Aromatic compounds are both common growth substrates for microorganisms and prominent environmental pollutants. The crucial step in their degradation is overcoming the resonance energy that stabilizes the ring structure. The classical strategy for degradation comprises an attack by oxygenases that hydroxylate and finally cleave the ring with the help of activated molecular oxygen. Here, we describe three alternative strategies used by microorganisms to degrade aromatic compounds. All three of these methods involve the use of CoA thioesters and ring cleavage by hydrolysis. However, these strategies are based on different ring activation mechanisms that consist of either formation of a non-aromatic ring-epoxide under oxic conditions, or reduction of the aromatic ring under anoxic conditions using one of two completely different systems.

Anoxic metabolism of many aromatic compounds proceeds via the common intermediate benzoylCoA. Benzoyl-CoA is dearomatized by benzoyl-CoA reductase (dearomatizing) in a two-elcctron reduction step, possibly yielding cyclohex-1 ,S-diene-l-carboxyl-CoA. This process has to overcome a high activation energy and is considered a biological Birch reduction. The central, aromatic-ring-reducing enzyme was investigated for the first time in the denitrifying bacterium YhuuercL a~~rnaticu strain K172. A spectrophotometric assay was developed which was strictly dependent on MgATP. both with cell extract and with purified enzyme. The oxygen-sensitive new enzyme was purified 35-fold with 20% yicld under anaerobic conditions in the presence of 0.25 mM dithionite. It had ii native molecular mass of approximately 170 kDa and consisted of four subunits a,b,c,d of 48, 45, 38 and 32 kDa. The oligomer composition of the protein most likely is abcd. The ultraviolet/visible spectrum of the protein as isolated, but without dithionite, was characteristic for an iron-sulfur protein with an absorption maxiinuni at 279 nm and a broad shoulder at 390 nm. The estimated molar absorption coefficient at 390 nm was 35000 M-l cm-'. Reduction of the cnzyrne by dithionite resulted in a decrease of absorbance at 390 nm, and the colour turned from greenish-brown to red-brown. The cnzyrne contained 10.8 2 1.5 mol Fe and 10.5 1 1.5 mol acid-labile sulfur/mol. Besides zinc (0.5 mol/mol protein) no other metals nor selenium could be detected in significant amounts. The enzyme preparation contained a flavin or tlavin-like conipound; the estimated content was 0.3 mol/mol enzymc. The enzyme reaction required MgATP and a strong reductant such as Ti(II1). The reaction catalyzed i s : benzoyl-CoA f 2 Ti(lI1) f n ATP -nonaromatic acyl-CoA + 2 Ti(TV) + n ADP + n P,. The estimated number n of ATP molecules hydrolyzcdl two electrons transferred in benzoyKoA reduction is 2-4. In the absence of benzoyl-CoA the enzyme exhibited oxygen-sensitive ATPase activity. The enzyme was specific for Mg' ' -ATP, other nucleoside triphosphates being inactive (< 1 %). Mg" could be substituted to some cxtent by MnZ+, Fez+ and less efficiently by Co' ' . Benzoate was not reduced, whereas some fluoro, hydroxy, amino and methyl analogues of the activated benzoic acid were reduced, albeit at much lower rate; the products remain to be identified. The specific activity with reduced methyl viologen as the electron donor was 0.55 pmol min-' mg-' corresponding to a catalytic number of 1.6 s I. The apparent K , values under the assay conditions (0.5 mM for both reduced and oxidized methyl viologen) of benzoyl-CoA and ATP were IS pM and 0.6 mM, respectively. The enzyme was inactivated by ethylene, hipyridyl and, in higher concentrations, by acetylene. Benzoyl-CoA reductase also catalyzed the ATP-dependent two-electron reduction of hydroxylamine ( K , 0.15 mM) and azide. Some of the properties of the enzyme are reminiscent of those of nitrogenase which similarly overcomes the high activation en...

SummaryThe degradation of aromatic compounds follows different biochemical principles in aerobic and anaerobic microorganisms. While aerobes dearomatize and cleave the aromatic ring by oxygenases, facultative anaerobes utilize an ATP-dependent ring reductase for the dearomatization of the activated key intermediate benzoyl-coenzyme A (CoA). In this work, the aromatic metabolism was studied in the obligately anaerobic model organism Geobacter metallireducens . The gene coding for a putative carboxylic acid-CoA ligase was heterologously overexpressed and the gene product was characterized as a highly specific benzoate-CoA ligase catalysing the initial step of benzoate metabolism. However, no evidence for the presence of an ATP-dependent benzoyl-CoA reductase as observed in facultative anaerobes was obtained. In a proteomic approach benzoate-induced proteins were identified; the corresponding genes are organized in two clusters comprising 44 genes. Induction of representative genes during growth on benzoate was confirmed by reverse transcription polymerase chain reaction. The results obtained suggest that benzoate is activated to benzoyl-CoA, which is then reductively dearomatized to cyclohexa-1,5-diene-1-carbonyl-CoA, followed by β β β β -oxidation reactions to acetyl-CoA units, as in facultatively anaerobic bacteria. However, in G. metallireducens the process of reductive benzene ring dearomatization appears to be catalysed by a set of completely different protein components comprising putative molybdenum and selenocysteine containing enzymes.