Differential induction of enzymes involved in anaerobic metabolism of aromatic substrates was studied in the denitrifying bacterium Thauera aromatica. This metabolism is divided into (1) peripheral reactions transforming the aromatic growth substrates to the common intermediate benzoyl-CoA, (2) the central benzoyl-CoA pathway comprising ring-reduction of benzoyl-CoA and subsequent beta-oxidation to 3-hydroxypimelyl-CoA, and (3) the pathway of beta-oxidation of 3-hydroxypimelyl-CoA to three acetyl-CoA and CO2. Regulation was studied by three methods. 1. Determination of protein patterns of cells grown on different substrates. This revealed several strongly substrate-induced polypeptides that were missing in cells grown on benzoate or other intermediates of the respective metabolic pathways. 2. Measurement of activities of known enzymes involved in this metabolism in cells grown on different substrates. The enzyme pattern found is consistent with the regulatory pattern deduced from simultaneous adaptation of cells to utilisation of other aromatic substrates. 3. Immunological detection of catabolic enzymes in cells grown on different substrates. Benzoate-CoA ligase and 4-hydroxybenzoate-CoA ligase were detected only in cells yielding the respective enzyme activity. However, presence of the subunits of benzoyl-CoA reductase and 4-hydroxybenzoyl-CoA reductase was also recorded in some cell batches lacking enzyme activity. This possibly indicates an additional level of regulation on protein level for these two reductases.

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. Here two new enzymes, which convert this intermediate to the noncyclic product 3-hydroxypimelyl-CoA, were purified from T. aromatica and studied. 6-Hydroxycyclohex-1-ene-1-carbonyl-CoA dehydrogenase is an NAD + -specific b-hydroxyacyl-CoA dehydrogenase that catalyzes 6-hydroxycyclohex-1-ene-1-carbonyl-CoA + NAD + 3 6-oxocyclohex-1-ene-1-carbonyl-CoA + NADH + H + . 6-Oxocyclohex-1-ene-1-carbonyl-CoA hydrolase acts on the b-oxoacyl-CoA compound and catalyzes the addition of one molecule of water to the double bound and the hydrolytic C±C cleavage of the alicyclic ring, 6-oxocyclohex-1-ene-1-carbonyl-CoA + 2 H 2 O 3 3-hydroxypimelyl-CoA. The genes for both enzymes, had and oah, were cloned, had was overexpressed in Escherichia coli and the recombinant protein was purified. Hence, presumably all enzymes of the central benzoyl-CoA pathway of anaerobic aromatic metabolism from this organism have now been purified and studied and the corresponding genes have been cloned and sequenced.

Many aromatic compounds can be metabolized by bacteria under anoxic conditions via benzoyl-CoA as the common intermediate. The central pathway of benzoyl-CoA metabolism is initiated by an ATPdriven reduction of the aromatic ring producing cyclohexa-1,5-diene-1-carboxyl-CoA. The 1,5-dienoylCoA intermediate is thought to be transformed to 6-hydroxycyclohex-1-ene-1-carboxyl-CoA by a specific dienoyl-CoA hydratase catalyzing the formal addition of water to one of the double bonds.This dienoyl-CoA hydratase was detected in the denitrifying bacterium Thauera aromatica after anaerobic growth with benzoate. Substrate and product were confirmed and a convenient spectrophotometric assay was developed. The equilibrium concentrations of substrate and product were almost equal. Enzyme activity was induced after anoxic growth with benzoate, in contrast to acetate. The enzyme of 28 kDa was purified from T. aromatica and was found to be highly specific for the cyclic 1,5-dienoyl-CoA. A second 29-kDa enoyl-CoA hydratase acted on crotonyl-CoA; this highly active enoyl-CoA hydratase also acted slowly on cyclohex-1-ene-1-carboxyl-CoA. The regulation of expression of dienoyl-CoA hydratase activity, the kinetic constants, the substrate specificity, and the specific activity of the enzyme in cell extract provide evidence that dienoyl-CoA hydratase is the second enzyme of the central benzoyl-CoA pathway of anaerobic aromatic metabolism in T. aromatica.Extracts of Rhodopseudomonas palustris contained high activity of cyclohex-1-ene-1-carboxyl-CoA hydratase, but no 1,5-dienoyl-CoA hydratase activity. It appears that a variant of the benzoyl-CoA pathway is operating in R. palustris in which hydration of the 1,5-dienoyl-CoA does not take place. Rather, cyclohex-1-ene-1-carboxyl-CoA is hydrated to 2-hydroxycyclohexane-1-carboxyl-CoA.Keywords : benzoyl-CoA reductase; enoyl-CoA hydratase; dienoyl-CoA hydratase; cyclohexa-1,5-diene-1-carboxyl-CoA hydratase.Aromatic compounds represent an important class of biomolecules that end up in anoxic zones; aromatic xenobiotic compounds are being added to these potential substrates for microbial growth. It is well established that various bacteria can grow on water-soluble, non-polymeric aromatic compounds in the complete absence of oxygen. Hence, the anaerobic metabo- Abbreviations. Dienoyl-CoA hydratase, cyclohexa-1,5-diene-1-carboxyl-CoA hydratase ; dienoyl-CoA, cyclohexa-1,5-diene-1-carboxylCoA ; enoyl-CoA hydratase, cyclohex-1-ene-1-carboxyl-CoA hydratase ; enoyl-CoA, cyclohex-1-ene-1-carboxyl-CoA. Note that enoyl-CoA hydratase is not specific for the cyclic enoyl-CoA substrate but also acts on other enoyl-CoA compounds such as crotonyl-CoA. Compound (I),

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-hydroxybenzoylCoA 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 ␤-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.Phenolic compounds comprise a large and diverse group of organic, water-soluble compounds that can serve as growth substrates for microorganisms. In recent years, it has been established that bacteria can make use of these compounds as carbon and energy source both aerobically and under anoxic conditions. Aerobic metabolism of phenolic compounds requires molecular oxygen and oxygenases for the cleavage of the aromatic ring (for a recent review, see reference 19). Anaerobic metabolism differs in several aspects; most importantly, it is by definition an oxygen-independent process. Phenolic compounds such as phenol or o-cresol are converted to the corresponding hydroxybenzoic acids 4-hydroxybenzoate and 3-methyl-4-hydroxybenzoate by para carboxylation (reviewed in references 18, 21, and 36). Hydroxybenzoic acids are also formed from other aromatic compounds by bacteria; e.g., p-cresol is oxidized to 4-hydroxybenzoate, and m-cresol is oxidized to 3-hydroxybenzoate (7, 33).It appears that there are at least four ways to metabolize hydroxybenzoic acids further under an...