coli K12 mutants with a deletion of the paaF, paaG, paaH, paaJ or paaZ gene are unable to grow with phenylacetate as carbon source.. The paaG and paaZ mutants also converted phenylacetat
Trang 1Functional genomics by NMR spectroscopy
Wael Ismail1, Magdy El-Said Mohamed1, Barry L Wanner2, Kirill A Datsenko2, Wolfgang Eisenreich3, Felix Rohdich3, Adelbert Bacher3and Georg Fuchs1
1
Mikrobiologie, Institut fu¨r Biologie II, Universita¨t Freiburg, Germany;2Department of Biological Sciences, Purdue University, West Lafayette, IN, USA;3Lehrstuhl fu¨r Organische Chemie undBiochemie, Technische Universita¨t Mu¨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-13C8]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 C6compounds from the aromatic ring was
shown using [ring-13C6]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 W mutant 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 coliK12 using NMR and multiply13C-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-14C]Phenylalanine was from Amersham-Pharmacia Bio-tech (Freiburg, Germany), and [1-14C]phenylacetic acid was from American Radiolabeled Chemicals (Ko¨ln, Germany)
L-[U-13C9]phenylalanine and L-[ring-13C6]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: Georg.Fuchs@biologie.uni-freiburg.de,
Web site: http://www.biologie.uni-freiburg.de
Abbreviation: HMQC, heteronuclear multiple-quantum correlation.
(Received 14 March 2003, revised 29 April 2003,
accepted 22 May 2003)
Trang 2(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 of13C-labeled phenylacetate
Reaction mixtures containing 0.1M phosphate, pH 6.5,
40 mg L-[U-13C9]phenylalanine or L-[ring-13C6
]phenyl-alanine, 740 kBq [U-14C9]phenylalanine, and 2 UL-amino
acid oxidase (Fluka, Neu-Ulm, Germany) in a total volume
of 60 mL were incubated at 37Cfor 3 h A solution
(12 mL) containing 1% H2O2 in 6M 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 mM) or
glycerol (10 mM) as carbon and energy source in a
phosphate-buffered mineral salt medium supplemented
with vitamins, as described previously [7,10] Cultures were
incubated at 37Cwith shaking (180 r.p.m.) Cells were
harvested in the exponential growth phase (A5780.4–0.6) by
centrifugation at 10 000 g for 10 min Growth of E coli
K12 with 2-hydroxyphenylacetate (at 1 and 5 mM
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 mMphenylacetate and 1 mM
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 coliK12 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 K12 by P1 using
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 of13C-labeled phenylacetate Mutants were grown in phosphate-buffered mineral salt medium supplemented with vitamins [10] containing 10 mM
glycerol, 1 mM isopropyl thio-b-D-galactoside, and 5 mM
phenylacetate Cells were harvested by centrifugation, washed with 30 mMNH4HCO3, pH 7.3, and resuspended
in the same buffer (1.5 g cells per 15 mL) [U-13C8 ]Pheny-lactate or [ring-13C6]phenylacetate (6 mg), 111 kBq [1-14C]phenylacetate or [U-14C]phenylacetate, and glycerol (final concentration, 0.3 mM) were added to 15 mL of cell suspension The mixtures were incubated at 30Cunder shaking (180 r.p.m.) Samples were retrieved at intervals and centrifuged The supernatants were analyzed by HPLC and lyophilized
HPLC
A reversed-phase C18column (RP-C18, Grom-Sil octadecyl silane-4 hydrophilic; end capped; particle size, 5 lm;
120· 4 mm; Grom, Herrenberg, Germany) was equili-brated with 50 mMpotassium 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
5 min The flow rate was 1 mLÆmin)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 D2O and methanol-d4 1D
13C-NMR spectra and 2D INADEQUATE
measured at 125.6 MHz using a Bruker DRX 500 spectrometer equipped with a dual 13C/1H probe head 2D gradient-enhanced HMQCand 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.
Trang 3deletion mutant with promoter fusion in E coli BW25113
and transferred it into E coli K12 by P1 [12] To avoid
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-14C]phenylacetate, 1–3 mM[U-13C8
]phenyl-acetate or [ring-13C6]phenylacetate, and 0.3 mM glycerol
Isopropyl thio-b-D-galactoside was added for expression
of downstream genes, as appropriate The expected
con-sumption of phenylacetate was confirmed by HPLCof
the culture fluid using online liquid-scintillation counting
for detection (data not shown)
Metabolites in the culture supernatants derived from [14C/13C]phenylacetate were detected by HPLC They were subsequently analyzed by13C-NMR spectroscopy with high selectivity and sensitivity because of the multiple
13C-labeling In 1D13C-NMR spectra, the signals of these metabolites appeared as complex multiplets because of multiple 13C13Ccoupling Carbon–carbon connectivities were gleaned from 2D spectra of the totally 13C-labeled metabolites
Metabolites frompaaG and paaZ mutants
13C-NMR spectra of supernatants from paaG and paaZ mutants were dominated by eight13C-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 CH2COOH 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-13C8 ]phenylace-tate (5, R ¼ H, Fig 3)
Table 1 NMR data of13C-labeled products from [U-13C 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
2-Hydroxy[U-13C 8 ]phenylacetate (5)
4 157.22 65.0, 66.8(3,5), 9.0(7), 1.6(8,6)
ring-1,2-Dihydroxy-1,2-dihydro[U-13C 8 ]phenylacetate or its c-lactone (1)
cis-D3-Dehydro[U-13C 6 ]adipate (3)
3-Hydroxy[U-13C 6 ]adipate (4) or its lactone
6 181.17 48.0(5), 4.2(3), 1.2 (4,2)
Trang 4Besides 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 HMQCspectroscopy
(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 ofpaaF and paaH mutants
A set of six intense13C-NMR signals was detected in the
supernatants of the paaF and paaH mutants which had been
incubated with [U-13C8]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 the13Cspin system by simulation (Table 1) identified the metabolite
as 3-hydroxy[U-13C6]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-13C6]phenylacetate The same set of six coupled signals as in the experiment with [U-13C8 ]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-13C 8 ]phenylacetate (cf Table 2) Bold lines connect 13C-labeled carbon atoms.
Table 2 Product patterns observed in supernatants of paa gene-deficient mutants of E coli given [U-13C 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
Trang 5Fig 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 13C-labeled olefinic atom
(42.5 Hz), as well as a 13C-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-13C6]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, or paaZ 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 13C-labeled phenylacetate yielded several uni-formly 13C-labeled metabolites (Fig 3, Table 2), which were identified by 13C-NMR spectroscopy without prior purification This approach was possible by the sensitivity and selectivity enhancement due to multiple13C-labeling of the precursor The deconvolution of the13Cspin systems by heteronuclear correlation spectroscopy was greatly facilita-ted by the contiguous 13Clabeling 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-13C 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)
[ring-13C 6 ]phenylacetate.13Ccoupling patterns
are indicated.
Trang 6principles 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 paaZmutants accumulated C8compounds rather than C6 compounds This suggests a role for the PaaG and PaaZ proteins early in the pathway The other major finding is that C6dicarboxylic acids formed later in the pathway are derived from the aromatic ring carbons of phenylacetyl-CoA This indicates that removal of the original C2 side chain yields a C6 intermediate via an open-chain C8
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 (D3,D2-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 D3,D2 isomeriza-tion of double bonds in 8 and/or addiisomeriza-tion 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.
Trang 7C–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]
paaABCDEmutants 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 PaaABCmay
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, in which the paaZ 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 C8 triene, which becomes
rearranged to 2,4,6-cycloheptatriene-1-one with the release
of CoASH and a C1unit
paaFand 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 palustrisand 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-D3-trans-D2-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, Geobacillussp., 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 Biochemie der Enzyme (University
of Freiburg) to G.F., and the U S National Science Foundation to B.L.W.
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