benzoyl-CoA Reductase Research Articles

Selenocysteine-containing proteins in anaerobic benzoate metabolism of Desulfococcus multivorans.

The sulfate-reducing bacterium Desulfococcus multivorans uses various aromatic compounds as sources of cell carbon and energy. In this work, we studied the initial steps in the aromatic metabolism of this strictly anaerobic model organism. An ATP-dependent benzoate coenzyme A (CoA) ligase (AMP plus PPi forming) composed of a single 59-kDa subunit was purified from extracts of cells grown on benzoate. Specific activity was highest with benzoate and some benzoate derivatives, whereas aliphatic carboxylic acids were virtually unconverted. The N-terminal amino acid sequence showed high similarities with benzoate CoA ligases from Thauera aromatica and Azoarcus evansii. When cultivated on benzoate, cells strictly required selenium and molybdenum, whereas growth on nonaromatic compounds, such as cyclohexanecarboxylate or lactate, did not depend on the presence of the two trace elements. The growth rate on benzoate was half maximal with 1 nM selenite present in the growth medium. In molybdenum- and/or selenium-depleted cultures, growth on benzoate could be induced by addition of the missing trace elements. In extracts of cells grown on benzoate in the presence of [75Se]selenite, three radioactively labeled proteins with molecular masses of approximately 100, 30, and 27 kDa were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The 100- and 30-kDa selenoproteins were 5- to 10-fold induced in cells grown on benzoate compared to cells grown on lactate. These results suggest that the dearomatization process in D. multivorans is not catalyzed by the ATP-dependent Fe-S enzyme benzoyl-CoA reductase as in facultative anaerobes but rather involves unknown molybdenum- and selenocysteine-containing proteins.

Substrate binding and reduction of benzoyl-CoA reductase: evidence for nucleotide-dependent conformational changes.

Benzoyl-CoA reductase (BCR) from the denitrifying bacterium Thauera aromatica catalyzes the ATP driven two-electron reduction of the aromatic moiety of benzoyl-CoA (BCoA) to a nonaromatic cyclic diene (2 ATP/2 e(-)). The enzyme contains two similar but nonidentical ATP-binding sites of the acetate kinase/sugar kinase/Hsp70/actin family. To obtain further insights into the overall catalytic cycle of BCR, the binding affinities and stoichiometries of all substrates as well as their effects on reduction kinetics were studied by stopped-flow UV/vis spectroscopy, freeze-quench EPR spectroscopy, and equilibrium dialysis. BCR bound maximally two nucleotides and a single BCoA. The binding of a single nucleotide induced a molecular switch (BCR --> BCR) as evidenced as follows: (i) the reduction rate of BCR by sulfoxide radical anion was significantly decreased in the nucleotide-bound state, (ii) the binding of BCoA to the reduced enzyme strictly depended on bound nucleotides, and (iii) the nucleotide binding affinities increased up to 60-fold compared to the steady-state values. The "ATP-binding switch" is distinguished from the previously described "low-spin/high-spin switch" of a [4Fe-4S] cluster which strictly depends on ATP hydrolysis. The two nucleotide binding sites were occupied sequentially; binding constants of the two sites differed by a factor of 10-40. The kinetic data obtained suggest that the ATP-binding switch is a rather fast process (>100 s(-)(1)) with a switch equilibrium constant of 54 +/- 10. In contrast, the reverse switch back of the MgADP-bound enzyme (BCR --> BCR) is considered rate-limiting in the overall catalytic cycle of BCR (4 +/- 1 s(-)(1)). The binding of nucleotides did not affect the redox potential of the [4Fe-4S](+1/+2) clusters; the switch is rather considered to alter the kinetics of internal electron transfer. Implications for the overall catalytic cycle of benzoyl-CoA reductase are discussed and compared with other ATP-hydrolyzing enzymes.

Properties of 2-oxoglutarate:ferredoxin oxidoreductase from Thauera aromatica and its role in enzymatic reduction of the aromatic ring.

Benzoyl coenzyme A (benzoyl-CoA) reductase is a key enzyme in the anaerobic metabolism of aromatic compounds catalyzing the ATP-driven reductive dearomatization of benzoyl-CoA. The enzyme from Thauera aromatica uses a reduced 2[4Fe-4S] ferredoxin as electron donor. In this work, we identified 2-oxoglutarate:ferredoxin oxidoreductase (KGOR) as the ferredoxin reducing enzyme. KGOR activity was increased 10- to 50-fold in T. aromatica cells grown under denitrifying conditions on an aromatic substrate compared to that of cells grown on nonaromatic substrates. The enzyme was purified from soluble extracts by a 60-fold enrichment with a specific activity of 4.8 micromol min(-1) mg(-1). The native enzyme had a molecular mass of 200 +/- 20 kDa (mean +/- standard deviation) and consisted of two subunits with molecular masses of 66 and 34 kDa, suggesting an (alphabeta)(2) composition. The UV/visible spectrum was characteristic for an iron-sulfur protein; the enzyme contained 8.3 +/- 0.5 mol of Fe, 7.2 +/- 0.5 mol of acid-labile sulfur, and 1.6 +/- 0.2 mol of thiamine diphosphate (TPP) per mol of protein. The high specificity for 2-oxoglutarate and the low K(m) for ferredoxin ( approximately 10 microM) indicated that both are the in vivo substrates of the enzyme. KGOR catalyzed the isotope exchange between (14)CO(2) and C(1) of 2-oxoglutarate, representing a typical reversible partial reaction of 2-oxoacid oxidoreductases. The two genes coding for the two subunits of KGOR were found adjacent to the gene cluster coding for enzymes and ferredoxin of the catabolic benzoyl-CoA pathway. Sequence comparisons with other 2-oxoacid oxidoreductases indicated that KGOR from T. aromatica belongs to the Halobacterium type of 2-oxoacid oxidoreductases, which lack a ferredoxin-like module which contains two additional [4Fe-4S](1+/2+) clusters/monomer. Using purified KGOR, ferredoxin, and benzoyl-CoA reductase, the 2-oxoglutarate-driven reduction of benzoyl-CoA was shown in vitro. This demonstrates that ferredoxin acts as an electron shuttle between the citric acid cycle and benzoyl-CoA reductase by coupling the oxidation of the end product of the benzoyl-CoA pathway, acetyl-CoA, to the reduction of the aromatic ring.

A Birch-like mechanism in enzymatic benzoyl-CoA reduction: a kinetic study of substrate analogues combined with an ab initio model.

Benzoyl-CoA reductase from the anaerobic bacterium Thauera aromatica catalyzes the ATP-driven two-electron reduction of the aromatic moiety of benzoyl-CoA. A Birch mechanism involving alternate one-electron and one-proton transfer steps to the aromatic ring was previously proposed for benzoyl-CoA reductase. Due to the high redox barrier, the first electron transfer step yielding a radical anion is considered the rate-limiting step in this reaction. Focusing on the mechanism of substrate reduction, this work combines the kinetic analysis of a number of substrate analogues with a model based on the ab initio calculated electron density of the radical anion of benzoyl-CoA, a transition state model of the proposed Birch mechanism. Both K(m) and k(cat) of ortho-substituted benzoyl-CoA increased in parallel with the substituent's acceptor strength (F > Cl = H > OH > NH(2)). Among the isomers of monofluorobenzoyl-CoA, reduction rates decreased in the following order: ortho > meta > para; the K(m) values increased in the following order: meta > ortho > para. Five-ring heteroaromatic acid thiol esters were reduced in the following order: thiophene > furan > pyrrole; the 2-isomers are reduced much faster than the 3-isomers. Most of these results could be rationalized by the model. A Hammett plot indicated that the reaction mechanism is only slightly polar, suggesting the involvement of a partial protonation of the carbonyl oxygen of benzoyl-CoA and/or a simultaneous transfer of the first electron and proton. Surprisingly, benzoyl-CoA reductase exhibited a hydrogen kinetic isotope effect on k(cat) for pyridine-2-carbonyl-CoA (2.1) but only a negligible one for benzoyl-CoA (1.2), indicating that pyridine-2-carbonyl-CoA reduction proceeds according to a varied mechanism.

Mechanism of ATP-driven electron transfer catalyzed by the benzene ring-reducing enzyme benzoyl-CoA reductase.

Benzoyl-CoA reductase (BCR) from the bacterium Thauera aromatica catalyzes the two-electron reduction of benzoyl-CoA (BCoA) to a nonaromatic cyclic diene. In a process analogous to enzymatic nitrogen reduction, BCR couples the electron transfer to the aromatic ring to a stoichiometric hydrolysis of 2 ATP/2e(-). Reduced but not oxidized BCR hydrolyzes ATP to ADP. In this work, purified BCR was shown to catalyze an isotope exchange from [(14)C]ADP to [(14)C]ATP, which was approximately 15% of the ATPase activity in the presence of equimolar amounts of ADP and ATP. In accordance, BCR (alpha beta gamma delta-composition) autophosphorylated its gamma-subunit when incubated with [gamma-(32)P]ATP. Formation of the enzyme-phosphate was independent of the redox state, whereas only dithionite-reduced BCR catalyzed a dephosphorylation associated with the ATPase activity. This finding suggests that the ATPase- and autophosphatase-partial activities of BCR exhibit identical redox dependencies. BCoA or the nonphysiological electron-accepting substrate hydroxylamine stimulated the redox-dependent effects; the rates of both the overall ATPase and the autophosphatase activities of reduced BCR were increased 6-fold. In contrast, BCoA and hydroxylamine had no effect on oxidized and phosphorylated BCR. The reactivity of the phosphoamino acid fits best with a phosphoamidate or acylphosphate linkage. The results obtained suggest a mechanism of ATP hydrolysis-driven electron transfer, which differs from that of nitrogenase by the transient formation of a phosphorylated enzyme.

Single turnover EPR studies of benzoyl-CoA reductase.

Benzoyl-CoA reductase (BCR) catalyzes the ATP-driven transport of two electrons from a reduced 2[4Fe-4S] ferredoxin to the aromatic ring of benzoyl-CoA. A mechanism involving radical species and very low potential electrons similar to the Birch reduction of aromatics has been suggested for this reaction. The redox centers of BCR have previously been identified, by EPR- and Mössbauer spectroscopy, to be three cysteine-ligated [4Fe-4S] clusters [Boll et al. (2000) J. Biol. Chem. 275, 31857-31868] with redox potentials more negative than -500 mV. In this work, the catalytic cycle of BCR was studied by freeze-quench experiments; the dithionite reduced enzyme was rapidly mixed with equimolar amounts of benzoyl-CoA and excess MgATP plus dithionite, and subjected to EPR spectroscopic analysis. The turnover period of the enzyme under the conditions used was 3 s. The total S = (1)/(2) spin concentration increased 3-fold very rapidly (within approximately 25 ms). In the course of a single turnover the extent of enzyme reduction decreased again, finally reaching the starting value. An increased magnetic interaction of [4Fe-4S] clusters and the rise of an S = (7)/(2) high-spin EPR signal occurred as second simultaneous and transient events (at approximately 200 ms). Previous work showed that binding of the nucleotide affects the magnetic interaction of [4Fe-4S] clusters, whereas hydrolysis of MgATP is required for the switch to high-spin EPR signals. Finally, two novel transient EPR signals with an isotropic line-shape developed maximally in the late phase of the catalytic cycle ( approximately 1-2 s). These signals differed from those of typical free radicals by shifted g values at g = 2.015 and g = 2.033 and by an unusually fast relaxation rate, suggesting an interaction of these paramagnetic species with [4Fe-4S](+1) clusters. On the basis of these results, we present a proposal for a catalytic cycle involving radical species.

Anaerobic metabolism of 3-hydroxybenzoate by the denitrifying bacterium Thauera aromatica.

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.

EPR and Mössbauer Studies of Benzoyl-CoA Reductase

Benzoyl-CoA reductase catalyzes the two-electron transfer from a reduced ferredoxin to the aromatic ring of benzoyl-CoA; this reaction is coupled to stoichiometrical ATP hydrolysis. A very low reduction potential (less than -1 V) is required for the first electron transfer to the aromatic ring. In this work the nature of the redox centers of purified benzoyl-CoA reductase from Thauera aromatica was studied by EPR and Mössbauer spectroscopy. The results obtained indicated the presence of three [4Fe-4S] clusters. Redox titration studies revealed that the reduction potentials of all three clusters were below -500 mV. The previously reported S = 7/2 state of the enzyme during benzoyl-CoA-independent ATPase activity (Boll, M., Albracht, S. J. P., and Fuchs, G. (1997) Eur. J. Biochem. 244, 840-851) was confirmed by Mössbauer spectroscopy. Inactivation by oxygen was associated with the irreversible conversion of part of the [4Fe-4S] clusters to [3Fe-4S] clusters. Acetylene stimulated the benzoyl-CoA-independent ATPase activity and induced novel EPR signals with g(av) >2. The presence of simple cubane clusters in benzoyl-CoA reductase as the sole redox-active metal centers demonstrates novel aspects of [4Fe-4S] clusters since they adopt the role of elemental sodium or lithium which are used as electron donors in the analogous chemical Birch reduction of aromatic rings.

Nonaromatic Products from Anoxic Conversion of Benzoyl-CoA with Benzoyl-CoA Reductase and Cyclohexa-1,5-diene-1-carbonyl-CoA Hydratase

The enzymes benzoyl-CoA reductase and cyclohex-1, 5-diene-1-carbonyl-CoA hydratase catalyzing the first steps of benzoyl-CoA conversion under anoxic conditions were purified from the denitrifying bacterium, Thauera aromatica. Reaction products obtained with [ring-(13)C(6)]benzoyl-CoA and [ring-(14)C]benzoyl-CoA as substrates were analyzed by high pressure liquid chromatography and by NMR spectroscopy. The main product obtained with titanium(III) citrate or with reduced [8Fe-8S]-ferredoxin from T. aromatica as electron donors was identified as cyclohexa-1, 5-diene-1-carbonyl-CoA. The cyclic diene was converted into 6-hydroxycyclohex-1-ene-1-carbonyl-CoA by the hydratase. Assay mixtures containing reductase, hydratase, and sodium dithionite or a mixture of sulfite and titanium(III) citrate as reducing agent afforded cyclohex-2-ene-1-carbonyl-CoA and 6-hydroxycylohex-2-ene-1-carbonyl-CoA. The potential required for the first electron transfer to the model compound S-ethyl-thiobenzoate yielding a radical anion was determined by cyclic voltammetry as -1.9 V versus a standard hydrogen electrode. The energetics of enzymatic ring reduction of benzoyl-CoA are discussed.

Initial steps in the fermentation of 3-hydroxybenzoate by Sporotomaculum hydroxybenzoicum.

The anaerobic bacterium Sporotomaculum hydroxybenzoicum ferments 3-hydroxybenzoate to acetate, butyrate, and CO2. 3-Hydroxybenzoate was activated to 3-hydroxybenzoyl-CoA in a CoA-transferase reaction with acetyl-CoA or butyryl-CoA as CoA donors. 3-Hydroxybenzoyl-CoA was reductively dehydroxylated, forming benzoyl-CoA. This reaction was measured in cell-free extracts with cob(I)alamin as low-potential electron donor. No evidence was obtained that cob(I)alamin is the physiological electron donor; however, inhibitor studies indicated involvement of a strong nucleophile in the reaction. Benzoate was degraded by dense cell suspensions without a lag phase until an in situ deltaG' value <-25 kJ mol(-1) was reached. Benzoyl-CoA reductase was not detected. Enzyme activities for all reaction steps from glutaryl-CoA to butyryl-CoA, and ATP formation via acetate kinase were detected in cell-free extracts. Glutaconyl-CoA decarboxylase is likely to act as a primary sodium ion pump.

Iron sulfur clusters in benzoyl CoA reductase

The enzyme benzoyl-CoA reductase (BCR) has been studied by Mossbauer spectroscopy in fields up to 7 T and at temperature down to 4.2 K. It has been shown that the oxidized BCR contains three diamagnetic [4Fe-4S]2+ centers. Treatment of BCR with dithionite or deazaflavin reduces only one of the three centers from [4Fe-4S]2+ to [4Fe-4S]1+. The latter exhibits Mossbauer spectra at 4.2 K in applied fields characteristic for Fe2.5+Fe2.5+ (S=9/2) and Fe2+Fe2+ (S=4) pairs, both coupled antiferromagnetically to total spin S=1/2.

Two distinct pathways for anaerobic degradation of aromatic compounds in the denitrifying bacterium Thauera aromatica strain AR-1.

Denitrifying bacteria degrade many different aromatic compounds anaerobically via the well-described benzoyl-CoA pathway. We have shown recently that the denitrifiers Azoarcus anaerobius and Thauera aromatica strain AR-1 use a different pathway for anaerobic degradation of resorcinol (1,3-dihydroxybenzene) and 3,5-dihydroxybenzoate, respectively. Both substrates are converted to hydroxyhydroquinone (1,2,4-trihydroxybenzene). In the membrane fraction of T. aromatica strain AR-1 cells grown with 3,5-dihydroxybenzoate, a hydroxyhydroquinone-dehydrogenating activity of 74 nmol min(-1)(mg protein)-1 was found. This activity was significantly lower in benzoate-grown cells. Benzoate-grown cells were not induced for degradation of 3,5-dihydroxybenzoate, and cells grown with 3,5-dihydroxybenzoate degraded benzoate only at a very low rate. With a substrate mixture of benzoate plus 3,5-dihydroxybenzoate, the cells showed diauxic growth. Benzoate was degraded first, while complete degradation of 3,5-dihydroxybenzoate occurred only after a long lag phase. The 3,5-dihydroxybenzoate-oxidizing and the hydroxyhydroquinone-dehydrogenating activities were fully induced only during 3,5-dihydroxybenzoate degradation. Synthesis of benzoyl-CoA reductase appeared to be significantly lower in 3,5-dihydroxybenzoate-grown cells as shown by immunoblotting. These results confirm that T. aromatica strain AR-1 harbors, in addition to the benzoyl-CoA pathway, a second, mechanistically distinct pathway for anaerobic degradation of aromatic compounds. This pathway is inducible and subject to catabolite repression by benzoate.

6-Hydroxycyclohex-1-ene-1-carbonyl-CoA dehydrogenase and 6-oxocyclohex-1-ene-1-carbonyl-CoA hydrolase, enzymes of the benzoyl-CoA pathway of anaerobic aromatic metabolism in the denitrifying bacterium Thauera aromatica.

Benzoyl-CoA is a common intermediate in the anaerobic bacterial metabolism of many aromatic substrates. Two enzymes and ferredoxin of the central benzoyl-CoA pathway in Thauera aromatica have been purified so far. Benzoyl-CoA reductase reduces the aromatic ring with reduced ferredoxin yielding cyclohexa-1,5-diene-1-carbonyl-CoA [Boll, M. & Fuchs, G. (1995) Eur. J. Biochem. 234, 921-933]. Dienoyl-CoA hydratase subsequently adds one molecule of water and thereby produces 6-hydroxycyclohex-1-ene-1-carbonyl-CoA [Laempe, D., Eisenreich, W., Bacher, A., & Fuchs, G. (1998) Eur. J. Biochem. 255, 618-627]. 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 beta-hydroxyacyl-CoA dehydrogenase that catalyzes 6-hydroxycyclohex-1-ene-1-carbonyl-CoA + NAD(+) --> 6-oxocyclohex-1-ene-1-carbonyl-CoA + NADH + H(+). 6-Oxocyclohex-1-ene-1-carbonyl-CoA hydrolase acts on the beta-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-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.

Radical species in the catalytic pathways of enzymes from anaerobes

Radicals are reactive intermediates of growing importance in enzymatic catalysis. There are reactions in which neutral radicals participate and those where radical anions are involved. The former class is illustrated by lysine 2,3-aminomutase and also by enzymes dependent on coenzyme B12, that catalyse carbon skeleton rearrangements (e.g. glutamate mutase). A substrate-based radical for both lysine 2,3-aminomutase and glutamate mutase has been characterised by EPR spectroscopy. Representatives of the second class are 2-hydroxyglutaryl-CoA dehydratase, benzoyl-CoA reductase, DNA photolyase and chorismate synthase, all of which may generate the radical anion by one-electron reduction. 4-Hydroxybutyryl-CoA dehydratase, pyruvate formate lyase, and the coenzyme B12-dependent eliminases (ribonucleotide reductase, ethanolamine ammonia lyase and diol dehydratase) could be examples of radical anion formation by one-electron oxidation. The electron-rich ketyl-like radical anions cause the elimination of an adjacent group. The advantages of using radicals as intermediates in enzymatic transformations are their high reactivity and special properties. However, this reactivity includes rapid bimolecular combination with dioxygen and radicals are therefore primarily utilised as intermediates by anaerobic organisms.

Anaerobic metabolism of aromatic compounds via the benzoyl-CoA pathway

Aromatic compounds are important growth substrates for microorganisms. They form a large group of diverse compounds including lignin monomers, amino acids, quinones, and flavonoids. Aerobic aromatic metabolism is characterized by the extensive use of molecular oxygen which is essential for the hydroxylation and cleavage of aromatic ring structures. The anaerobic metabolism of low molecular mass soluble aromatic compounds requires, of necessity, a quite different strategy. In most known cases, aromaticity is broken by reduction and the ring is subsequently opened hydrolytically. A small number of different central aromatic intermediates can be reduced, the most common of which is benzoyl-CoA, a compound that is formed as a central intermediate in the degradation of a large number of aromatic growth substrates. This review concentrates on the anaerobic aromatic metabolism via the benzoyl-CoA pathway. The peripheral pathways that transform growth substrates to benzoyl-CoA include various types of novel reactions, for example carboxylation of phenolic compounds, reductive elimination of ring substituents like hydroxyl or amino groups, oxidation of methyl substituents, O-demethylation reactions and shortening of aliphatic side chains. The central benzoyl-CoA pathway differs in several aspects in the denitrifying, phototrophic and fermenting bacteria studied. In denitrifying and phototrophic bacteria it starts with the two-electron reduction of benzoyl-CoA to a cyclic dienoyl-CoA driven by the hydrolysis of two molecules of ATP to ADP+P i. This ring reduction is catalyzed by benzoyl-CoA reductase and requires a low-potential ferredoxin as an electron donor. In Rhodopseudomonas palustris the cyclic diene is further reduced to cyclohex-1-ene-1-carboxyl-CoA. In the denitrifying species Thauera aromatica, the cyclic diene is hydrated to give 6-hydroxycyclohex-1-ene-1-carboxyl-CoA. Subsequent β-oxidation results in the formation of a cyclic β-oxo compound, followed by hydrolytic carbon ring opening yielding 3-hydroxypimelyl-CoA in the case of T. aromatica and pimelyl-CoA in the case of R. palustris. These intermediates are further β-oxidized via glutaryl-CoA; final products are 3 acetyl-CoA and 1 CO 2. In fermenting bacteria benzoyl-CoA may possibly be reduced to the level of cyclohex-1-ene-1-carboxyl-CoA in an ATP-independent reaction. The genes coding for the enzymes of the central benzoyl-CoA pathway have been cloned and sequenced from R. palustris, T. aromatica, and Azoarcus evansii. Sequence analyses of the genes support the concept that phototrophic and denitrifying bacteria use two slightly different pathways to metabolize benzoyl-CoA. The gene sequences have in some cases been very helpful for the identification of possible catalytic mechanisms that were not obvious from initial characterizations of purified enzymes.

Anaerobic metabolism of aromatic compounds via the benzoyl-CoA pathway

Aromatic compounds are important growth substrates for microorganisms. They form a large group of diverse compounds including lignin monomers, amino acids, quinones, and flavonoids. Aerobic aromatic metabolism is characterized by the extensive use of molecular oxygen which is essential for the hydroxylation and cleavage of aromatic ring structures. The anaerobic metabolism of low molecular mass soluble aromatic compounds requires, of necessity, a quite different strategy. In most known cases, aromaticity is broken by reduction and the ring is subsequently opened hydrolytically. A small number of different central aromatic intermediates can be reduced, the most common of which is benzoyl-CoA, a compound that is formed as a central intermediate in the degradation of a large number of aromatic growth substrates. This review concentrates on the anaerobic aromatic metabolism via the benzoyl-CoA pathway. The peripheral pathways that transform growth substrates to benzoyl-CoA include various types of novel reactions, for example carboxylation of phenolic compounds, reductive elimination of ring substituents like hydroxyl or amino groups, oxidation of methyl substituents, O-demethylation reactions and shortening of aliphatic side chains. The central benzoyl-CoA pathway differs in several aspects in the denitrifying, phototrophic and fermenting bacteria studied. In denitrifying and phototrophic bacteria it starts with the two-electron reduction of benzoyl-CoA to a cyclic dienoyl-CoA driven by the hydrolysis of two molecules of ATP to ADP+Pi. This ring reduction is catalyzed by benzoyl-CoA reductase and requires a low-potential ferredoxin as an electron donor. In Rhodopseudomonas palustris the cyclic diene is further reduced to cyclohex-1-ene-1-carboxyl-CoA. In the denitrifying species Thauera aromatica, the cyclic diene is hydrated to give 6-hydroxycyclohex-1-ene-1-carboxyl-CoA. Subsequent β-oxidation results in the formation of a cyclic β-oxo compound, followed by hydrolytic carbon ring opening yielding 3-hydroxypimelyl-CoA in the case of T. aromatica and pimelyl-CoA in the case of R. palustris. These intermediates are further β-oxidized via glutaryl-CoA; final products are 3 acetyl-CoA and 1 CO2. In fermenting bacteria benzoyl-CoA may possibly be reduced to the level of cyclohex-1-ene-1-carboxyl-CoA in an ATP-independent reaction. The genes coding for the enzymes of the central benzoyl-CoA pathway have been cloned and sequenced from R. palustris, T. aromatica, and Azoarcus evansii. Sequence analyses of the genes support the concept that phototrophic and denitrifying bacteria use two slightly different pathways to metabolize benzoyl-CoA. The gene sequences have in some cases been very helpful for the identification of possible catalytic mechanisms that were not obvious from initial characterizations of purified enzymes.

Genes coding for the benzoyl-CoA pathway of anaerobic aromatic metabolism in the bacterium Thauera aromatica.

Many aromatic compounds are anaerobically oxidized to CO2 via benzoyl-CoA as the common aromatic intermediate. In Thauera aromatica, the central benzoyl-CoA pathway comprises the ATP-driven two-electron reduction of the benzene ring; this reaction uses a ferredoxin as electron donor and is catalyzed by benzoyl-CoA reductase. The first intermediate, cyclohex-1,5-diene-1-carboxyl-CoA, is subsequently hydrated by dienoyl-CoA hydratase to 6-hydroxycyclohex-1-ene-1-carboxyl-CoA. Formation of the main product produced by cell extracts, 3-hydroxypimelyl-CoA, requires at least two further steps; the oxidation of a hydroxyl group and the hydrolytic carbon ring cleavage of a CoA-activated beta-oxoacid. In addition, enoyl-CoA hydratase may come into play. A cluster of eight adjacent genes, which are transcribed in the same direction and may form an operon, was found in this bacterium. The cluster codes for proven and postulated enzymes of the benzoyl-CoA pathway. The genes for the enzymes code for ferredoxin, four subunits of benzoyl-CoA reductase, dienoyl-CoA hydratase, 6-hydroxycyclohex-1-ene-1-carboxyl-CoA dehydrogenase (NAD+), and the ring hydrolyzing enzyme. The deduced amino acid sequences of these proteins were 35-86% similar to the corresponding sequences found in Rhodopseudomonas palustris. Benzoyl-CoA reductase subunits exhibit distinct similarities with 2-hydroxyglutaryl-CoA dehydratase and its ATP-hydrolysing activase protein of Acidaminococcus fermentans as well as with open reading frames of unknown function in other bacteria. Conversion of benzoyl-CoA to 3-hydroxypimelyl-CoA can be explained by a minimal model of the benzoyl-CoA pathway assuming the four enzymes whose genes were characterized and an additional enoyl-CoA hydratase. In R. palustris the dienoyl-CoA hydratase gene is lacking suggesting the operation of a modified benzoyl-CoA pathway with cyclohex-1-ene-1-carboxyl-CoA as intermediate.

Differential induction of enzymes involved in anaerobic metabolism of aromatic compounds in the denitrifying bacterium Thauera aromatica.

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.

Anaerobic metabolism of L-phenylalanine via benzoyl-CoA in the denitrifying bacterium Thauera aromatica.

The anaerobic metabolism of phenylalanine was studied in the denitrifying bacterium Thauera aromatica, a member of the beta-subclass of the Proteobacteria. Phenylalanine was completely oxidized and served as the sole source of cell carbon. Evidence is presented that degradation proceeds via benzoyl-CoA as the central aromatic intermediate; the aromatic ring-reducing enzyme benzoyl-CoA reductase was present in cells grown on phenylalanine. Intermediates in phenylalanine oxidation to benzoyl-CoA were phenylpyruvate, phenylacetaldehyde, phenylacetate, phenylacetyl-CoA, and phenylglyoxylate. The required enzymes were detected in extracts of cells grown with phenylalanine and nitrate. Oxidation of phenylalanine to benzoyl-CoA was catalyzed by phenylalanine transaminase, phenylpyruvate decarboxylase, phenylacetaldehyde dehydrogenase (NAD+), phenylacetate-CoA ligase (AMP-forming), enzyme(s) oxidizing phenylacetyl-CoA to phenylglyoxylate with nitrate, and phenylglyoxylate:acceptor oxidoreductase. The capacity for phenylalanine oxidation to phenylacetate was induced during growth with phenylalanine. Evidence is provided that alpha-oxidation of phenylacetyl-CoA is catalyzed by a membrane-bound enzyme. This is the first report on the complete anaerobic degradation of an aromatic amino acid and the regulation of this process.

Benzoyl-CoA reductase (dearomatizing), a key enzyme of anaerobic aromatic metabolism. A study of adenosinetriphosphatase activity, ATP stoichiometry of the reaction and EPR properties of the enzyme.

An enzyme was recently described, benzoyl-CoA reductase (dearomatizing), which catalyses the ATP-driven reduction of the aromatic ring of benzoyl-CoA yielding a non-aromatic CoA thioester, ADP and phosphate [Boll, M. & Fuchs, G. (1995) Eur. J. Biochem. 234, 921-933]. The 170-kDa enzyme consists of four different subunits and contains approximately 12 Fe and acid-labile sulfur/mol. Benzoyl-CoA reductase exhibits ATPase activity in the absence of substrate. It is shown that only the reduced form of this iron-sulfur protein has ATPase activity. ATPase activity is reversibly lost when the enzyme is oxidized by thionine; reduction of the enzyme fully restores ATPase and ring-reduction activity. 2 mol ATP are hydrolyzed/2 mol electrons transferred in the course of the reaction. The product ADP acts as competitive inhibitor (Ki = 1.1 mM) for ATP in benzoyl-CoA reduction; ADP inhibits ATPase activity to the same extent as ring-reduction activity. EPR investigation of the dithionite-reduced enzyme suggested the presence of two separate [2Fe-2S] clusters and two interacting [4Fe-4S] clusters. Addition of MgATP to the reduced enzyme resulted in a new isotropic signal at g = 5.15 and a weak signal at g = 12; in controls with MgADP only a minor signal at g = 5.15 was observed. The positions, shapes and temperature dependencies of these MgATP-induced signals are indicative for excited states of a S = 7/2 spin multiplet. The [2Fe-2S] signals were not affected by ATP, but one of the [4Fe-4S] clusters became slowly oxidized. Addition of both benzoyl-CoA and MgATP resulted in a major oxidation of the iron-sulfur clusters accompanied by the appearance of some minor signals of unknown origin in the g = 2.037-1.96 region. Neither the benzoyl-CoA plus MgATP-oxidized nor the thionine-oxidized enzyme showed the ATP-dependent formation of the high-spin signals of the reduced enzyme. At present we hypothesize that the S = 7/2 signal is due to an ATP-induced change of one of the [4Fe-4S] clusters. The data suggest that hydrolysis of MgATP is required to activate the enzyme; in the absence of substrate the energy involved in this activation dissipates. MgATP-driven formation of this excited state of the reduced enzyme rather than transfer of electrons from the reduced enzyme to the aromatic substrate appears to be the rate-limiting step in the catalytic cycle. We suggest that the excited state is required to overcome the high activation energy associated with the loss of the aromatic character and/or to render ring reduction irreversible.