica. Cells grown with this substrate were adapted to grow with benzoate but not with 4-hydroxybenzoate. Vice versa, 4-hydroxybenzoate-grown cells did not utilize 3-hydroxybenzoate. The first step in 3-hydroxybenzoate metabolism is a coenzyme A (CoA) thioester formation, which is catalyzed by an inducible 3-hydroxybenzoate-CoA ligase. The enzyme was purified and characterized. Further metabolism of 3-hydroxybenzoyl-CoA by cell extract required MgATP and was coupled to the oxidation of 2 mol of reduced viologen dyes per mol of substrate added. Purification of the 3-hydroxybenzoyl-CoA reducing enzyme revealed that this activity was due to benzoyl-CoA reductase, which reduced the 3-hydroxy analogue almost as efficiently as benzoyl-CoA. The further metabolism of the alicyclic dienoyl-CoA product containing the hydroxyl substitution obviously required additional specific enzymes. Comparison of the protein pattern of 3-hydroxybenzoate-grown cells with benzoate-grown cells revealed several 3-hydroxybenzoate-induced proteins; the N-terminal amino acid sequences of four induced proteins were determined and the corresponding genes were identified and sequenced. A cluster of six adjacent genes contained the genes for substrate-induced proteins 1 to 3; this cluster may not yet be complete. Protein 1 is a short-chain alcohol dehydrogenase. Protein 2 is a member of enoyl-CoA hydratase enzymes. Protein 3 was identified as 3-hydroxybenzoate-CoA ligase. Protein 4 is another member of the enoyl-CoA hydratases. In addition, three genes coding for enzymes of beta-oxidation were present. The anaerobic 3-hydroxybenzoate metabolism here obviously combines an enzyme (benzoyl-CoA reductase) and electron carrier (ferredoxin) of the general benzoyl-CoA pathway with enzymes specific for the 3-hydroxybenzoate pathway. This raises some questions concerning the regulation of both pathways.

Project description:Genes involved in the anaerobic metabolism of phenol in the denitrifying bacterium Thauera aromatica have been studied. The first two committed steps in this metabolism appear to be phosphorylation of phenol to phenylphosphate by an unknown phosphoryl donor ("phenylphosphate synthase") and subsequent carboxylation of phenylphosphate to 4-hydroxybenzoate under release of phosphate ("phenylphosphate carboxylase"). Both enzyme activities are strictly phenol induced. Two-dimensional gel electrophoresis allowed identification of several phenol-induced proteins. Based on N-terminal and internal amino acid sequences of such proteins, degenerate oligonucleotides were designed to identify the corresponding genes. A chromosomal DNA segment of about 14 kbp was sequenced which contained 10 genes transcribed in the same direction. These are organized in two adjacent gene clusters and include the genes coding for five identified phenol-induced proteins. Comparison with sequences in the databases revealed the following similarities: the gene products of two open reading frames (ORFs) are each similar to either the central part and N-terminal part of phosphoenolpyruvate synthases. We propose that these ORFs are components of the phenylphosphate synthase system. Three ORFs showed similarity to the ubiD gene product, 3-octaprenyl-4-hydroxybenzoate carboxy lyase; UbiD catalyzes the decarboxylation of a 4-hydroxybenzoate analogue in ubiquinone biosynthesis. Another ORF was similar to the ubiX gene product, an isoenzyme of UbiD. We propose that (some of) these four proteins are involved in the carboxylation of phenylphosphate. A 700-bp PCR product derived from one of these ORFs cross-hybridized with DNA from different Thauera and Azoarcus strains, even from those which have not been reported to grow with phenol. One ORF showed similarity to the mutT gene product, and three ORFs showed no strong similarities to sequences in the databases. Upstream of the first gene cluster, an ORF which is transcribed in the opposite direction codes for a protein highly similar to the DmpR regulatory protein of Pseudomonas putida. DmpR controls transcription of the genes of aerobic phenol metabolism, suggesting a similar regulation of anaerobic phenol metabolism by the putative regulator.

Project description:The anaerobic metabolism of phenol in the beta-proteobacterium Thauera aromatica proceeds via carboxylation to 4-hydroxybenzoate and is initiated by the ATP-dependent conversion of phenol to phenylphosphate. The subsequent para carboxylation of phenylphosphate to 4-hydroxybenzoate is catalyzed by phenylphosphate carboxylase, which was purified and studied. This enzyme consists of four proteins with molecular masses of 54, 53, 18, and 10 kDa, whose genes are located adjacent to each other in the phenol gene cluster which codes for phenol-induced proteins. Three of the subunits (54, 53, and 10 kDa) were sufficient to catalyze the exchange of 14CO2 and the carboxyl group of 4-hydroxybenzoate but not phenylphosphate carboxylation. Phenylphosphate carboxylation was restored when the 18-kDa subunit was added. The following reaction model is proposed. The 14CO2 exchange reaction catalyzed by the three subunits of the core enzyme requires the fully reversible release of CO2 from 4-hydroxybenzoate with formation of a tightly enzyme-bound phenolate intermediate. Carboxylation of phenylphosphate requires in addition the 18-kDa subunit, which is thought to form the same enzyme-bound energized phenolate intermediate from phenylphosphate with virtually irreversible release of phosphate. The 54- and 53-kDa subunits show similarity to UbiD of Escherichia coli, which catalyzes the decarboxylation of a 4-hydroxybenzoate derivative in ubiquinone (ubi) biosynthesis. They also show similarity to components of various decarboxylases acting on aromatic carboxylic acids, such as 4-hydroxybenzoate or vanillate, whereas the 10-kDa subunit is unique. The 18-kDa subunit belongs to a hydratase/phosphatase protein family. Phenylphosphate carboxylase is a member of a new family of carboxylases/decarboxylases that act on phenolic compounds, use CO2 as a substrate, do not contain biotin or thiamine diphosphate, require K+ and a divalent metal cation (Mg2+or Mn2+) for activity, and are strongly inhibited by oxygen.

Project description:A gene, badH, whose predicted product is a member of the short-chain dehydrogenase/reductase family of enzymes, was recently discovered during studies of anaerobic benzoate degradation by the photoheterotrophic bacterium Rhodopseudomonas palustris. Purified histidine-tagged BadH protein catalyzed the oxidation of 2-hydroxycyclohexanecarboxyl coenzyme A (2-hydroxychc-CoA) to 2-ketocyclohexanecarboxyl-CoA. These compounds are proposed intermediates of a series of three reactions that are shared by the pathways of cyclohexanecarboxylate and benzoate degradation used by R. palustris. The 2-hydroxychc-CoA dehydrogenase activity encoded by badH was dependent on the presence of NAD(+); no activity was detected with NADP(+) as a cofactor. The dehydrogenase activity was not sensitive to oxygen. The enzyme has apparent K(m) values of 10 and 200 microM for 2-hydroxychc-CoA and NAD(+), respectively. Western blot analysis with antisera raised against purified His-BadH identified a 27-kDa protein that was present in benzoate- and cyclohexanecarboxylate-grown but not in succinate-grown R. palustris cell extracts. The active form of the enzyme is a homotetramer. badH was determined to be the first gene in an operon, termed the cyclohexanecarboxylate degradation operon, containing genes required for both benzoate and cyclohexanecarboxylate degradation. A nonpolar R. palustris badH mutant was unable to grow on benzoate or cyclohexanecarboxylate but had wild-type growth rates on succinate. Cells blocked in expression of the entire cyclohexanecarboxylate degradation operon excreted cyclohex-1-ene-1-carboxylate into the growth medium when given benzoate. This confirms that cyclohex-1-ene-1-carboxyl-CoA is an intermediate of anaerobic benzoate degradation by R. palustris. This compound had previously been shown not to be formed by Thauera aromatica, a denitrifying bacterium that degrades benzoate by a pathway that is slightly different from the R. palustris pathway. 2-Hydroxychc-CoA dehydrogenase does not participate in anaerobic benzoate degradation by T. aromatica and thus may serve as a useful indicator of an R. palustris-type benzoate degradation pathway.

Dataset Information

Benzoate-coenzyme A ligase from Thauera aromatica: an enzyme acting in anaerobic and aerobic pathways.

Publications

Benzoate-coenzyme A ligase from Thauera aromatica: an enzyme acting in anaerobic and aerobic pathways.

Similar Datasets

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