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Anaerobic activation of p-cymene in denitrifying betaproteobacteria: methyl group hydroxylation versus addition to fumarate.

ABSTRACT: The betaproteobacteria "Aromatoleum aromaticum" pCyN1 and "Thauera" sp. strain pCyN2 anaerobically degrade the plant-derived aromatic hydrocarbon p-cymene (4-isopropyltoluene) under nitrate-reducing conditions. Metabolite analysis of p-cymene-adapted "A. aromaticum" pCyN1 cells demonstrated the specific formation of 4-isopropylbenzyl alcohol and 4-isopropylbenzaldehyde, whereas with "Thauera" sp. pCyN2, exclusively 4-isopropylbenzylsuccinate and tentatively identified (4-isopropylphenyl)itaconate were observed. 4-Isopropylbenzoate in contrast was detected with both strains. Proteogenomic investigation of p-cymene- versus succinate-adapted cells of the two strains revealed distinct protein profiles agreeing with the different metabolites formed from p-cymene. "A. aromaticum" pCyN1 specifically produced (i) a putative p-cymene dehydrogenase (CmdABC) expected to hydroxylate the benzylic methyl group of p-cymene, (ii) two dehydrogenases putatively oxidizing 4-isopropylbenzyl alcohol (Iod) and 4-isopropylbenzaldehyde (Iad), and (iii) the putative 4-isopropylbenzoate-coenzyme A (CoA) ligase (Ibl). The p-cymene-specific protein profile of "Thauera" sp. pCyN2, on the other hand, encompassed proteins homologous to subunits of toluene-activating benzylsuccinate synthase (termed [4-isopropylbenzyl]succinate synthase IbsABCDEF; identified subunits, IbsAE) and protein homologs of the benzylsuccinate ?-oxidation (Bbs) pathway (termed BisABCDEFGH; all identified except for BisEF). This study reveals that two related denitrifying bacteria employ fundamentally different peripheral degradation routes for one and the same substrate, p-cymene, with the two pathways apparently converging at the level of 4-isopropylbenzoyl-CoA.

SUBMITTER: Strijkstra A

PROVIDER: S-EPMC4249252 | biostudies-literature | 2014 Dec

REPOSITORIES: biostudies-literature

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Anaerobic activation of p-cymene in denitrifying betaproteobacteria: methyl group hydroxylation versus addition to fumarate.

Strijkstra Annemieke A Trautwein Kathleen K Jarling René R Wöhlbrand Lars L Dörries Marvin M Reinhardt Richard R Drozdowska Marta M Golding Bernard T BT Wilkes Heinz H Rabus Ralf R

Applied and environmental microbiology 20140926 24

The betaproteobacteria "Aromatoleum aromaticum" pCyN1 and "Thauera" sp. strain pCyN2 anaerobically degrade the plant-derived aromatic hydrocarbon p-cymene (4-isopropyltoluene) under nitrate-reducing conditions. Metabolite analysis of p-cymene-adapted "A. aromaticum" pCyN1 cells demonstrated the specific formation of 4-isopropylbenzyl alcohol and 4-isopropylbenzaldehyde, whereas with "Thauera" sp. pCyN2, exclusively 4-isopropylbenzylsuccinate and tentatively identified (4-isopropylphenyl)itaconat ...[more]

PMID: 25261521

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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.

Dataset Information

Anaerobic activation of p-cymene in denitrifying betaproteobacteria: methyl group hydroxylation versus addition to fumarate.

Publications

Anaerobic activation of p-cymene in denitrifying betaproteobacteria: methyl group hydroxylation versus addition to fumarate.

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