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Anaerobic bacterial metabolism of hydrocarbons.

It was generally believed until ten years ago that hydrocarbons released into the environment, e.g. from petroleum spills, can only be degraded by bacteria and fungi when oxygen is present. Hydrocarbons are chemically inert compounds, which are always transformed to polar intermediates by the use of molecular oxygen as reactive cosubstrate under aerobic conditions. However, starting in 1990 many bacterial hydrocarbon degrading species have been isolated, which do not require molecular oxygen for growth. These bacteria are ubiquitous in the environment and belong to the denitrifying, metal ion or sulfate reducing bacteria. The hydrocarbon substrates used under anaerobic conditions include aliphatic hydrocarbons, such as alkanes and alkenes, and aromatic hydrocarbons, e. g. benzene, toluene, ethylbenzene or xylenes. Obviously, the anaerobic degradation pathways of these compounds must proceed differently than known from aerobic organisms. Initial studies on the biochemical basis of anaerobic metabolism of a few model compounds revealed that anaerobic hydrocarbon degraders have indeed developed new strategies to attack these recalcitrant substrates. Our research focusses mainly on the enzymes involved in anaerobic catabolism of alkylbenzenes, such as toluene, ethylbenzene and xylenes.

1. Pathway of toluene and xylene metabolism.

A novel biochemical reaction was identified as initial step of anaerobic toluene and xylene metabolism, namely the addition of the methyl group to the double bond of a fumarate cosubstrate. The product formed from toluene by this reaction, (R)-benzylsuccinate, is further degraded to benzoyl-CoA and succinate via specific enzymes of a ß-oxidation pathway (Fig. 1).

Fig 1 Heider
Figure 1: Anaerobic metabolism of toluene.

The pathway is initiated by benzylsuccinate synthase (BssABC), which catalyses the addition of the methyl group of toluene to a fumarate cosubstrate. The first intermediate (R)-benzylsuccinate is then activated to the CoA-thioester by a succinyl-CoA-dependent CoA-transferase (BbsEF), and benzylsuccinyl-CoA is oxidised via beta-oxidation to benzoyl-CoA and succinyl-CoA. This involves the consecutive action of benzylsuccinyl-CoA dehydrogenase (BbsG), and enoyl-CoA hydratase (BbsH), alcohol dehydrogenase (BbsCD), and a benzoylsuccinyl-CoA thiolase (BbsB). We currently purify the enzymes of this pathway from denitrifying bacteria which have been grown on toluene and nitrate as sole sources of cell carbon and energy. The key enzyme of anaerobic toluene metabolism, (R)-benzylsuccinate synthase, has been identified as a novel glycyl radical enzyme, and a possible reaction mechanism has been postulated (Fig. 2). Our goal is to proof the catalytic mechanisms and structure/function relationships of these enzymes. Similar enzyme reactions are studied in other anaerobic bacteria (phototrophic and sulfate-reducing bacteria). Fumarate addition reactions, which occur via similar catalytic mechanisms, appear to be widespread in anoxic metabolic pathways of hydrocarbons. In addition to several methyl-substituted aromatic compounds (e. g. cresols or 2-methylnaphthalene), a similar reaction also initiates anaerobic alkane degradation. Therefore, our results provide foundations to develop more effective methods in bioremediation of many hydrocarbon-contaminated sites, as well as biotechnological applications.

Fig 2 Heider
Figure 2: Postulated reaction mechanism of benzylsuccinate synthase.

The glycyl radical of the enzyme is part of an active site, which removes a hydrogen atom from the methyl group of toluene and generates a benzyl radical intermediate. This radical can readily add to the double bond of fumarate, generating a benzylsuccinyl radical. Finally, the product radical is converted to benzylsuccinate, rendering the enzyme again in the active, radical-containing state.

2. Pathway of ethylbenzene and propylbenzene metabolism

Altough the aromatic hydrocarbons ethylbenzene and n-propylbenzene are chemically very similar to toluene, they are degraded via a completely different pathway. The initial metabolic step is catalysed by ethylbenzene dehydrogenase, a novel molybdenum/iron-sulfur/heme enzyme. It oxidises the methylene group of ethylbenzene independently of oxygen, generating (S)-1-phenylethanol as first intermediate (Fig. 3). The same enzyme is apparently also oxidising n-propylbenzene. Further metabolism of (S)-1-phenylethanol proceeds via oxidation to acetophenone, which is then carboxylated at the methyl group (Fig. 3). Further metabolic steps lead to formation of benzoyl-CoA as central intermediate of anaerobic aromatic catabolism. Our goal is to characterise the enzymes involved in the pathway to understand better the biochemical principles of these reactions.

Figure 3: Anaerobic metabolism of ethylbenzene.

The pathway is initiated by ethylbenzene dehydrogenase (EBDH), a periplasmic molybdenum-enzyme, which catalyses oxidation of the methylene group of ethylbenzene and n-propylbenzene. The first intermediate (S)-1-phenylethanol is then oxidised to acetophenone by an alcohol dehydrogenase (PEDH), and acetophenone is carboxylated to benzoylactetate by an as-yet unknown carboxylase (APC). The proposed further steps are activation to a CoA-thioester and thiolytic cleavage.

Zuletzt aktualisiert: 19.09.2008 · Patricia Wagner

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