Download degradation: a process unique to prokaryotes

Shedding light on anaerobic benzene ring
degradation: a process unique to prokaryotes?
C S Harwood and J Gibson
J. Bacteriol. 1997, 179(2):301.
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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
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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.
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FIG. 1. Schematic view of the degradation of representative aromatic compounds to form 4-OHbenzoyl-CoA or benzoyl-CoA as central intermediates.
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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-
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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-
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FIG. 4. Conversion of cyclohexanecarboxylic acid and 4-OHBen to intermediates of the anaerobic benzoate pathway.
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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
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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.).
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still being explored. Recent emphasis has been on aromatic
hydrocarbon-degrading isolates, with good reason. Aromatic
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reported for species from other metabolic groups. R. palustris
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