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Shedding light on anaerobic benzene ring degradation: a process unique to prokaryotes? C S Harwood and J Gibson J. Bacteriol. 1997, 179(2):301. These include: CONTENT ALERTS Receive: RSS Feeds, eTOCs, free email alerts (when new articles cite this article), more» Information about commercial reprint orders: http://journals.asm.org/site/misc/reprints.xhtml To subscribe to to another ASM Journal go to: http://journals.asm.org/site/subscriptions/ Downloaded from http://jb.asm.org/ on February 21, 2013 by PENN STATE UNIV Updated information and services can be found at: http://jb.asm.org/content/179/2/301.citation JOURNAL OF BACTERIOLOGY, Jan. 1997, p. 301–309 0021-9193/97/$04.0010 Copyright q 1997, American Society for Microbiology Vol. 179, No. 2 MINIREVIEW Shedding Light on Anaerobic Benzene Ring Degradation: a Process Unique to Prokaryotes? CAROLINE S. HARWOOD1* AND JANE GIBSON2 biochemical process that is radically different from the oxygenrequiring strategies used by microbes to catalyze aromatic compound degradation under aerobic conditions (35). The anaerobic degradation of aromatic compounds is a major unsolved problem in microbial physiology. Here we present a status report on one aspect of this problem, focusing on enzymatic and molecular studies of benzoate and 4-hydroxybenzoate (4-OHBen) degradation. INTRODUCTION Studies of anaerobic benzene ring degradation date back 50 years, but research on this topic proceeded only sporadically until about 10 years ago. At that time, concerns about toxic industrial wastes converged with a realization that many polluted environments are anaerobic to stimulate a renewed and sustained interest in the anaerobic metabolism of aromatic compounds. It is now clear that a wide variety of aromatic compounds can be completely degraded by bacteria in the complete absence of oxygen (14, 21, 24, 31, 32, 55). Increasing numbers of bacterial strains, representing most known modes of anaerobic energy metabolism, including phototrophy, denitrification, and sulfate or iron reduction, as well as fermentation, are now being isolated and studied in pure culture for their abilities to degrade a range of aromatic hydrocarbons, phenols, and halogenated aromatics. The global recycling of naturally occurring aromatic compounds derived from lignin, an aromatic polymer that makes up 25% or more of the woody tissues of plants, is also catalyzed by anaerobic bacteria. As far as is known, the anaerobic degradation of aromatic compounds is a talent restricted to bacteria. It must be remembered, however, that the metabolic capabilities of anaerobic fungi have only recently begun to attract the interest they deserve and are little known. Anaerobic aromatic ring degradation has not yet been reported in members of the archaea. A major area of interest has been reactions that modify benzene ring substituents, particularly dehalogenation (49, 58) and methyl group modification of, for example, toluene and cresols (7, 13, 14) as these effectively convert toxic compounds to nontoxic derivatives. Other modifications including removal of hydroxyl, methoxyl, amino, and acyl groups are also being studied (24, 55, 57) (Fig. 1). Although in most cases, the enzymes involved in these conversions have been examined only superficially, it is evident that many catalyze reactions unique to anaerobic aromatic degradation (24). Studies with members of the range of different bacteria involved have shown that preliminary modifications of substituted aromatic rings lead to the production of benzoate, usually as its coenzyme A (CoA) thioester, and often via 4-hydroxybenzoyl CoA (4-OHbenzoylCoA) (Fig. 1). Benzoyl-CoA is also a hypothesized intermediate in the degradation of ethylbenzenes and xylene (53). The benzoate pathway is then the principal route of aromatic ring cleavage leading to the complete oxidation of diverse aromatic substrates. Anaerobic attack on the extremely stable structure of the benzene ring proceeds by a ring reduction mechanism, a BENZENE RING REDUCTION AND CLEAVAGE The benzoate pathway. Most studies of anaerobic benzoate degradation have been carried out with the phototrophic bacterium Rhodopseudomonas palustris or with two denitrifying species, Thauera aromatica and Azoarcus evansii (formerly Pseudomonas sp. strains K 172 and KB 740) (3). R. palustris, a purple nonsulfur phototroph, generates its ATP from light via cyclic photophosphorylation when it is growing anaerobically. Under these conditions, benzoate and other aromatic acids, as well as a suite of alicylic and fatty acids, serve as carbon sources (36). These compounds are incorporated into cell material with close to 100% efficiency after their degradation into acetyl-CoA or related metabolic intermediates. Denitrifiers, by contrast, utilize aromatic compounds as a source of both carbon and energy and generate ATP from benzoate by employing oxidative phosphorylation with nitrate as the terminal electron acceptor. Despite their differing mechanisms of energy generation, those aspects of aromatic acid degradation that have been studied in phototrophic and denitrifying species show many points in common, suggesting that the strategies and pathways employed in mobilizing the aromatic nucleus share fundamental similarities. A pathway for benzoate degradation involving reduction of the aromatic ring as the initial resonance-relieving step was proposed in 1969 by Dutton and Evans (17) and independently by Guyer and Hegeman (34), based on work with R. palustris. The experimental approaches used included isotope dilution studies, in which suspected intermediates of the pathway were added exogenously to cell suspensions of R. palustris and then tested for their ability to trap radioactive material generated from 14C-labeled benzoate (17). In parallel work, R. palustris mutants blocked in anaerobic growth on benzoate were isolated and tested for their abilities to degrade suspected pathway intermediates (34). Recent reevaluation of the benzoate pathway has confirmed that many of the originally proposed reactions are catalyzed by benzoate-grown cells of R. palustris (52) and T. aromatica (9). However, the proposed intermediates occur in the form of CoA thioesters rather than as free acids. Also, the ring reduction step yields a product that is less reduced than that origi- * Corresponding author. Mailing address: Department of Microbiology, University of Iowa, Iowa City, IA 52242. Phone: (319) 335-7783. Fax: (319) 335-7679. E-mail: [email protected]. 301 Downloaded from http://jb.asm.org/ on February 21, 2013 by PENN STATE UNIV Department of Microbiology and Center for Biocatalysis and Bioprocessing, The University of Iowa, Iowa City, Iowa 52242,1 and Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 148532 302 MINIREVIEW nally proposed, and this appears to be followed by an additional, previously undescribed, two-electron reduction. A pathway incorporating current experimental data bearing on benzoate degradation, which combines features of the original scheme of Dutton and Evans (17) as well as some reactions recently proposed by Koch and coworkers (44), is shown in Fig. 2. As currently understood, the benzoate pathway can be divided into several phases: (i) CoA thioester formation; (ii) ring reduction; (iii) introduction of a carbonyl group; (iv) ring opening; and (v) a b-oxidation sequence leading to the conversion of the remainder of the molecule to acetyl-CoA, a mainstream metabolic product (Fig. 2 and 3). It is important to point out that very few of the enzymatic activities demonstrated in benzoate-grown cells have actually been shown to be essential for benzoate degradation, and so it is possible that some of the currently hypothesized steps represent side reactions or reactions of minor importance that are not part of the mainstream metabolite flow from benzoate. The sequence of reactions that occurs immediately following ring reduction is still uncertain. Nuclear magnetic resonance and mass spectrometric methods have identified three isomers of cyclohexadienecarboxyl-CoA, any or all of which could be the initial two-electron reduction product formed from benzoyl-CoA. Gibson and Gibson (33) identified cyclohex-1,4- and 2,5-dienecarboxylates in alkali-treated extracts of R. palustris growing exponentially on benzoate, while Koch et al. (44) detected the formation of cyclohex-1,5-diene-1-carboxyl-CoA from [13C]benzoyl-CoA in cell extracts of T. aromatica and R. palustris. 6-Hydroxycyclohex-1-ene-1-carboxyl-CoA was also detected as a metabolite generated from [13C]benzoyl-CoA in these experiments and could be a pathway intermediate formed by hydration of cyclohex-1,5-diene-1-carboxyl-CoA. As an extension of this, it has been suggested that the reactions leading to ring cleavage may involve substrates with substituents in both positions 2 and 6 (44), rather than in position 2 only, as originally proposed. Corroborating evidence for or against this has been difficult to obtain because the proposed disubstituted intermediates are not easily synthesized and so are not available for use in physiological studies. Continued application of combined biochemical and genetic approaches, such as those described below, will be required before a definitive description of the benzoate pathway can be presented. Enzymes. CoA plays an important general role in acyl group transfer in metabolism. The CoA moiety is likely to be important for acyl group recognition and binding by both biosynthetic and degradative enzymes involved in diverse metabolic processes. In the case of anaerobic benzoate degradation, the CoA modification probably also plays a critical role in providing a point of entry for electrons into the benzene ring, so that the very high energy barrier (30 kcal/mol) to ring reduction can be overcome (11). The best-studied benzoate degradation enzyme is benzoateCoA ligase, which catalyzes the first step of the pathway. This enzyme is stable in air and easily assayed (30). Benzoate-CoA ligases have been purified from R. palustris (26) and A. evansii (1) grown anaerobically on benzoate. Both enzymes have a high affinity for benzoate with Kms of about 1 and 10 mM for the R. palustris and the A. evansii proteins, respectively. The two enzymes are also quite specific for benzoate and have significant activity at low concentrations with only a few additional substrates, namely, fluorobenzoates, and in the case of the R. palustris enzyme, cyclohex-2,5- and cyclohex-1,4-dienecarboxylates (28). The R. palustris enzyme is a monomer (56 kDa), and the A. evansii enzyme is a homodimer (120 kDa). The physicochemical properties of the benzoate-CoA ligases resemble those of other enzymes belonging to the general group of carboxylate:CoAH ligases (AMP forming) (62). This includes enzymes that function in fatty acid degradation in mammalian systems and bacteria, as well as aromatic carboxylate-CoA ligases that are involved in pathways for the biosynthesis of flavonoids and lignins in higher plants. The gene encoding the R. palustris benzoate-CoA ligase has been cloned and sequenced. Its deduced amino acid sequence has between 25 and 30% identity to those of ligases from sources as varied as Escherichia coli, methanogens, and parsley (20). A unique feature of the benzoate pathway is aromatic ring reduction. This is a difficult step to catalyze because of the high resonance energy that stabilizes the aromatic ring. This suggests that benzoyl-CoA reductase will have unusual characteristics, and this has, in fact, been found to be the case. This FIG. 2. Current version of the anaerobic benzoate degradation pathway. The sequence of reactions includes those originally proposed (17, 34) and also several reactions proposed by Koch et al. (44), based on the identification of cyclohex-1,5-diene-1-carboxyl-CoA and 6-hydroxycyclohex-1-ene-1-carboxyl-CoA as products of benzoyl-CoA reduction in cell extracts of R. palustris and T. aromatica. Metabolites from experiments of Dutton and Evans with whole cells of R. palustris (17) (‡) and compounds detected as products in cell-free reactions (44) (†) or detected in soluble intracellular contents of R. palustris cells growing exponentially on benzoate (33) (p) are indicated. Solid arrows indicate enzymatic activities that have been detected in benzoate-grown R. palustris or T. aromatica cells (9, 26, 37, 52). Dashed arrows indicate hypothetical enzymatic reactions. Downloaded from http://jb.asm.org/ on February 21, 2013 by PENN STATE UNIV FIG. 1. Schematic view of the degradation of representative aromatic compounds to form 4-OHbenzoyl-CoA or benzoyl-CoA as central intermediates. J. BACTERIOL. 303 MINIREVIEW VOL. 179, 1997 Downloaded from http://jb.asm.org/ on February 21, 2013 by PENN STATE UNIV 304 MINIREVIEW FIG. 3. Proposed steps in the anaerobic conversion of pimelyl-CoA, the hypothesized ring cleavage intermediate of benzoate degradation, to three molecules of acetyl-CoA and one molecule of carbon dioxide. Modified from Gallus and Schink (25). graded by cells when energetic circumstances become more favorable. The degradation of the seven-carbon aliphatic compound, either pimelyl-CoA or 3-hydroxypimelyl-CoA, that is the ring cleavage product, has so far received only cursory attention but really should be considered as a part of the benzoate degradation pathway. A proposed pimelyl-CoA degradation sequence is shown in Fig. 3 (8, 25). Although most of the enzymes of this sequence do not appear to have been directly assayed in the phototrophs or denitrifiers, glutaryl-CoA dehy- Downloaded from http://jb.asm.org/ on February 21, 2013 by PENN STATE UNIV enzyme has recently been purified from T. aromatica by Boll and Fuchs (9), who have determined that the reaction catalyzed is a two-electron reduction of benzoyl-CoA that appears to be energized by the hydrolysis of two ATP molecules. This is of interest from a mechanistic point of view because benzoylCoA reductase is the only enzyme other than nitrogenase that catalyzes a reduction reaction accompanied by a stoichiometric consumption of ATP. As in the case of nitrogenase, ATP is probably required to generate a biological electron donor of sufficiently low potential to accomplish the reduction reaction (9, 11). T. aromatica benzoyl-CoA reductase has a native molecular mass of 170 kDa and is composed of four subunits of 48, 45, 38, and 32 kDa. It contains 12 Fe and 12 acid-labile sulfur atoms, probably arranged as two 2Fe-2S and two 4Fe-4S centers. The enzyme also has the spectral properties of a flavoprotein, although the identity of the prosthetic group is not known. It is rapidly inactivated by exposure to oxygen. Major questions that are as yet unanswered include the identity of the nonaromatic product(s) formed and the identity of the natural electron donor. It is probable that the product(s) of the benzoyl-CoA reductase is one or more of the cyclohexadienecarboxyl-CoA molecules shown in Fig. 2, but the identity of the isomer(s) formed has not been reported. Although the natural electron donor has not been identified, ferredoxins are strong candidates because of their known role as donors of low potential electrons. T. aromatica contains large quantities of ferredoxin-like compounds (9), as does R. palustris. Enzymatic activities required for conversion of the partially reduced compound cyclohex-1-ene-1-carboxyl-1-CoA to the postulated ring cleavage product pimelyl-CoA have been detected in extracts of benzoate-grown cells of R. palustris (52) (Fig. 2). The cyclohex-1-ene-1-carboxyl-CoA hydratase and 2-hydroxycyclohexanecarboxyl-CoA dehydrogenase reactions appear, at least superficially, to correspond mechanistically to those which occur as part of the b-oxidation sequence for metabolism of long-chain fatty acids in E. coli and mammals (50). However, the ring-opening step in aromatic degradation by R. palustris is hydrolytic and therefore distinct from the thiolytic cleavage that leads to acetyl-CoA formation in the classical b-oxidation pathway. An enzyme catalyzing this reaction, 2-ketocyclohexanecarboxyl-CoA hydrolase, has been purified from R. palustris (51). The enzyme, like the two preceding it in the reaction sequence shown in Fig. 2, is stable in air. The hydrolase is a homotetramer of 35-kDa subunits and does not appear to have bound cofactors or metal ions. Although it has been found to cleave only 2-ketocyclohexanecarboxylCoA, a range of related potential substrates remain to be tested. The fully reduced alicyclic acid, cyclohexanecarboxylate, was at one time proposed to be an intermediate of the anaerobic benzoate pathway (17, 34). However, the current view is that this compound feeds into the benzoate pathway from the side at the level of cyclohex-1-ene-1-carboxyl-CoA (see Fig. 4). A CoA ligase that reacts with cyclohexanecarboxylate has been partially purified (45), and a dehydrogenase that has properties similar to other acyl CoA dehydrogenases and that can catalyze the conversion of cyclohexanecarboxyl-CoA to cyclohex-1-ene1-carboxyl-CoA is under study (27). A constitutively expressed thioesterase able to hydrolyze the CoA moiety from cyclohexanecarboxyl-CoA has also been purified, and Küver et al. (45) have suggested that transient production of cyclohexanecarboxylate may be one mechanism used by cells to dispose of reducing equivalents that are generated during the later stages of aromatic compound degradation (see Fig. 3 and 4). The fully reduced compound could then be reassimilated and de- J. BACTERIOL. VOL. 179, 1997 drogenase, which catalyzes a key step in the sequence, has been purified from T. aromatica and A. evansii (37). The purified enzyme, which is a homotetramer, is bifunctional and has a flavin adenine dinucleotide (FAD)-dependent glutaconyl-CoA decarboxylase activity as well as a glutaryl-CoA dehydrogenase activity. Crude extracts of R. palustris also have glutaryl-CoA dehydrogenase and glutaconyl-CoA decarboxylase activities that are three- to sixfold higher in benzoate-grown cells than in acetate-grown cells (37). METABOLISM OF 4-HYDROXYBENZOATE As is the case for the anaerobic degradation of all aromatic and alicyclic carboxylic acids, metabolism of 4-OHBen is initiated by thioesterification of the carboxylate group with CoA (Fig. 4). 4-Hydroxybenzoate-CoA (4-OHBen-CoA) ligases have been purified from R. palustris and T. aromatica, and the gene (hbaA) encoding the R. palustris enzyme has been cloned and sequenced (6, 28). The two enzymes differ from each other in size (the T. aromatica enzyme is a 48-kDa monomer, and the R. palustris enzyme is a 117-kDa homodimer) and substrate specificity. The T. aromatica enzyme is quite specific for 4-OHBen, whereas the R. palustris enzyme is equally active with 4-OHBen and benzoate and has good activity with a number of other aromatic substrates and cyclohexadienecarboxylates (28). Neither of the 4-OHBen-CoA ligases has as high an affinity for its primary substrate as the purified benzoate-CoA 305 ligases; the Km of the T. aromatica enzyme for 4-OHBen is 37 mM, and that of the R. palustris enzyme for 4-OHBen is 120 mM. Nucleotide sequence comparisons show that the 4-OHBen-CoA and benzoate-CoA ligases from R. palustris share 50% amino acid identity. 4-OHBen-CoA ligase mutants are unable to grow on 4-OHBen, indicating that 4-OHBen-CoA formation is an essential step in anaerobic 4-OHBen degradation and that R. palustris cells synthesize just one enzyme that can activate this compound (28). 4-OHbenzoyl-CoA is converted to benzoyl-CoA by a reductive dehydroxylase. This enzymatic activity has been measured in extracts of R. palustris and the denitrifying species A. evansii, and 4-OHbenzoyl-CoA reductase (dehydroxylating) has been purified from T. aromatica (10). The enzyme is inactivated by oxygen and catalyzes the dehydroxylation of 4-OHbenzoylCoA by two successive one-electron transfer reactions (41). As with the benzoyl-CoA reductase, reduced viologen dyes serve as artificial electron donors in activity assays; the natural electron donor has not yet been identified. The reductase is composed of three subunits of 75, 35, and 17 kDa in an a2b2g2 configuration. The native enzyme has 12 Fe and 12 S atoms, suggesting that iron-sulfur centers participate in electron transfer. Investigators tested for molybdenum but did not find significant amounts of this transition metal in the purified enzyme (10). The genes that encode the three subunits of the 4-OHbenzoyl-CoA reductase from R. palustris are adjacent to and divergently transcribed from the gene encoding the 4-OHBenCoA ligase (29). Disruption of the first gene in the three-gene (hbaBCD) sequence with an antibiotic resistance cassette resulted in the generation of a mutant that was unable to grow on 4-OHBen and that lacked the ability to dehydroxylate 4-OHbenzoyl-CoA to benzoyl-CoA. The hbaBCD genes specify polypeptides (17.5, 82.6, and 34.5 kDa) with sizes similar to those of the three subunits of the 4-OHbenzoyl-CoA reductase purified from T. aromatica. The deduced amino acid sequences of the HbaBCD polypeptides show significant similarities (25 to 30% amino acid identities) to a group of molybdenumcontaining iron-sulfur proteins that are involved in aerobic CO oxidation and xanthine and nicotine hydroxylation. Consistent with this is the observation that R. palustris cultures require added molybdate for optimal growth on 4-OHBen, but not for growth on benzoate (29). Although amino acid similarities between 4-OHbenzoylCoA reductase and aerobic hydroxylases suggest a common evolutionary origin that should provide clues about enzyme mechanism, there are also important differences between the two types of enzymes. The most obvious of these are the extreme sensitivity of the reductase to oxygen, its requirement for a very low potential electron donor, and also for a CoAmodified substrate. Reductive dehydroxylases probably have wide importance in anaerobic aromatic degradation. Many lignin monomers, including compounds like caffeate, coumarate, and ferulate, are degraded under anaerobic conditions to form 4-hydroxybenzoate as a common intermediate. Also, phenol present in anaerobic environments appears to be carboxylated, activated with CoA, and then dehydroxylated as first steps to degradation (14, 24, 32). MOLECULAR STUDIES Molecular approaches to defining various aspects of anaerobic aromatic compound degradation have been applied primarily in studies with R. palustris and mostly in the last few years. The first structural genes were identified by immuno- Downloaded from http://jb.asm.org/ on February 21, 2013 by PENN STATE UNIV FIG. 4. Conversion of cyclohexanecarboxylic acid and 4-OHBen to intermediates of the anaerobic benzoate pathway. MINIREVIEW 306 MINIREVIEW J. BACTERIOL. REGULATION The levels of enzymes involved in aromatic acid degradation are modulated by the two major environmental cues of carbon source and oxygen availability. The enzymes required for the conversion of cyclohex-1-ene-1-carboxyl-CoA to pimelyl-CoA are induced by growth on benzoate to levels that are 3- to 10-fold higher than those observed in succinate-grown cells (52). Benzoate-CoA ligase, CHC-CoA ligase, and 4-OHBenCoA ligase, all of which can activate benzoate with CoA, are induced by growth on benzoate (20, 45). The latter two ligases are also, of course, induced by their cognate substrates, 4-OHBen in the case of 4-OHBen-CoA ligase and cyclohexanecarboxylate in the case of CHC-CoA ligase. The influence of various growth conditions on the syntheses of the benzoate-CoA and 4-OHBen-CoA ligases from R. palustris has been studied by immunoblot assays using polyclonal antisera specific to each of the enzymes (28, 43). This approach obviates the potential problem of measuring an activity in a crude extract that may represent the sum of two or more different enzymes catalyzing the same reaction. Much higher levels of benzoate-CoA ligase protein were synthesized by cells grown on aromatic compounds than by cells grown on succinate or pimelate. Cells grown on succinate in the presence of a variety of ring-substituted compounds, such as 2-aminobenzoate and 4-methylbenzoate, that are not metabolized by R. palustris also expressed the ligase. This indicates that aromatic acids can directly elicit synthesis of benzoate-CoA ligase without being modified. R. palustris is a facultative organism and can switch between aerobic and anaerobic growth modes. 4-OHBen supports both anaerobic (phototrophic) and aerobic (heterotrophic) growth of R. palustris, and the aerobic pathway utilized appears to be a typical oxygenase-initiated, meta-ring fission pathway (38) that is completely different from the anaerobic pathway of 4-OHBen degradation. The switch from the aerobic to the anaerobic pathway, both of which are induced by 4-OHBen, appears to depend, at least in part, on the R. palustris protein designated AadR (for anaerobic aromatic degradation regulator) (16). aadR mutants are completely blocked in anaerobic growth on 4-OHBen but grow normally aerobically with this substrate. Mutant cells also grow extremely slowly on benzoate, but other aspects of anaerobic growth are unimpaired; growth on nonaromatic compounds is normal, and nitrogen fixation is also not affected by the aadR mutation (16). Sequence comparisons indicate that AadR belongs to the Crp-Fnr-FixK family of transcriptional regulators, members of which modulate expression of genes for sugar utilization (Crp), anaerobic respiration (Fnr), and nitrogen fixation (FixK) in gram-negative bacteria (22). AadR is similar in size to other family members and shares many amino acid sequence features in common with them. It resembles Fnr in having four cysteine residues near the N terminus and one in the middle of the protein. In Fnr, conserved cysteines appear to participate in the formation of an iron sulfur center that may sense changes in redox potential. A change in oxidation state has been hypothesized to result in a conformational change in the protein to a transcriptionally active form (4, 42). Amino acid similarities and the aadR phenotype raise the obvious suggestion that AadR may also function to sense redox potential. However, the apparent specialized function of AadR as a regulator specific to anaerobic aromatic degradation is in striking contrast to the global regulatory function of Fnr, and AadR may also respond to an aromatic effector molecule. One of several likely target genes for AadR transcriptional activation is hbaA. aadR mutants synthesize only constitutive levels of the hbaA product, 4-OHBen-CoA ligase, even in the presence of 4-OHBen, and a nucleotide sequence that closely resembles an Fnr-like consensus binding site lies about 35 bases upstream of the hbaA transcriptional start site (15). Denitrifying bacteria also switch between aerobic and anaerobic aromatic degradation pathways. In contrast to R. palustris and most other bacteria, the denitrifying species A. evansii uses unusual aerobic degradation pathways that include CoA thioesters as intermediates. The degradation of both 2-aminobenzoate and benzoate is initiated by CoA thioesterification. 2-Aminobenzoyl-CoA is then hydroxylated by a mixed-function oxygenase-reductase (12), whereas benzoyl-CoA is hydroxylated by an FAD-dependent monooxygenase (2). The ligases that initiate aerobic degradation are synthesized only under aerobic conditions. Ligase isozymes specific for anaerobic benzoate and 2-aminobenzoate degradation are synthesized under anoxic growth conditions and thus are likely to be regulated by oxygen availability or redox potential (1, 2). Regulatory proteins required for the differential expression of ligase isozymes or other anaerobic aromatic degradation enzymes have not yet been identified in denitrifying species. FUTURE DIRECTIONS Studies of anaerobic aromatic compound degradation are still in the beginning stages. Anaerobic aromatic-degrading Downloaded from http://jb.asm.org/ on February 21, 2013 by PENN STATE UNIV screening with antisera raised against purified benzoate and 4-OHBen-CoA ligases (20, 28). The two ligase genes, badA and hbaA, lie within 6 kb of each other on the R. palustris chromosome and are surrounded by genes that also participate in anaerobic benzoate and 4-OHBen degradation. Although this gene cluster, which appears to be about 30 kb in size, is still incompletely characterized, a number of open reading frames have been defined in this area by nucleotide sequence analysis (19). Comparisons of the sequences of some of these open reading frame sequences with those of genes catalogued in DNA databases has led to predictions of gene function that can sometimes then be confirmed by analyzing the phenotypes of constructed mutants. Such an approach worked well for the identification and assignment of genes encoding the 4-OHbenzoyl-CoA reductase mentioned above. The use of site-directed mutations does not always give clear-cut results, however. For example, this approach has revealed an example of redundancy in the number of R. palustris genes that encode ligases able to thioesterify benzoate with CoA. By constructing and analyzing the phenotypes of ligase mutants and by carrying out competition experiments involving the addition of mixtures of aromatic and alicylic acids to ligase assay mixtures, Egland et al. (20) determined that R. palustris has three ligases that can catalyze the initial step of benzoate degradation and support phototrophic utilization of this compound for growth. These include BadA, the originally purified benzoate-CoA ligase, as well as 4-OHBen-CoA and cyclohexanecarboxylate-CoA (CHC-CoA) ligases. The high affinity of the BadA protein for benzoate (Km , 1 mM) suggests that it is the principal enzyme that initiates anaerobic benzoate degradation. The other two ligases have substantially higher Kms for benzoate (about 500 mM) (28, 45) but can nevertheless function to support wild-type growth rates of cells on benzoate when it is present at 100 mM and, possibly lower, concentrations (20). The finding that R. palustris has two back-up systems to ensure the initial activation of benzoate underscores the importance of this reaction in anaerobic aromatic degradation. VOL. 179, 1997 307 following reduction of the aromatic ring. The question of how the benzoate pathway is regulated has hardly been touched, and the possibility that cells synthesize and maintain transport systems for the efficient acquisition of aromatic compounds has not been addressed. Larger issues relate to the operation of the anaerobic benzoate and 4-OHBen degradation pathways in different organisms. Comparisons of R. palustris and T. aromatica indicate that there are extensive similarities among the key reactions of benzoate and 4-OHBen degradation, but the degree of differences either in biochemistry or at the genetic level is not at all clear. The picture is even murkier when one considers other metabolic groups. So far, very little biochemical work has been done on benzoate degradation in sulfate-reducing or ironreducing bacteria, although there would seem to be no theoretical reason why these organisms could not degrade benzoate according to the schemes shown in Fig. 2 and 3. By contrast, it is difficult to see how the benzoate pathway, as formulated for phototrophic and denitrifying bacteria, can provide fermentative bacteria (which for thermodynamic reasons can degrade benzoate only when grown in coculture with a hydrogen-utilizing organism such as a methanogen [39]) with sufficient ATP to support growth. If the energetics of the pathway are defined in terms of ATP consumed and regenerated by substrate-level phosphorylation, the investment in thioesterification and reduction (four ATP molecules) exceeds the return from conversion of benzoate to acetate (three ATP molecules). While this negative balance may be acceptable to microorganisms that can generate additional ATP by anaerobic respiration or photophosphorylation, it clearly cannot support a purely fermentative existence. ATP generation from energy conserved as an ion gradient during decarboxylation (of, for example, glutaconyl-CoA) is a possible additional feature of the energetics of aromatic-compound-fermenting bacteria. In general, work on aromatic compound degradation by various metabolic groups, but principally phototrophic and denitrifying bacteria, has proceeded along parallel rather than integrated courses. So far, combined analytical biochemical and molecular approaches have not generally been brought to bear on specific aspects of benzoate and 4-OHBen degradation in a coordinated way. However, now that the outlines of the process have been established and areas of uncertainty have been defined, rapid progress in pulling all threads together can be predicted in the next few years. ACKNOWLEDGMENTS Work from our laboratories was supported by the Department of Energy (Division of Energy Biosciences) (DE-FG02-86ER13495 to J.G. and DE-FG02-95ER20184 to C.S.H.) and by the U.S. Army Research Office (DAAH04-95-1-0124 and -0315 to C.S.H.). REFERENCES 1. Altenschmidt, U., B. Oswald, and G. Fuchs. 1991. Purification and characterization of benzoate-coenzyme A ligase and 2-aminobenzoate-coenzyme A ligases from a denitrifying Pseudomonas sp. J. Bacteriol. 173:5494–5501. 2. Altenschmidt, U., B. Oswald, E. Steiner, H. Herrmann, and G. Fuchs. 1993. New aerobic benzoate oxidation pathway via benzoyl-coenzyme A and 3-hydroxybenzoyl-coenzyme A in a denitrifying Pseudomonas sp. J. Bacteriol. 175:4851–4858. 3. Anders, H.-J., A. Kaetzke, P. Kämpfer, W. Ludwig, and G. Fuchs. 1995. Taxonomic position of aromatic-degrading denitrifying pseudomonad strains K 172 and KB 740 and their description as new members of the genera Thauera, as Thauera aromatica sp. nov., and Azoarcus, as Azoarcus evansii sp. nov., respectively, members of the beta subclass of the Proteobacteria. Int. J. Syst. Bacteriol. 45:327–333. 4. Becker, S., G. Holighaus, T. Gabrielczyk, and G. Unden. 1996. O2 as the regulatory signal for FNR-dependent gene regulation in Escherichia coli. J. Bacteriol. 178:4515–4521. 5. Beller, H. R., A. M. Spormann, P. K. Sharma, J. R. Cole, and M. Reinhard. Downloaded from http://jb.asm.org/ on February 21, 2013 by PENN STATE UNIV bacteria are being isolated at a rapid rate, and the range of aromatic compounds that can be degraded by pure cultures is still being explored. Recent emphasis has been on aromatic hydrocarbon-degrading isolates, with good reason. Aromatic hydrocarbons, including benzene, toluene, ethylbenzene, and xylenes (known collectively as BTEX), comprise about 15% of gasoline and are also produced in huge amounts (megatons per year) as bulk chemicals for industrial use as solvents and starting materials for the production of pesticides, plastics, and synthetic fibers (59). In view of these large quantities, the release of substantial amounts of aromatic hydrocarbons into the environment, even by accident, is inevitable. A major avoidable avenue of contamination is underground gasoline storage tanks. It is estimated that as many as 35% of the 1.4 million tanks in the United States are leaking (60). Contamination of groundwater by BTEX is particularly problematic because these compounds are relatively soluble in water and can migrate large distances in aquifers. Benzene, a proven carcinogen, is the most soluble of the aromatic hydrocarbons and also poses the greatest health hazard. Pure cultures of anaerobic toluene-degrading bacteria include about 20 strains of denitrifiers (3, 14, 53, 63), most, if not all, of which appear to be species of the closely related genera Azoarcus and Thauera, one iron-reducing species (Geobacter metallireducens [47]), and two strains of sulfate-reducing bacteria (5, 54). Only a few m-xylene-utilizers and one ethylbenzene-utilizing strain have been described, all of which are denitrifying bacteria (23, 53, 56). A pure culture of an anaerobic benzene-degrading strain is much sought after but yet to be obtained. Stable benzene-degrading enrichment cultures have, however, been described (18, 46, 48). At this stage in the development of the field, it may in some senses be premature to focus on just a few strains for enzymological and molecular studies. On the other hand, benzoate and 4-OHBen are intermediates common to the degradation of a large number of structurally diverse aromatic compounds, and an understanding of the details of benzene ring dehydroxylation, reduction, and subsequent steps, as well as identification of the genes involved, may facilitate work on other aromatic compounds, including aromatic hydrocarbons. Because it is versatile, easy to grow, and amenable to genetic approaches, R. palustris offers a number of advantages as a model system for the study of anaerobic aromatic compound degradation, while comparable genetic accessibility has not yet been reported for species from other metabolic groups. R. palustris can utilize a wide range of substrates, including lignin monomers and chlorobenzoates (36, 40, 61), and should prove useful for determining the genetic basis for the removal of aliphatic side chains, demethoxylation, and dehalogenation. However, its substrate range does not extend to some toxic compounds, including phenol, toluene, and p-cresol, that are utilized by T. aromatica K 172 (3). The major insight of reductive attack on the aromatic nucleus remains intact after almost 30 years, but several aspects of the anaerobic benzoate pathway are still poorly understood. For example, the isomeric structure(s) of the initial benzoylCoA reduction product(s) is undefined, and the mechanistic details of the enzymatic reaction catalyzed by the benzoyl-CoA reductase, particularly the role of ATP, have yet to be studied. The substrate specificity of the ring-opening 2-ketocyclohexanecarboxyl-CoA hydrolase, essential for growth on benzoate or cyclohexanecarboxylate, is unexplored, and the originally postulated sequence of reactions leading from cyclohex-1-ene-1carboxyl-CoA has not been shown unequivocally to be in a unique and essential pathway. 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