Báo cáo khoa học: Functional genomics by NMR spectroscopy Phenylacetate catabolism in Escherichia coli docx

Functional genomics by NMR spectroscopy
Phenylacetate catabolism in
Escherichia coli
Wael Ismail
1
, Magdy El-Said Mohamed
1
, Barry L. Wanner
2
, Kirill A. Datsenko
2
, Wolfgang Eisenreich
3
,
Felix Rohdich
3
, Adelbert Bacher
3
and Georg Fuchs
1
1
Mikrobiologie, Institut fu
¨
r Biologie II, Universita
¨
t Freiburg, Germany;
2
Department of Biological Sciences, Purdue University,
West Lafayette, IN, USA;
3
Lehrstuhl fu

¨
r Organische Chemie und Biochemie, Technische Universita
¨
tMu
¨
nchen, Germany
Aerobic metabolism of phenylalanine in most bacteria
proceeds via oxidation to phenylacetate. Surprisingly, the
further metabolism of phenylacetate has not been elucida-
ted, even in well studied bacteria such as Escherichia coli.
The only committed step is the conversion of phenylacetate
into phenylacetyl-CoA. The paa operon of E. coli encodes
14 polypeptides involved in the catabolism of phenylacetate.
We have found that E. coli K12 mutants with a deletion of
the paaF, paaG, paaH, paaJ or paaZ gene are unable to
grow with phenylacetate as carbon source. Incubation of a
paaG mutant with [U-
13
C
8
]phenylacetate yielded ring-1,2-
dihydroxy-1,2-dihydrophenylacetyl lactone as shown by
NMR spectroscopy. Incubation of the paaF and paaH
mutants with phenylacetate yielded D3-dehydroadipate and
3-hydroxyadipate, respectively. The origin of the carbon
atoms of these C
6
compounds from the aromatic ring was
shown using [ring-
13

C
6
]phenylacetate. The paaG and paaZ
mutants also converted phenylacetate into ortho-hydroxy-
phenylacetate, which was previously identified as a dead end
product of phenylacetate catabolism. These data, in
conjunction with protein sequence data, suggest a novel
catabolic pathway via CoA thioesters. According to this,
phenylacetyl-CoA is attacked by a ring-oxygenase/reductase
(PaaABCDE proteins), generating a hydroxylated and
reduced derivative of phenylacetyl-CoA, which is not
re-oxidized to a dihydroxylated aromatic intermediate, as in
other known aromatic pathways. Rather, it is proposed that
this nonaromatic intermediate CoA ester is further metabo-
lized in a complex reaction sequence comprising enoyl-CoA
isomerization/hydration, nonoxygenolytic ring opening,
and dehydrogenation catalyzed by the PaaG and PaaZ
proteins. The subsequent b-oxidation-type degradation of
the resulting CoA dicarboxylate via b-ketoadipyl-CoA to
succinyl-CoA and acetyl-CoA appears to be catalyzed by
the PaaJ, PaaF and PaaH proteins.
Keywords: aromatic metabolism; phenylacetate; phenyl-
acetyl-CoA oxygenase; phenylalanine metabolism.
The aerobic catabolism of aromatic compounds in micro-
organisms has been studied in some detail. Hayaishi [1] was
the first to show the formation of hydroxylated products by
mono-oxygenases and dioxygenases. The resulting aromatic
vicinal dihydroxy derivatives can be cleaved by dioxygen-
ases between the hydroxy groups (ortho cleavage) or
adjacent to one hydroxy group (meta cleavage).

The bacterial metabolism of phenylalanine generally
proceeds via phenylacetate. Surprisingly, phenylacetate
metabolism is still largely unknown, even in Escherichia
coli, despite many efforts [2–10]. The genomes of several
proteobacteria, including E. coli, Pseudomonas putida and
Azoarcus evansii, contain clusters of 11–16 genes believed to
be involved in the catabolism of phenylacetate [2,5,6,9,10]
(Fig. 1). The paaK gene of E. coli and orthologous genes in
other bacteria specify a CoA ligase catalysing the conversion
of phenylacetate into phenylacetyl-CoA, which is the first
and only committed intermediate in the catabolic pathway
[3,4,7]. The use of substrate CoA thioesters is unprecedented
in aerobic aromatic metabolism, which may explain why
this pathway has been overlooked. Recombinant expression
of the paaABCDEK genes allows an E. coli Wmutant
lacking the paa genes to convert phenylacetate into
2-hydroxyphenylacetate but not to catabolize phenyl-
acetate. Because 2-hydroxyphenylacetate cannot be meta-
bolized by E. coli, it is believed to be a dead end product of
phenylacetate metabolism [5,6].
This paper describes catabolic studies on mutants of
E. coli K12 using NMR and multiply
13
C-labeled phenyl-
acetate samples. This has led to the proposal of a new
phenylacetate catabolic pathway, allowing putative func-
tions to be assigned to most of the paa catabolic genes.
Materials and methods
Materials
[U-

14
C]Phenylalanine was from Amersham-Pharmacia Bio-
tech (Freiburg, Germany), and [1-
14
C]phenylacetic acid was
from American Radiolabeled Chemicals (Ko
¨
ln, Germany).
L
-[U-
13
C
9
]phenylalanine and
L
-[ring-
13
C
6
]phenylalanine
were purchased from Cambridge Isotope Laboratories
Correspondence to G. Fuchs, Mikrobiologie, Institut Biologie II,
Scha
¨
nzlestrasse 1, D-79104 Freiburg, Germany.
Fax: + 49 761 203 2626, Tel.: + 49 761 203 2649,
E-mail: ,
Web site:
Abbreviation: HMQC, heteronuclear multiple-quantum correlation.
(Received 14 March 2003, revised 29 April 2003,

accepted 22 May 2003)
Eur. J. Biochem. 270, 3047–3054 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03683.x
(Andover, MA, USA). Oligonucleotides were from IDT
(Coralville, IA, USA). Qiaprep Spin Miniprep Kit (for
plasmid DNA isolation) and Qiaquick Gel Extraction Kit
(for gel purification of DNA fragments and PCR products)
were supplied by Qiagen (Hilden, Germany).
Synthesis of
13
C-labeled phenylacetate
Reaction mixtures containing 0.1
M
phosphate, pH 6.5,
40 mg
L
-[U-
13
C
9
]phenylalanine or
L
-[ring-
13
C
6
]phenyl-
alanine, 740 kBq [U-
14
C
9

]phenylalanine, and 2 U
L
-amino
acid oxidase (Fluka, Neu-Ulm, Germany) in a total volume
of 60 mL were incubated at 37 °C for 3 h. A solution
(12 mL) containing 1% H
2
O
2
in 6
M
NaOH was added.
After 5 min at room temperature, the pH was adjusted to
3.0 by the addition of HCl, and the mixture was extracted
three times with equal volumes of ethyl acetate. The solvent
was evaporated under reduced pressure. The yield was 50%.
Bacteria, media and growth conditions
Wild-type E. coli K12 (DSM 498) and E. coli BW25113 [11]
were grown aerobically with phenylacetate (5 m
M
)or
glycerol (10 m
M
) as carbon and energy source in a
phosphate-buffered mineral salt medium supplemented
with vitamins, as described previously [7,10]. Cultures were
incubated at 37 °C with shaking (180 r.p.m.). Cells were
harvested in the exponential growth phase (A
578
0.4–0.6) by

centrifugation at 10 000 g for 10 min. Growth of E. coli
K12 with 2-hydroxyphenylacetate (at 1 and 5 m
M
concen-
trations) was tested in the same medium. The ability of
different mutants to grow on phenylacetate was checked in
the same medium containing 5 m
M
phenylacetate and 1 m
M
isopropyl thio-b-
D
-galactoside (to induce expression of the
genes located downstream of the deleted gene).
Construction of mutants
Genes paaF, paaH, paaI, paaJ and paaZ were targeted in
E. coli K12 as previously described [11]. All gene mutations
were verified by PCR and sequencing (Microbiology and
Molecular Genetics Core Facility at Harvard Medical
School, Boston, MA, USA). Deletion of the paaG gene
required promoter fusion in E. coli BW25113 [11] and then
transfer of the mutation into E. coli K12byP1using
published procedures [12].
Complementation assays
The paaH gene of wild-type E. coli K12 was amplified and
cloned into the expression vector pLA35 [13] using standard
protocols [14,15]. Integration of the recombinant vector into
the chromosome of the paaH mutant was carried out as
described [13].
Metabolic transformation of

13
C-labeled phenylacetate
Mutants were grown in phosphate-buffered mineral salt
medium supplemented with vitamins [10] containing 10 m
M
glycerol, 1 m
M
isopropyl thio-b-
D
-galactoside, and 5 m
M
phenylacetate. Cells were harvested by centrifugation,
washed with 30 m
M
NH
4
HCO
3
, pH 7.3, and resuspended
in the same buffer (1.5 g cells per 15 mL). [U-
13
C
8
]Pheny-
lactate or [ring-
13
C
6
]phenylacetate (6 mg), 111 kBq
[1-

14
C]phenylacetate or [U-
14
C]phenylacetate, and glycerol
(final concentration, 0.3 m
M
) were added to 15 mL of cell
suspension. The mixtures were incubated at 30 °C under
shaking (180 r.p.m.). Samples were retrieved at intervals
and centrifuged. The supernatants were analyzed by HPLC
and lyophilized.
HPLC
A reversed-phase C
18
column (RP-C
18
,Grom-Siloctadecyl
silane-4 hydrophilic; end capped; particle size, 5 lm;
120 · 4 mm; Grom, Herrenberg, Germany) was equili-
brated with 50 m
M
potassium phosphate, pH 4, containing
8% (v/v) acetonitrile for 15 min and then developed with a
linear gradient of 8–40% acetonitrile in the same buffer for
5min.The flowratewas 1mLÆmin
)1
. The effluent was
monitored photometrically (260 nm) and by online liquid-
scintillation counting. The retention times for phenylacetate,
phenylacetyl-CoA and 2-hydroxyphenylacetate were 20, 21

and 12 min, respectively.
NMR spectroscopy
The lyophilized samples were dissolved in 0.5 mL of
a 1 : 1 (v/v) mixture of D
2
O and methanol-d
4
.1D
13
C-NMR spectra and 2D INADEQUATE
1
spectra were
measured at 125.6 MHz using a Bruker DRX 500
spectrometer equipped with a dual
13
C/
1
H probe head.
2D gradient-enhanced HMQC and HMQC-COSY experi-
ments were performed with an AV 500 spectrometer
equipped with a triple-resonance inverse cryo probe head.
Acquisition and processing parameters were according to
standard Bruker software (
XWINNMR
). Spectral simula-
tions were performed with
NMRSIM
software (Bruker,
Karlsruhe, Germany).
Results

Mutagenesis and mutant phenotype
Mutants with deletions of the genes paaF, paaH, paaI,
paaJ and paaZ (Fig. 1) were constructed using PCR
fragments as described elsewhere [11]. For unknown
reasons, it was not possible to delete the paaG gene
directly in E. coli K12. Therefore, we constructed the
Fig. 1. Organization of paa gene cluster in E. coli which codes for
aerobic phenylacetate metabolism. The only proven function of a
catabolic gene product is that of PaaK, phenylacetate-CoA ligase. The
following functions are putative: PaaABCD, phenylacetyl-CoA
oxygenase; PaaE, oxygenase reductase; PaaF, enoyl-CoA hydratase/
isomerase; PaaG, enoyl-CoA hydratase/isomerase; PaaH, 3-hydroxy-
acyl-CoA dehydrogenase; PaaI, unknown, low similarity to thioest-
erase; PaaJ, b-ketothiolase; PaaZ, unknown, putative ring-cleavage
enzyme, aldehyde dehydrogenase domain (N-terminus), MaoC-like
protein domain (C-terminus). PaaX and PaaY may be involved in
regulation.
3048 W. Ismail et al.(Eur. J. Biochem. 270) Ó FEBS 2003
deletion mutant with promoter fusion in E. coli BW25113
and transferred it into E. coli K12byP1[12].Toavoid
polar effects, deletions were either in-frame or the
lacUV5 promoter was introduced to drive expression
of the respective downstream gene(s). Chromosomal
regions modified were verified by PCR amplification and
sequencing.
Except for the paaI mutant, all mutants were unable to
grow on phenylacetate as sole carbon and energy source in
mineral salt medium within 48 h. A plasmid carrying an
intact paaH gene restored the growth of the DpaaH mutant
on phenylacetate as sole carbon and energy source. All

wild-type and mutant strains grew on succinate, but none
grew on adipate.
Transformation of labeled phenylacetate into labeled
products by the mutants
Mutant cell suspensions were incubated in buffer contain-
ing traces of [U-
14
C]phenylacetate, 1–3 m
M
[U-
13
C
8
]phenyl-
acetate or [ring-
13
C
6
]phenylacetate, and 0.3 m
M
glycerol.
Isopropyl thio-b-
D
-galactoside was added for expression
of downstream genes, as appropriate. The expected con-
sumption of phenylacetate was confirmed by HPLC of
the culture fluid using online liquid-scintillation counting
for detection (data not shown).
Metabolites in the culture supernatants derived from
[

14
C/
13
C]phenylacetate were detected by HPLC. They were
subsequently analyzed by
13
C-NMR spectroscopy with high
selectivity and sensitivity because of the multiple
13
C-labeling. In 1D
13
C-NMR spectra, the signals of these
metabolites appeared as complex multiplets because of
multiple
13
C
13
C coupling. Carbon–carbon connectivities
were gleaned from 2D spectra of the totally
13
C-labeled
metabolites.
Metabolites from
paaG
and
paaZ
mutants
13
C-NMR spectra of supernatants from paaG and paaZ
mutants were dominated by eight

13
C-coupled signals. Six
signals in the range 117–157 p.p.m. (Table 1) suggested a
benzenoid ring with one downfield-shifted signal
(157.2 p.p.m., Fig. 2A), indicating a phenolic hydroxy
group. Two signals (40.9 and 179.1 p.p.m.) suggested the
presence of a CH
2
COOH side chain. The carbon
connectivities were gleaned from correlation patterns
detected by HMQC-COSY experiments (Table 1).
Numerical simulation was used for an in depth study of
the complex coupling patterns (for an example, see
Fig. 2B). On this basis, the structure of the major
metabolite was assigned as 2-hydroxy[U-
13
C
8
]phenylace-
tate (5,R¼ H, Fig. 3).
Table 1. NMR data of
13
C-labeled products from [U-
13
C
8
]phenylacetate in paa gene-deficient mutants of E. coli. nd, Not determined.
Position
Chemical shifts (p.p.m.)
Coupling constants, J

CC
(Hz)
Correlation patterns
d
13
C d
1
H HMQC HMQC-COSY
2-Hydroxy[U-
13
C
8
]phenylacetate (5)
1 179.07 52.1(2)
2 40.86 3.52 52.4(1), 43.3(3) 2 2
3 124.54 43.6(2), 65 (4, 8)
4 157.22 65.0, 66.8(3,5), 9.0(7), 1.6(8,6)
5 117.15 6.76 5 5, 6
6 128.91 7.04 55.8, 57.5(5, 7), 6.8 6 6, 5, 7
7 120.57 6.74 56.2(6, 8), 8.7(4) 7 7, 6, 8
8 131.69 7.05 57(3, 7) 8 8, 7
ring-1,2-Dihydroxy-1,2-dihydro[U-
13
C
8
]phenylacetate or its c-lactone (1)
1 177.94 50.2(2)
2 41.62 2.91, 2.83 50.2(1), 37.8(3) 2, 2¢ 2, 2¢
3 73.46 nd
6 126.25 6.11 67.7(7), 50.2(5), 5.1 6 6, 5, 7

7 122.14 5.86(d) 67.3(6), 45.1(8), 6.9 7 7, 8, 6
4 129.33 5.91(d) 67.0(5), 46.6(3), 6.9 4 4, 5
5 123.10 5.99 nd 5 5, 4, 6
8 85.54 5.23 44.9(7), 36.5(3), 4.2(6) 8 8, 7
cis-D3-Dehydro[U-
13
C
6
]adipate (3)
1/6 nd
2/5 34.76 2.95 51.6(1/6), 42.5(3/4) 2/5 2/5, 3/4
3/4 125.31 5.60 3/4 3/4, 2/5
3-Hydroxy[U-
13
C
6
]adipate (4) or its lactone
1 176.82 50.2(2)
2 42.36 2.52 52.0(1), 39.9(3), 1.2 (6,4) 2 2, 3
3 79.81 4.86 39.8(2), 32.1(4), 4.2(6) 3 3, 2, 4
4 27.01 2.34, 1.88 32.1(3.5), 3.2(1) 4, 4¢ 4, 5, 3
5 28.53 2.59, 2.54 48.0(6), 32.7(4) 5, 5¢ 5, 4
6 181.17 48.0(5), 4.2(3), 1.2 (4,2)
Ó FEBS 2003 Bacterial phenylacetate and phenylalanine metabolism (Eur. J. Biochem. 270) 3049
Besides the signal set described above, an additional
set of eight minor signals was detected in the sample
from the paaG-deficient mutant. Their relative intensities
were  2% compared with the signals of 2-hydroxy-
phenylacetate (Table 2). Four signals had chemical shifts
typical of olefinic carbon atoms (122.1, 123.1, 126.5 and

129.3 p.p.m.; Table 1); they were all correlated to directly
attached protons as shown by 2D HMQC spectroscopy
(Fig. 4). Carbon connectivities established on the basis of
the coupling constants and the correlation pattern in
HMQC-COSY experiments (Table 1) identified the com-
pound as a conjugated cyclohexadiene derivative. The
carbon atoms resonating at 85.5 and 73.5 p.p.m. are likely
to carry heteroatom substituents. The carbon atom
resonating at 85.5 p.p.m. has an attached proton (
1
H-
NMR signal at 5.23 p.p.m.), whereas the signal at
73.46 p.p.m. represents a quaternary carbon atom. On
this basis, the structure can be assigned as ring-1,2-
dihydroxy-1,2-dihydrophenylacetyl lactone (1, Fig. 3; note
the different carbon numbering of 1 in the figure). A
structurally similar lactone (2, Fig. 3) has been found in
the Caribbean sponge, Aplysina cauliformis [16].
Metabolites of
paaF
and
paaH
mutants
A set of six intense
13
C-NMR signals was detected in the
supernatants of the paaF and paaH mutants which had been
incubated with [U-
13
C

8
]phenylacetate. Two carbon atoms
resonated at chemical shifts typical of carboxylic groups
(181.2 and 176.8 p.p.m.). Four signals were detected in the
region for aliphatic carbons; one of these had a chemical
shift (79.8 p.p.m.) characteristic of a carbon atom carrying
an OH or OR residue. The detailed analysis of the
13
C spin
system by simulation (Table 1) identified the metabolite
as 3-hydroxy[U-
13
C
6
]adipate (4) or the cognate lactone
(Fig. 3).
To identify the precursor carbon atoms that are lost in the
formation of 4, we incubated cells of the paaH-deficient
mutant with [ring-
13
C
6
]phenylacetate. The same set of six
coupled signals as in the experiment with [U-
13
C
8
]phenyl-
acetate was observed. As an example, the signals of C6 and
C1 from the two different experiments are displayed in

Fig. 2.
13
C-NMR signal of ring-C2 of 2-hydroxy[U-
13
C
8
]phenylacetate.
(A) Detected in the experiment with the paaG-deficient mutant of
E. coli; (B) simulated signals on the basis of the chemical shifts and
coupling constants summarized in Table 1.
Fig. 3. Compounds observed in supernatants of E. coli mutants given
[U-
13
C
8
]phenylacetate (cf. Table 2). Bold lines connect
13
C-labeled
carbon atoms.
Table 2. Product patterns observed in supernatants of paa gene-deficient
mutants of E. coli given [U-
13
C
8
]phenylacetate. Relative amounts of
product are shown estimated from
13
C-NMR signal intensities referred
to an arbitrary value of 100 for the major product.
Mutant

Product
134 5
paaZ – – – 100
paaG 2 – – 100
paaH – – 100 20
paaF – 3 100 3
3050 W. Ismail et al.(Eur. J. Biochem. 270) Ó FEBS 2003
Fig. 5. The data show that the formation of 3-hydroxy-
adipate (4) proceeds by the loss of the acetyl side-chain
carbon atoms of phenylacetate.
A set of two relatively weak signals ( 3% signal
intensity compared with the signals of 3-hydroxyadipate)
was observed in the experiments with the paaF-deficient
mutant. One signal had a chemical shift typical of olefinic
carbon atoms (125.3 p.p.m.), and the other one
(34.76 p.p.m.) was found in the frequency range typical of
aliphatic carbon atoms. The coupling constants of the latter
signal indicated an attached
13
C-labeled olefinic atom
(42.5Hz),aswellasa
13
C-labeled carboxylate group
(51.6 Hz); the expected signal for the carboxylate carbon
was not observed directly because of signal overlap. The
detailed analysis of the coupling pattern by spectral
simulation suggests an inherently symmetrical metabolite
(Fig. 6). All spectroscopic data, as well as a comparison
with published NMR data for adipate derivatives [17],
support the assignment of the minor metabolite as cis-D3-

dehydro[U-
13
C
6
]adipate (3,Fig.3).
The supernatant of the paaF mutant also showed the
signal set of 2-hydroxyphenylacetate at a relative intensity of
 3% compared with that of the major metabolite 4
(Table 2).
Discussion
We have shown in this work that E. coli K12 mutants with
deletion of the paaF, paaG, paaH, paaJ,orpaaZ gene are
unable to use phenylacetate as carbon source. In contrast,
deletion of the paaI gene did not impair growth on
phenylacetate and phenylacetate consumption. This sug-
gests that the paaI gene product is not essential for
phenylacetate metabolism or can be substituted by the
translation product of another similar gene that is consti-
tutively expressed. The function of the PaaI protein is not
known.
Incubation of the paaF, paaG, paaH or paaZ mutant with
multiply
13
C-labeled phenylacetate yielded several uni-
formly
13
C-labeled metabolites (Fig. 3, Table 2), which
were identified by
13
C-NMR spectroscopy without prior

purification. This approach was possible by the sensitivity
and selectivity enhancement due to multiple
13
C-labeling of
the precursor. The deconvolution of the
13
C spin systems by
heteronuclear correlation spectroscopy was greatly facilita-
ted by the contiguous
13
C labeling of the metabolites.
Moreover, this experimental approach minimizes the risk of
artefact formation by decomposition of chemically unstable
metabolites. The mutant phenotypes and the observed
products (Table 2) led us to propose a working hypothesis
for further studies (Fig. 7). The conversion of phenylacetate
[6] into phenylacetyl-CoA [7] is established (see the Intro-
duction). Although details of the pathway remain unknown,
the available data cannot be reconciled with conventional
Fig. 4. Part of an HMQC spectrum of ring-1,2-dihydroxy-1,2-
dihydro[U-
13
C
8
]phenylacetate (1) or its c-lactone.
Fig. 5.
13
C-NMR signals of 3-hydroxy-
[U-
13

C
6
]adipate (4) or its lactone observed in the
supernatant of a paaH-deficient mutant of
E. coli given (A) [U-
13
C
8
]phenylacetate or (B)
[rin g-
13
C
6
]phenylacetate.
13
C coupling patterns
are indicated.
Ó FEBS 2003 Bacterial phenylacetate and phenylalanine metabolism (Eur. J. Biochem. 270) 3051
principles of aromatic metabolism. Therefore we postulate a
new strategy in which CoA thioesters are used throughout
the pathway and the ring is cleaved nonoxygenolytically
at the stage of a nonaromatic CoA ester intermediate.
Moreover, we postulate the presence of an unprecedented
phenylacetyl-CoA oxygenase/reductase.
One major finding of this study was that both paaG and
paaZ mutants accumulated C
8
compounds rather than C
6
compounds. This suggests a role for the PaaG and PaaZ

proteins early in the pathway. The other major finding is
that C
6
dicarboxylic acids formed later in the pathway are
derived from the aromatic ring carbons of phenylacetyl-
CoA. This indicates that removal of the original C
2
side
chain yields a C
6
intermediate via an open-chain C
8
intermediate. The formation of 1 by the paaG mutant
may suggest that phenylacetyl-CoA is attacked by phenyl-
acetyl-CoA (di)oxygenase/reductase (PaaABCDE), adding
molecular oxygen and reducing the intermediate, possibly to
a cis-dihydrodiol derivative of phenylacetyl-CoA [8]. Fur-
ther metabolism of this intermediate appears to be blocked
in the paaG mutant, suggesting that the PaaG protein uses
this nonaromatic product of the oxygenase/reductase as
substrate. Artificial formation of the lactone 1 from 8 may
be facilitated by the CoA thioesterification of the carboxy
group. Likewise, formation of 5 from 8 by various mutants
(Table 2) may be explained by water elimination from
accumulated 8 resulting in re-aromatization and subsequent
enzyme-catalyzed or spontaneous hydrolysis of the thioester
bond. Wild-type and mutant E. coli strains are unable to use
2-hydroxyphenylacetate as carbon source, probably because
the compound and its CoA derivative are dead end products
rather than intermediates of the pathway under study.

Sequence similarity (40% identity and 11% of conserva-
tive exchange by comparison with ChcB of Streptomyces
collinus) of the PaaG protein with members of the enoyl-
CoA hydratase/isomerase family [18,19] suggests a similar
function. ChcB (D
3
,D
2
-enoyl-CoA isomerase) catalyzes the
isomerization of cyclohex-1-ene-1-carbonyl-CoA and cyclo-
hex-2-ene-1-carbonyl-CoA. Thus, ring opening may be
preceded by a reversible PaaG-catalyzed D
3
,D
2
isomeriza-
tion of double bonds in 8 and/or addition of water.
The enzyme may even play a role in C–C cleavage as
Fig. 6.
13
C-NMR signals of cis-D3-
dehydro[U-
13
C
6
]adipate (3). (A) Detected in
the experiment with the paaF-deficient mutant
of E. coli; (B) simulated for the AA¢MM¢XX¢
spin system using the chemical shifts and
coupling constants summarized in Table 1.

Fig. 7. Proposed outline of the pathway of aerobic metabolism of
phenylacetate in E. coli. For details see text.
3052 W. Ismail et al.(Eur. J. Biochem. 270) Ó FEBS 2003
C–C-cleaving enoyl-CoA hydratases are known [20].
Re-aromatization of the product of phenylacetyl-CoA
oxygenase/reductase 8 by a cis-diol dehydrogenase, as is
common in aromatic pathways, is unlikely because no such
gene could be found in the paa gene cluster.
Formation of the (di)hydroxylated and reduced deriva-
tive 8 from 7 is proposed to be catalysed by a protein
complex specified by the paaABCDE genes. Based on
sequence similarities, the paaABCDE genes may jointly
specify a five-subunit oxygenase/reductase enzyme com-
plex using phenylacetyl-CoA as substrate [2,5,6,9,10].
paaABCDE mutants cannot grow with phenylacetate but
convert it into phenylacetyl-CoA by the catalytic action
of the PaaK protein [5,6]. Putative orthologs of
paaABCD genes are found in numerous proteobacteria
believed to catalyse the degradation of phenylacetate, but
only the PaaE protein shows similarity to enzymes of
micro-organisms outside that group. PaaABC may func-
tion as terminal oxygenase, and the small protein PaaD
may be required as an additional component, as is found
in some oxygenases. The similarity of the PaaE protein
to various oxidoreductases (2Fe-2S ferredoxin flavo-
proteins) indicates that it functions as a reductase which
delivers electrons from NAD(P)H to the oxygenase
components.
Tentatively, we suggest that the ring opening affords an
aldehyde which is converted into a carboxylic acid by the

PaaZ protein. The N-terminal part of PaaZ is similar to
various aldehyde dehydrogenases, e.g. succinate semialde-
hyde dehydrogenase GabD of E. coli (23.7% identity and
12.7% conserved exchange). The C-terminal part shows
similarity to MaoC-like proteins, the function of which are
unknown. It was recently shown that a mutant of Azoarcus
evansii,inwhichthepaaZ ortholog pacL was disrupted by
integration of a resistance cassette, excreted 2,4,6-cyclo-
heptatriene-1-one (9); the pacL gene in this organism
contains only the aldehyde dehydrogenase domain [21].
One can envisage elimination of water from 8 yielding
the corresponding conjugated C
8
triene, which becomes
rearranged to 2,4,6-cycloheptatriene-1-one with the release
of CoASH and a C
1
unit.
paaF and paaH mutants transform phenylacetate into the
open-chain dicarboxylic acid derivatives 3 and 4, respect-
ively. Consequently, the PaaF and PaaH proteins appear to
catalyse reactions in the downstream part of phenylacetate
degradation. In the absence of the PaaF and PaaH proteins,
catabolism of phenylacetate is proposed to be terminated at
the level of 10 or its dehydration product, which are
converted into 4 and 3, respectively, by spontaneous or
enzyme-catalyzed hydrolysis.
The PaaH protein has sequence similarity to 3-hydroxy-
acyl-CoA dehydrogenases. This suggests that it catalyzes the
dehydrogenation of 3-hydroxyadipyl-CoA [10], affording

3-ketoadipyl CoA [11] which could then be thiolytically
cleaved by the PaaJ protein with formation of acetyl-CoA
and succinyl-CoA [12]. The PaaJ protein is similar to
b-ketoadipyl-CoA thiolases. No orthologs of the PaaH or
PaaJ protein appear to exist in the E. coli genome.
The PaaF protein has sequence similarity (44% identity
and 11% conserved exchange) to BadK of Rhodopseudo-
monas palustris and to proteins of the enoyl-CoA hydratase
(isomerase) family (crotonase family) [18,19]. BadK
catalyzes the reversible addition of water to cyclohex-1-
ene-1-carbonyl-CoA forming 2-hydroxycyclohexane-1-car-
bonyl-CoA. Many enoyl-CoA hydratases of this type have
cis-D
3
-trans-D
2
-enoyl-CoA isomerase activity, in addition to
the enoyl-CoA hydratase activity [18,19].
The accumulated products 3, 4 and 5 are devoid of
CoA moieties, which they have probably lost by enzyme-
catalysed or nonenzymatic hydrolysis of catabolic inter-
mediates. Enzyme-catalysed hydrolysis of CoA derivatives
may serve as a salvage reaction to avoid the breakdown
of intermediary metabolism due to depletion of the CoA
pool in situations where CoA derivatives cannot be
metabolized further. Interestingly, the PaaI protein shows
low similarity to thioesterases, and cell extracts are
notorious for CoA thioesterase activity, which greatly
impairs enzyme studies using CoA thioesters. paaI
mutants could still grow with phenylacetate, indicating

that the PaaI protein does not serve an essential function
in the pathway itself.
The postulated phenylacetate pathway has considerable
similarity to the catabolism of benzoate in Azoarcus evansii,
Geobacillus sp., and probably other bacteria [20,22]. In both
cases, CoA thioesters are used throughout the pathway. The
aromatic substrate is first transformed into the CoA thio-
ester, followed by ring oxygenation, isomerization, nonoxy-
genolytical ring cleavage, and subsequent b-oxidation
to b-ketoadipyl-CoA. This intermediate is finally cleaved
into acetyl-CoA and succinyl-CoA, as in the conventional
b-ketoadipate pathway. A variant of this principle exists in
the catabolism of 2-aminobenzoate in Azoarcus evansii and
related bacteria. A mono-oxygenase/reductase rather than
a dioxygenase/reductase transforms 2-aminobenzoyl-CoA
into a nonaromatic monohydroxylated product [20,23,24].
Acknowledgements
We thank W. Buckel, University of Marburg, for suggesting the
enzymatic conversion of phenylalanine into phenylacetate, and R.
Bru
¨
ckner, University of Freiburg, for initial synthesis of [
13
C]phenyl-
acetate. This work was financially supported by the Deutsche
Forschungsgemeinschaft (Bonn), the Fonds der Chemischen Industrie
(Frankfurt), the Graduiertenkolleg ÔBiochemie der EnzymeÕ (University
of Freiburg) to G.F., and the U. S. National Science Foundation to
B.L.W.
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