Previous Article | Next Article 
Journal of Bacteriology, October 2000, p. 5448-5453, Vol. 182, No. 19
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Identification of a Serine Hydrolase Which
Cleaves the Alicyclic Ring of Tetralin
M. J.
Hernáez,1
E.
Andújar,1
J. L.
Ríos,2
S. R.
Kaschabek,3
W.
Reineke,3 and
E.
Santero1,*
Departamento de Genética,
Facultad de Biología, Universidad de
Sevilla,1 and Instituto de la Grasa,
CSIC,2 Seville, Spain, and Chemische
Mikrobiologie, Bergische Universität-Gesamthochschule
Wuppertal, Wuppertal, Germany3
Received 13 April 2000/Accepted 28 June 2000
 |
ABSTRACT |
A gene designated thnD, which is required for
biodegradation of the organic solvent tetralin by Sphingomonas
macrogoltabidus strain TFA, has been identified. Sequence
comparison analysis indicated that thnD codes for a
carbon-carbon bond serine hydrolase showing highest similarity to
hydrolases involved in biodegradation of biphenyl. An insertion mutant
defective in ThnD accumulates the ring fission product which results
from the extradiol cleavage of the aromatic ring of dihydroxytetralin.
The gene product has been purified and characterized. ThnD is an
octameric thermostable enzyme with an optimum reaction temperature at
65°C. ThnD efficiently hydrolyzes the ring fission intermediate of
the tetralin pathway and also 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic
acid, the ring fission product of the biphenyl
meta-cleavage pathway. However, it is not active towards
the equivalent intermediates of meta-cleavage pathways of
monoaromatic compounds which have small substituents in C-6. When ThnD
hydrolyzes the intermediate in the tetralin pathway, it cleaves a C-C
bond comprised within the alicyclic ring of tetralin instead of
cleaving a linear C-C bond, as all other known hydrolases of
meta-cleavage pathways do. The significance of this
activity of ThnD for the requirement of other activities to mineralize
tetralin is discussed.
| |
INTRODUCTION |
Tetralin
(1,2,3,4-tetrahydronaphthalene) is a toxic organic solvent since its
accumulation in the cell membranes because of its lipophilic character
may lead to changes in their structure and function (35, 36)
and also because of the formation of toxic hydroperoxides in the cell
(12). Tetralin is produced for industrial purposes from
naphthalene by catalytic hydrogenation or from anthracene by cracking,
and it is widely used as a degreasing agent and solvent for fats,
resins, and waxes; as a substitute for turpentine in paints, lacquers,
and shoe polishes; and also in the petrochemical industry in connection
with coal liquefaction (15).
Tetralin is a bicyclic molecule composed of an aromatic and an
alicyclic moiety which share two carbon atoms. In principle, initial
transformation of tetralin may involve metabolization of either the
aromatic or the alicyclic ring, thus rendering the corresponding
alicyclic or aromatic intermediate. However, since pathways known to
metabolize aromatic rings are quite different from those acting on
alicyclic rings (7, 38), mineralization of tetralin could
require the recruitment of two types of metabolic pathways. In spite of
this interesting characteristic, very little is known about tetralin
utilization by bacteria, and just a few bacterial strains able to grow
on tetralin as the only carbon and energy source have been reported
(33). By identifying accumulated intermediates, several
reports suggest that some bacteria, such as Pseudomonas
stutzeri AS39 (31), initially hydroxylate and further
oxidize the alicyclic ring, while others, such as
Corynebacterium sp. strain C125 (34), initially
dioxygenate the aromatic ring which is cleaved in the extradiol
position (meta-cleavage pathway). A strain designated TFA,
which is able to grow by using tetralin as the only carbon and energy
source, was recently isolated and ascribed to the species
Sphingomonas macrogoltabidus. Initial characterization of
this strain showed that TFA cannot grow by using other aromatic
compounds such as naphthalene, biphenyl, or xylenes as carbon and
energy sources (22). Physical and genetic analysis of
mutants led to the identification of a genomic region involved in
tetralin biodegradation which comprises two divergent operons
(22). Identification of thnC, coding for an
extradiol dioxygenase, and characterization of the reaction catalyzed
by its gene product indicated that biodegradation of tetralin by strain
TFA also involves an initial metabolization of the aromatic ring by a
meta-cleavage catabolic pathway (2). However, a
complete biodegradation pathway has not yet been established.
An important step in meta-cleavage pathways is the
hydrolysis of the ring fission product. Substrate specificity of
hydrolases catalyzing this step may be a key determinant of the
selectivity of the pathway with respect to the degradation of various
aromatic compounds (13, 14). These enzymes belong to the
group of 
-ketolases (EC 3.7.1.-), which hydrolyze carbon-carbon
bonds of -diketones. C-C bond hydrolytic cleavage has been
considered a relatively rare enzymatic reaction type and has been
little studied in spite of its potential in organic synthesis
(27). However, characterization of catabolic
meta-cleavage pathways of different aromatic compounds in
the last years has led to the identification of an increasing number of
hydrolases catalyzing C-C bond cleavage in different gram-positive and
gram-negative bacterial species. Some of them have been purified and
characterized (3, 5, 8, 10, 13, 18, 25, 32).
Hydrolases involved in catabolic meta-cleavage pathways of
aromatic compounds are a class of -ketolases (27) which
hydrolyze linear C-C bonds of vinylogous 1,5-diketones formed by the
dioxygenative meta-cleavage of activated arenes. This
results in production of a vinylpyruvate and a carboxylate. Sequence
comparisons of some of these hydrolases indicate that they are members
of the 
/-hydrolase fold superfamily of enzymes (8,
26), which contain a catalytic triad with the configuration
nucleophile-acid-histidine in order of amino acid sequence. In
vinylogous -ketolases and other hydrolytic enzymes, the catalytic
triad serine-aspartate-histidine is well conserved, and mutagenesis
studies on XylF, the hydrolase of the TOL pathway, have shown that
these residues are critical for catalysis (8).
We report here the sequence analysis of a gene designated
thnD, which codes for a hydrolase involved in tetralin
biodegradation by S. macrogoltabidus strain TFA; the
overproduction, purification, and characterization of ThnD; and the
identification of the reaction product. Unlike all other known
hydrolases involved in meta-cleavage pathways of aromatic
compounds, which cleave linear C-C bonds, ThnD hydrolyzes a C-C bond
comprised in the alicyclic ring of the tetralin derivative, thus
yielding a single dicarboxylate product.
| |
MATERIALS AND METHODS |
Bacterial strains, plasmids, and growth conditions.
Escherichia coli DH5 (17) was used for cloning
and isolation of DNA for sequencing. NCM631/pIZ227 (16), a
strain producing the T7 RNA polymerase, was used to overproduce ThnD.
E. coli strains were routinely grown in Luria-Bertani
medium. Strain TFA and its mutant derivative K6 (22) were
grown in mineral medium (9) with tetralin in the vapor phase
and -hydroxybutyrate (1 g liter
1) as the carbon and
energy source.
Plasmids pIZ608, pIZ591, and pIZ578 have been described elsewhere
(2, 22). A 1,349-bp StuI-ClaI fragment
from pIZ608 was cloned into pBluescript II KS(+) (Stratagene) and
commercially sequenced (Boehringer Mannheim). The same
StuI-ClaI fragment was cloned into pIZ578
linearized with SmaI and ClaI to construct pIZ670. To construct pIZ671, a 104-bp deletion was generated in pIZ670
by oligonucleotide site-directed mutagenesis, as previously described
(23), using the oligonucleotide 5'GAAGGTCGTAGTGAAGC3'. The deletion of 104 bp was designed to join the second codon of thnD to the frame coding for a His10 tag located upstream.
Overexpression, purification, and electrophoretic
conditions.
For overexpression of thnD, E. coli NCM631/pIZ227 was transformed with pIZ670 or pIZ671. The
resulting transformants were grown in Luria-Bertani liquid medium at
26°C to reach an optical density at 600 nm of 0.7. They were then
induced with 1 mM isopropyl--D-thiogalactopyranoside (IPTG) overnight (10 to 12 h). Cells were harvested by
centrifugation, frozen in liquid nitrogen, broken with aluminum oxide
90 (Merck), and suspended in 0.01 volume of 20 mM Tris-HCl (pH 8.0)
plus 100 mM NaCl. Purification of the His-tagged ThnD was performed by affinity chromatography with a Co2+-bound resin, following
the instructions of the TALON metal affinity resin user manual
(Clontech Laboratories, Inc.). Imidazole (0.2 M) was used to elute the
protein. Sample preparation and sodium dodecyl sulfate-polyacrylamide
gel electrophoresis were performed essentially as described
(24). Gels were stained with GELCODE Blue stain reagent (Pierce).
Chemicals.
Dihydroxytetralin (DHT) was chemically
synthesized (2). Catechol, 3-methylcatechol,
4-methylcatechol, and 2,3-dihydroxybiphenyl were purchased from Aldrich
at the highest purity available. The corresponding ring fission
products were synthesized biologically by whole cells of
NCM631/pIZ227,pIZ591, which overproduce the broad-substrate-specificity
extradiol dioxygenase ThnC (2).
Activity assays.
One unit of enzyme activity was defined as
the amount of enzyme that converts 1 µmol of substrate per min.
Hydrolase activity towards the different ring fission products, which
are yellow compounds, was determined in 20 mM
Na2HPO4-KH2PO4 buffer
(pH 7.2), by measuring substrate consumed at 65°C. The extinction
coefficients used for the following substrates were
2-hydroxy-4-(2-oxocyclohexyl)-buta-2,4-dienoic acid (OCHBDA),
max = 336 nm, 
= 12.26 mM1 cm1 (2);
2-hydroxy-6-oxohexa-2,4-dienoic acid (HODA), max = 375 nm, = 36 mM1 cm1;
2-hydroxy-6-oxohepta-2,4-dienoic acid (6-methyl-HODA),
max = 388 nm, = 13.8 mM1
cm1; 2-hydroxy-5-methyl-6-oxohexa-2,4-dienoic acid
(5-methyl-HODA), max = 382 nm, = 28.1 mM1 cm1; and
2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (6-phenyl-HODA), max = 434 nm, = 13.2 mM1 cm1 (20). Protein
concentration was determined by the method of Bradford (4),
with bovine serum albumin as the standard. All assays were quantified
using a Beckman DU 640 spectrophotometer equipped with a thermojacketed
cuvette holder.
Molecular weight determination.
The relative molecular
weight of the native enzyme was determined by gel filtration through a
Pharmacia Biotech Sephacryl S-300 HR column (15-ml bed volume)
calibrated with horse pancreas ferritine (Mr,
440,000), bovine liver catalase (Mr, 232,000), rabbit muscle aldolase (Mr, 158,000), and
ovalbumin (Mr, 45,000) as reference proteins.
Samples of 60 µl were loaded to the column, which was equilibrated
with buffer containing 50 mM Tris-HCl (pH 8.0) and 100 mM NaCl. Protein
elution from the column was in the same buffer at a flow rate of 0.4 ml
min1. Fractions of 225 µl were collected and assayed
for hydrolase activity.
Identification of intermediates.
The products from the
hydrolase reaction were separated and detected by high-pressure liquid
chromatography (HP 1100 series; Hewlett-Packard, Waldbronn, Germany)
equipped with a diode array detector, using a reversed-phase column
(ODS Hypersil 5 µm, 250 by 2 mm; Hewlett-Packard).
To identify the hydrolase reaction product in the tetralin pathway,
induced whole cells of NCM631/pIZ227,pIZ591 were used
to produce high
amounts of the yellow compound from DHT. Cells
were removed by
centrifugation, and purified hydrolase was added
to the supernatant.
When the yellow compound turned over, the
supernatant was acidified to
pH 2, and the hydrolase product was
extracted with Bakerbond spe
extraction columns (Mallinckrodt
Baker B.V.), following the supplier's
instructions. The product
was eluted with 1/7.5 sample volume of
methanol. The solvent was
subsequently changed to dichloromethane in
order to just methylate
the carboxyl groups. The solution was
methylated with diazomethane
(
6). A sample of the methylated
product was concentrated to
dryness under a stream of N
2.
The residue was dissolved in methanol
and blown for 30 min with
hydrogen using PtO
2 as the catalyst
or trimethylsilylated
with
N,
O-bis-trimethylsilylfluoroacetamide
(
28). Afterwards the solution was filtered, concentrated
under
nitrogen and injected directly in the gas chromatography-mass
spectrometry (GC-MS) system. The resulting products were analyzed
by
GC-MS using a Fisons mass selective detector (MD800; VG Analytical,
Manchester, United Kingdom) with a DB-5 MS fused silica column
(inner
diameter, 30 m by 0.25 mm; 0.25-µm film thickness; J&W
Scientific, Folsom, Calif.). The column program temperature used
was
150°C (5-min isothermal) to 240°C (final hold, 15 min) at
4°C/min. The injector was 250°C, and the transfer line temperature
was 250°C. The carrier gas (helium) flow rate was 1 ml
min
1. The end of column was inserted directly into the
ion source
block. The mass spectra were generated in EI+ (electron
ionization
positive mode) at 70
eV.
Sequence analysis comparison.
The resulting sequence of
1,346 bp was initially compared to those in the databases using the
BLASTp and tBLASTn programs (1). Sequences which showed high
similarity to that of strain TFA were aligned using the ClustalX
program (37) with default parameters. A distance matrix and
a phylogenetic tree were constructed using the neighbor-joining method
(30) and visualized with the TreeView program.
Nucleotide sequence accession number.
The nucleotide
sequence reported here has been submitted to the DDBJ, EMBL, and
GenBank nucleotide sequence databases under accession no. AF204963.
| |
RESULTS AND DISCUSSION |
Cloning and sequencing of thnD and sequence analysis of
hydrolases.
A collection of nonpolar KIXX insertion mutants of
strain TFA unable to grow on tetralin was previously constructed
(22). When grown in the presence of -hydroxybutyrate plus
tetralin, mutant K6 produced a yellow pigment with two absorption peaks at 336 nm and 417 nm, which shifted depending on the pH. Absorption spectra of the accumulated compound at different pHs (data not shown)
matched those of OCHBDA, the identified product of the ring fission
reaction catalyzed by the extradiol dioxygenase ThnC (2).
This result suggested that in the mutant K6, the catabolic pathway of
tetralin was blocked in the step following meta-cleavage of
the aromatic ring. A 1,349-bp DNA fragment encompassing the insertion
site in the mutant K6 was subcloned from the plasmid pIZ608 and sequenced.
Translation of the nucleotide sequence in all possible reading frames
revealed the existence of a complete open reading frame
(ORF) of 284 amino acids followed by an ORF for which only a partial
sequence was
obtained (Fig.
1). Comparison of the
deduced amino
acid sequence of the complete ORF to those in the
databases revealed
that it was highly similar to hydrolases of
different
meta-cleavage
pathways of aromatic compounds.
Similarity was highest to BphD
encoded in the plasmid pNL1 of
Sphingomonas aromaticivorans F199
(
29) and to
EtbD from
Rhodococcus sp. strain RHA1 (
19), with
which it showed 53 and 51% identity, respectively. The incomplete
ORF
showed high similarity to hydratases of
meta-cleavage
pathways.
Therefore, the identified ORFs were designated
thnD and
thnE,
respectively (Fig.
1). Restriction
analysis of the sequence showed
that the KIXX cassette was inserted at
the second codon of
thnD in the mutant K6. Direction of
transcription of
thnD and
thnE is opposite to
that of the previously identified gene
thnC (
2)
(Fig.
1), thus confirming the existence of two divergent operons
involved in tetralin biodegradation. This was previously inferred
by
complementation analysis of mutants (
22).
View larger version (22K):
[in this window]
[in a new window]
|
FIG. 1.
Representation of the genomic region of strain TFA
involved in tetralin biodegradation, showing the divergent
thnC and thnD genes. Arrows represent divergent
operons. Triangles represent locations of KIXX insertions in mutants
unable to grow on tetralin as the only carbon source. aa, amino
acids.
|
|
A dendrogram resulting from the comparison of amino acid sequences of
hydrolases involved in catabolic
meta-cleavage pathways
of
aromatic compounds and other hydrolases showing significant
similarity
to ThnD is shown in Fig.
2. The enzymes
have been divided
in four groups according to their sequence similarity
relationships.
Except MhpC, all hydrolases of group I are involved in
biodegradation
of bicyclic molecules such as biphenyls, carbazole, or
tetralin.
On the other hand, most of the enzymes of group III are
involved
in biodegradation of monocyclic compounds, though DxnB, CarD
PSOM1,
and CarC PSCA10, comprising the highly divergent branch of group
III, are part of catabolic pathways of bicyclic molecules. Group
II is
represented by a single hydrolase involved in biodegradation
of
3-hydroxyphenylpropionic acid. Except CmtE, which is a C-C
bond
hydrolase, the heterogeneous group IV is composed of carbon-heteroatom
hydrolases such as carboxylesterases (represented by Nap),
enol-lactone
hydrolases involved in catabolic
ortho-cleavage
pathways (CatD
and PcaD), epoxide hydrolases (Eph), and the haloalkane
dehalogenase
DhlA. Except DhlA and epoxide hydrolases, all other
enzymes in
Fig.
2 are serine hydrolases since the catalytic triad
serine-aspartate-histidine
is conserved.
View larger version (38K):
[in this window]
[in a new window]
|
FIG. 2.
Dendrogram showing the best tree obtained by the
neighbor-joining method, from the alignment of 39 sequences showing
significant similarity to ThnD. Scale represents distance expressed as
percentage of divergence. The ThnD sequence is boxed. GenBank accession
numbers for the following other hydrolases are given parenthetically:
BphD RGA1 (D78322), BphD SPpNL1 (AF079317), CarD SPCB3 (AF060489), MhpC
ECCS520 (Y09555), PcbD PSDJ12 (D44550), HbpD PSHBP1 (U73900), BphD
PSKKS102 (M26433), BphD CTB356 (L34338), BphD PSKF715 (M33813), BphD
PSKF707 (D85851), BphD PSLB400 (X66123), BphD2 RERTA421 (AB014348),
BpdF RGM5 (U44891), BphD RERTA421 (D88016), HppC RGPWD1 (U89712), CumD
PSIP01 (D83955), IpbD PSRE204 (AF006691), TecF BUPS12 (U78099), TodF
PSF1 (Y18245), McbF RAJS705 (AJ006307), AtdD ACYAA (AB008831), DmpD
PSCF600 (X52805), NahN PSAN10 (AF039534), XylF PSpWW0 (M64747), EtbD1
RGA1 (AB004320), EtbD2 RGA1 (AB004321), XylF SPpNL1 (AF079317), CmpF
SPHV3 (Z84817), PhnD PSDJ77 (U83881), DxnB SPRW1 (X72850), CarD PSOM1
(AB001723), CarC PSCA10 (D89064), CmtE PSF1 (U24215), Nap BS168
(AB001488), CatD ACADP1 (AF009224), PcaD ACADP1 (L05770), Eph COC12
(AJ224332), EphX2 HS (L05779), DhlA XAGJ10 (M26950).
|
|
In general, sequence divergences of hydrolases of
meta-cleavage pathways were higher than that of the
corresponding extradiol
dioxygenases (
11). On the other
hand, as for extradiol dioxygenases,
sequences of hydrolases involved
in degradation of bicyclic compounds
and those involved in degradation
of monoaromatic compounds tend
to cluster together in two different
groups (I and III in Fig.
2). Therefore, sequence similarity
relationships among hydrolases
also reflect substrate specificity.
However, there are notable
exceptions, such as MhpC and HppC; CumD and
CmtE; and CarC, CarDPSOM1,
and CarDPSCB3. Hydrolases within each set of
enzymes hydrolyze
the same substrate, but their sequences show very low
similarity
and cluster in different groups (Fig.
2), which suggests
functional
convergence of some hydrolases. This and the overall high
divergence
found among these hydrolases are consistent with the view
that
members of the
/
-hydrolase fold family do not have to share
significant primary structure similarity in spite of having a
similar
mechanism of reaction (
26).
Comparison of ThnD to sequences in the databases did not show
similarity to other C-C bond hydrolases cleaving 1,3-diketones,
such as
fumarylacetoacetate hydrolases or kynureninases (
27).
Alignment of these sequences together with those in Fig.
2 showed
an
overall divergence of 80 to 95%. Therefore, C-C bond hydrolases
cleaving vinylogous 1,5-diketones are more related in sequence
to
C-heteroatom hydrolases (group IV) than to C-C bond hydrolases
cleaving
1,3-diketones, in spite of the latter being ascribed
to the same
biochemical group (EC 3.7.1.-).
Overproduction and purification of ThnD.
Involvement of ThnD
in degradation of tetralin was shown by the Thn phenotype
of the mutant K6 (22), which bears a nonpolar KIXX insertion
in thnD. The sequence of ThnD and the identification of
OCHBDA as the intermediate accumulated in the mutant K6 suggested that
ThnD catalyzes hydrolysis of the ring fission product of DHT. However,
to confirm that thnD codes for a hydrolase catalyzing the
step following aromatic ring cleavage, the catalysis reaction had to be
characterized. In order to identify, overproduce, and purify the
thnD gene product in a single step for subsequent analysis, the E. coli strain NCM631/pIZ227 was transformed with
plasmid pIZ670 or pIZ671. pIZ670 drives production of native ThnD,
while pIZ671 should drive production of an His10-tagged
ThnD, which also contains the peptide signal for the protease factor Xa.
The overproducing strain NCM631/pIZ227 bearing pIZ670 accumulated a
protein with an apparent molecular mass of 32 kDa (not
shown),
consistent with the molecular weight deduced from its
coding sequence
(32,040). When bearing the plasmid pIZ671, the
overproducing strain
accumulated to significantly higher amounts
a product of 35 kDa,
consistent with the predicted molecular weight
of the His-tagged
protein (34,293). Approximately half of the
His-tagged protein was
soluble and could be purified by affinity
chromatography with a
cobalt-bound resin (data not shown). Attempts
to remove the N-terminal
tail of the purified His-tagged protein
with the factor Xa were
unsuccessful, thus suggesting that its
signal sequence is occluded in
the native conformation of this
protein. Therefore, the His-tagged form
was used for subsequent
characterization.
Using both the
E. coli crude extract enriched in
native ThnD and the purified His-tagged ThnD, a spectrophotometric
hydrolase
activity assay was set, based on consumption of OCHBDA, which
was biologically produced from DHT by ThnC (
2).
Biochemical properties of ThnD.
Enzyme activity assays using
purified ThnD were run at different temperatures to estimate its
optimum reaction temperature. The rate of enzyme-dependent substrate
consumption increased with increasing temperature until a maximum
was reached at 65°C (not shown). Stability of the hydrolase
activity was also tested by incubating His-tagged ThnD at room
temperature and at 70°C and measuring activity at different times.
Activity of the enzyme was fully stable for 8 h at room
temperature, and heat treatment at 70°C for 2 h resulted in 78%
of the initial activity (not shown). These results indicate that ThnD
is a thermostable hydrolase efficiently working at high temperatures.
This is an interesting characteristic of ThnD given the potential
interest of this class of enzymes in organic synthesis (27).
Subunit composition of purified His-tagged ThnD was estimated by
calibrated gel filtration chromatography. The elution volume
of maximum
hydrolase activity corresponded to a size of 273 ±
9 kDa (not
shown), which indicated that active His-tagged ThnD
consists of eight
subunits. Oligomeric structure of hydrolases
involved in
meta-cleavage pathways appears to be variable, since
monomers (
5), dimers (
18,
25), tetramers
(
13,
32) and
octamers (
18) have been previously
described. Although the octameric
structure has been shown for the
His-tagged protein, this may
be the likely structure of native ThnD
since BphD from
Rhodococcus sp. strain RHA1, which showed
high similarity to ThnD and an optimum
reaction temperature at 65°C,
is also an octamer (
18).
Purified His-tagged ThnD was found to obey the classical
Michaelis-Menten kinetic. Assuming an octameric structure, the kinetic
parameters calculated for the His-tagged protein at its optimum
temperature were as follows: turnover number
(
kcat), 92.1 s
1;
Km, 26.3 µM; and
kcat/
Km, 35.23 × 10
5 M
1 s
1. Activity assays
using crude extracts enriched in native ThnD
showed that
Km for OCHBDA of native or His-tagged ThnD
was
the same, thus suggesting that the His tag does not dramatically
affect
ThnD
activity.
The capacity of ThnD to hydrolyze other ring fission products of
catabolic
meta-cleavage pathways of monocyclic or bicyclic
aromatic compounds was also tested. Consistently with its clustering
with BphD enzymes (Fig.
2), ThnD also efficiently hydrolyzed
6-phenyl-HODA
(Table
1), the ring fission product of
2,3-dihydroxybiphenyl,
which has a large substituent at C-6. Kinetic
parameters of the
reaction using this substrate were similar to those
shown using
OCHBDA, thus suggesting that ThnD could be proficient in
biphenyl
meta-cleavage pathways. Benzoate was identified as
a reaction
product of the hydrolysis of 6-phenyl-HODA (not shown), thus
suggesting
that the hydrolysis mechanism of ThnD is similar to that
previously
described for BphD (
32) or MhpC (
21)
hydrolases. ThnD very
poorly hydrolyzed 6-methyl-HODA, the ring fission
product of 3-methylcatechol,
consistent with the view that the size of
the substituent at C-6
is important for substrate affinity
(
32). The aldehydes HODA
and 5-methyl-HODA, the ring fission
products of catechol and 4-methylcatechol,
respectively, were
hydrolyzed even less
efficiently.
Identification of the hydrolysis product in the tetralin
pathway.
The hydrolysis reaction product(s) of the tetralin
pathway was methylated in order to protect the carboxylic groups. GC-MS of the methylated sample showed a major mass peak eluting at 16.75 min.
The mass spectrum taken at the top of the peak is shown in Fig.
3A. A weak ion at m/z 242 was
thought of as the molecular ion, whose molecular mass matches that of
the dimethyl ester of 2-hydroxydeca-2,4-dienedioic acid. A set of even
sequential fragments at m/z 210, 150, 122, and 94 shows
successive neutral losses of CH3OH, HCOOCH3,
CO, and CO, respectively, from the molecular ion and provides evidence
of two carboxymethylated groups and an additional C-O bond in the
molecule. Another set of odd key fragments at m/z 183, 151, and 123 corresponds to sequential elimination of CH3COO, CH3OH, and CO, respectively. Subsequent
ketene (CH2==CO) elimination gives the m/z 81 ion as the base peak of the spectrum. Loss of water at m/z
224 was not observed as could be expected if a hydroxyl group were
present. This process occurs predominantly by 1-4 elimination through a
six-membered intermediate. Absence of this ion may be interpreted as a
consequence of the high stability of conjugated double bonds in C-2 to
C-4.
View larger version (16K):
[in this window]
[in a new window]
|
FIG. 3.
Mass spectra of the product of hydrolysis modified by
methylation (A), methylation and trimethylsilylation (B), or
methylation and hydrogenation (C).
|
|
The presence of a hydroxyl group was confirmed by analyzing an aliquot
of the methylated sample which had been additionally
trimethylsilylated. The major peak eluted at 20.36 min and showed
a
mass spectrum (Fig.
3B) in which the molecular mass of the resulting
adduct appeared at
m/z 314. The ion at
m/z 299 evidenced the loss
of one methyl from the trimethylsilyl group, which
is typical
of trimethylsilylated compounds. The set of key fragment
ions
at
m/z 267 and 235 shows the sequential double
elimination of
CH
3OH from the
m/z 299 ion, while
m/z 193 represents the loss
of CH
3OH and the
trimethylsilyl group from this ion. The
m/z 199
ion
represents the cleavage at C-5-C-6 of the molecule containing
a double
bond in C-4-C-5. Subsequent elimination of trimethylsilyl
or methanol
from the last ion gives the
m/z 167 or 109, respectively.
Evidence for the presence of the trimethylsilyl group was provided
by
the couple of fragments at
m/z 73 and
m/z 75.
To ascertain the presence of unsaturated double bonds, a second aliquot
of the methylated sample was hydrogenated. The mass
chromatogram showed
a major peak eluting at 19.96 min whose mass
spectrum is shown in Fig.
3C. Instead of the molecular ion at
expected
m/z 246, the
highest ion found was at
m/z 228, which
should be
interpreted as a dehydration process suffered by the
alcohol prior to
being ionized in the ion source, due to a loss
of charge stabilization
around the conjugated double bonds once
they were eliminated from the
original compound. The
m/z 200 ion
is an ethylene expulsion,
which is frequently coupled to water
elimination. The spectrum of the
hydrogenated compound showed
a typical fragmentation pattern of methyl
esters, with a strong
ion at
m/z 74 (MacLafferty
rearrangement) as base peak of the
spectrum and the set of ions at
m/z 87, 101, 115, 129, 143, etc.,
from the linear
chain.
Taken together, these data indicate that hydrolysis of the ring fission
product in the tetralin pathway results in a single
dicarboxylic
product with a molecular weight of 214, which contains
one hydroxyl
group and two double bonds, one of which should be
in C-4. Since
hydrolysis of ring fission products in other biodegradation
pathways
involves cleavage of a linear C-C bond, thus releasing
two hydrolytic
products, it is obvious that the hydrolysis reaction
of OCHBDA
catalyzed by ThnD results in a significantly different
product.
However, this is consistent with the hydrolysis of the
C-5-C-6 bond of
the ring fission product described for other hydrolases.
Since C-5 and
C-6 of the vinylogous
-diketone in OCHBDA are part
of the alicyclic
ring (Table
1), hydrolysis of this
substrate
results in alicyclic ring opening, thus releasing a single
linear
compound instead of two hydrolytic products.
The ability of ThnD to cleave the alicyclic ring of tetralin has strong
implications for its catabolism, thus conferring unique
characteristics
on this pathway. In catabolic pathways of bicyclic
molecules such as
biphenyl or naphthalene, the two rings are degraded
at two distinct
stages, each requiring a complete set of enzymes
(
39). In
the tetralin pathway, the aromatic and the alicyclic
rings are opened
in two subsequent steps by an extradiol dioxygenase
and a hydrolase
activity (Fig.
4). Further
catabolism of the resulting
product catalyzed by potential
hydratase, aldolase, and aldehyde
dehydrogenase
activities, which are common activities in degradation
pathways of
aromatic compounds, would yield pyruvate and the dicarboxylic
acid
pimelate, which could then enter the general metabolic pathways
of the
bacteria. Although these activities have not yet been described
in the
strain TFA, the gene immediately downstream of
thnD
potentially
codes for a hydratase (Fig.
1). In turn, cleavage of the
alicyclic
ring of tetralin by ThnD makes unnecessary for mineralization
of tetralin the use of enzymatic activities involved in degradation
of
alicyclic rings. Thus, it appears that one set of enzymes typically
involved in degradation of one aromatic ring is sufficient to
degrade
both the aromatic and the alicyclic ring of tetralin.
| |
ACKNOWLEDGMENTS |
This work was supported by the Spanish Comisión
Interministerial de Ciencia y Tecnología, grant BIO96-0908; by
the European Union under the ENVIRONMENT Program, contract
EV5V-CT92-0192; and by fellowships of the Spanish Ministerio de
Educación to M.J.H. and to E.A.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Departamento de
Genética, Facultad de Biología, Universidad de Sevilla,
Ap. 1095, 41080 Seville, Spain. Phone: 34-95-4557106. Fax:
34-95-4557104. E-mail: esantero{at}cica.es.
| |
REFERENCES |
| 1.
|
Altschul, S. F.,
T. L. Madden,
A. A. Schäffer,
J. Zhang,
Z. Zhang,
W. Miller, and D. J. Lipman.
1997.
Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.
Nucleic Acids Res.
25:3389-3402[Abstract/Free Full Text].
|
| 2.
|
Andújar, E.,
M. J. Hernáez,
S. R. Kaschabek,
W. Reineke, and E. Santero.
2000.
Identification of an extradiol dioxygenase involved in tetralin biodegradation: gene sequence analysis, purification and characterization of the gene product.
J. Bacteriol.
182:789-795[Abstract/Free Full Text].
|
| 3.
|
Bayly, R. C., and D. di Berardino.
1978.
Purification and properties of 2-hydroxy-6-oxo-2,4-heptadienoate hydrolase from two strains of Pseudomonas putida.
J. Bacteriol.
134:30-37[Abstract/Free Full Text].
|
| 4.
|
Bradford, M. M.
1976.
A rapid and sensitive method for the quantitation of protein utilizing the principle of protein dye binding.
Anal. Biochem.
72:248-252[CrossRef][Medline].
|
| 5.
|
Bünz, P. V.,
R. Falchetto, and A. M. Cook.
1993.
Purification of two isofunctional hydrolases (EC 3.7.1.8) in the degradative pathway for dibenzofuran in Sphingomonas sp. strain RW1.
Biodegradation
4:171-178[CrossRef][Medline].
|
| 6.
|
Cohen, J. D.
1984.
Convenient apparatus for the generation of small amounts of diazomethane.
J. Chromatogr.
303:193[CrossRef].
|
| 7.
|
Dagley, S.,
W. C. Evans, and D. W. Ribbons.
1960.
New pathways in the oxidative metabolism of aromatic compounds by microorganisms.
Nature (London)
188:560-566[CrossRef][Medline].
|
| 8.
|
Díaz, E., and K. N. Timmis.
1995.
Identification of functional residues in a 2-hydroxymuconic semialdehyde hydrolase. A new member of the / hydrolase-fold family of enzymes which cleaves carbon-carbon bonds.
J. Biol. Chem.
270:6403-6411[Abstract/Free Full Text].
|
| 9.
|
Dorn, E.,
M. Hellwig,
W. Reineke, and H.-J. Knackmuss.
1974.
Isolation and characterization of a 3-chlorobenzoate degrading pseudomonad.
Arch. Microbiol.
99:61-70[CrossRef][Medline].
|
| 10.
|
Duggleby, C. J., and P. Williams.
1986.
Purification and properties of the 2-hydroxy-6-oxohepta-2,4-dienoate hydrolase (2-hydroxymuconic semialdehyde hydrolase) encoded by the TOL plasmid pWW0 from Pseudomonas putida mt-2.
J. Gen. Microbiol.
132:717-726.
|
| 11.
|
Eltis, L., and J. Bolin.
1996.
Evolutionary relationships among extradiol dioxygenases.
J. Bacteriol.
178:5930-5937[Abstract/Free Full Text].
|
| 12.
|
Ferrante, A. A.,
J. Augliera,
K. Lewis, and A. M. Klibanov.
1995.
Cloning of an organic solvent-resistance gene in Escherichia coli: the unexpected role of alkylhydroperoxide reductase.
Proc. Natl. Acad. Sci. USA
92:7617-7621[Abstract/Free Full Text].
|
| 13.
|
Furukawa, K.,
J. Hirose,
A. Suyama,
T. Zaiki, and S. Hayashida.
1993.
Gene components responsible for discrete substrate specificity in the metabolism of biphenyl (bph operon) and toluene (tod operon).
J. Bacteriol.
175:5224-5232[Abstract/Free Full Text].
|
| 14.
|
Furukawa, K.,
N. Tomizuka, and A. Kamibayashi.
1979.
Effect of chlorine substitution on the bacterial metabolism of various polychlorinated biphenyls.
Appl. Environ. Microbiol.
38:301-310[Abstract/Free Full Text].
|
| 15.
|
Gaydos, R. M.
1981.
Naphthalene, p. 698-719.
In
M. Grayson, and D. Eckroth (ed.), Kirk-Othmer encyclopedia of chemical technology, 3rd ed. John Wiley & Sons, Inc., New York, N.Y.
|
| 16.
|
Govantes, F.,
J. A. Molina-López, and E. Santero.
1996.
Mechanism of coordinated synthesis of the antagonistic regulatory proteins NifL and NifA of Klebsiella pneumoniae.
J. Bacteriol.
178:6817-6823[Abstract/Free Full Text].
|
| 17.
|
Hanahan, D.
1983.
Studies on transformation of Escherichia coli with plasmids.
J. Mol. Biol.
166:557-580[Medline].
|
| 18.
|
Hatta, T.,
T. Shimada,
T. Yoshihara,
A. Yamada,
E. Masai,
M. Fukuda, and H. Kiyohara.
1998.
Meta-fission product hydrolases from a strong PCB degrader Rhodococcus sp. RHA1.
J. Ferment. Bioeng.
85:174-179[CrossRef].
|
| 19.
|
Hauschild, J. E.,
E. Masai,
K. Sugiyama,
T. Hatta,
K. Kimbara,
M. Fukuda, and K. Yano.
1996.
Identification of an alternative 2,3-dihydroxybiphenyl 1,2-dioxygenase in Rhodococcus sp. strain RHA1 and cloning of the gene.
Appl. Environ. Microbiol.
62:2940-2946[Abstract].
|
| 20.
|
Heiss, G.,
A. Stolz,
A. E. Kuhm,
C. Müller,
J. Klein,
J. Altenbuchner, and H.-J. Knackmuss.
1995.
Characterization of a 2,3-dihydroxybiphenyl dioxygenase from the naphthalenesulfonate-degrading bacterium strain BN6.
J. Bacteriol.
177:5865-5871[Abstract/Free Full Text].
|
| 21.
|
Henderson, I. M., and T. D. Bugg.
1997.
Pre-steady-state kinetic analysis of 2-hydroxy-6-keto-nona-2,4-diene-1,9-dioic acid 5,6-hydrolase: kinetic evidence for enol/keto tautomerization.
Biochemistry
36:12252-12258[CrossRef][Medline].
|
| 22.
|
Hernáez, M. J.,
W. Reineke, and E. Santero.
1999.
Genetic analysis of biodegradation of tetralin by a Sphingomonas strain.
Appl. Environ. Microbiol.
65:1806-1810[Abstract/Free Full Text].
|
| 23.
|
Kunkel, T. A.
1985.
Rapid and efficient site-specific mutagenesis without phenotypic selection.
Proc. Natl. Acad. Sci. USA
82:488-492[Abstract/Free Full Text].
|
| 24.
|
Laemmli, U. K.
1970.
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227:680-685[CrossRef][Medline].
|
| 25.
|
Lam, W. W., and T. D. Bugg.
1997.
Purification, characterization, and stereochemical analysis of a C-C hydrolase: 2-hydroxy-6-keto-nona-2,4-diene-1,9-dioic acid 5,6-hydrolase.
Biochemistry
36:12242-12251[CrossRef][Medline].
|
| 26.
|
Ollis, D. L.,
E. Cheah,
M. Cygler,
B. Dijkstra,
F. Frolow,
S. M. Franken,
M. Harel,
S. J. Remington,
I. Silman,
J. Schrag,
J. L. Sussman,
K. H. Verschueren, and A. Goldman.
1992.
The alpha/beta hydrolase fold.
Protein Eng.
5:197-211[Abstract/Free Full Text].
|
| 27.
|
Pokorny, D.,
W. Steiner, and D. M. Ribbons.
1997.
-Ketolases forgotten hydrolytic enzymes?
Trends Biotechnol.
15:291-286.
|
| 28.
|
Poole, C. F.
1978.
Recent advances in the silylation of organic compounds for gas chromatography, p. 152-200.
In
K. Blau, and G. S. King (ed.), Handbook of derivatives for chromatography. Heyden, London, United Kingdom.
|
| 29.
|
Romine, M.,
L. Stillwell,
K.-K. Wong,
S. Thurston,
E. Sisk,
C. Sensen,
T. Gaasterland,
J. Fredrickson, and J. Saffer.
1999.
Complete sequence of a 184-kilobase catabolic plasmid from Sphingomonas aromaticivorans F199.
J. Bacteriol.
181:1585-1602[Abstract/Free Full Text].
|
| 30.
|
Saitou, N., and M. Nei.
1987.
The neighbor-joining method: a new method for reconstructing phylogenetic trees.
Mol. Biol. Evol.
4:406-425[Abstract].
|
| 31.
|
Schreiber, A. F., and U. K. Winkler.
1983.
Transformation of tetralin by whole cells of Pseudomonas stutzeri AS 39.
Eur. J. Appl. Microbiol. Biotechnol.
18:6-10.
|
| 32.
|
Seah, S. T.,
G. Terracina,
J. T. Bolin,
P. Riebel,
V. Snieckus, and L. Eltis.
1998.
Purification and preliminary characterization of a serine hydrolase involved in the microbial degradation of polychlorinated biphenyls.
J. Biol. Chem.
273:22943-22949[Abstract/Free Full Text].
|
| 33.
|
Sikkema, J., and J. A. M. de Bont.
1991.
Isolation and initial characterization of bacteria growing on tetralin.
Biodegradation
2:15-23.
|
| 34.
|
Sikkema, J., and J. A. M. de Bont.
1993.
Metabolism of tetralin (1,2,3,4-tetrahydronaphthalene) in Corynebacterium sp. strain C125.
Appl. Environ. Microbiol.
59:567-572[Abstract/Free Full Text].
|
| 35.
|
Sikkema, J.,
J. A. M. de Bont, and B. Poolman.
1994.
Interactions of cyclic hydrocarbons with biological membranes.
J. Biol. Chem.
269:8022-8028[Abstract/Free Full Text].
|
| 36.
|
Sikkema, J.,
B. Poolman,
W. N. Konings, and J. A. M. de Bont.
1992.
Effects of the membrane action of tetralin on the functional and structural properties of artificial and bacterial membranes.
J. Bacteriol.
174:2986-2992[Abstract/Free Full Text].
|
| 37.
|
Thompson, J. D.,
T. J. Gibson,
F. Plewniak,
F. Jeanmougin, and D. G. Higgins.
1997.
The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools.
Nucleic Acids Res.
25:4876-4882[Abstract/Free Full Text].
|
| 38.
|
Trudgill, P. W.
1984.
Microbial degradation of the alicyclic ring, p. 131-180.
In
D. T. Gibson (ed.), Microbial degradation of organic compounds. Marcel Dekker, New York, N.Y.
|
| 39.
|
Williams, P. A., and J. R. Sayers.
1994.
The evolution of pathways for aromatic hydrocarbon oxidation in Pseudomonas.
Biodegradation
5:195-217[CrossRef][Medline].
|
Journal of Bacteriology, October 2000, p. 5448-5453, Vol. 182, No. 19
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Lai, L., Xu, Z., Zhou, J., Lee, K.-D., Amidon, G. L.
(2008). Molecular Basis of Prodrug Activation by Human Valacyclovirase, an {alpha}-Amino Acid Ester Hydrolase. J. Biol. Chem.
283: 9318-9327
[Abstract]
[Full Text]
-
Seah, S. Y. K., Ke, J., Denis, G., Horsman, G. P., Fortin, P. D., Whiting, C. J., Eltis, L. D.
(2007). Characterization of a C--C Bond Hydrolase from Sphingomonas wittichii RW1 with Novel Specificities towards Polychlorinated Biphenyl Metabolites. J. Bacteriol.
189: 4038-4045
[Abstract]
[Full Text]
-
Martinez-Perez, O., Lopez-Sanchez, A., Reyes-Ramirez, F., Floriano, B., Santero, E.
(2007). Integrated Response to Inducers by Communication between a Catabolic Pathway and Its Regulatory System. J. Bacteriol.
189: 3768-3775
[Abstract]
[Full Text]
-
Mukerjee-Dhar, G., Shimura, M., Miyazawa, D., Kimbara, K., Hatta, T.
(2005). bph genes of the thermophilic PCB degrader, Bacillus sp. JF8: characterization of the divergent ring-hydroxylating dioxygenase and hydrolase genes upstream of the Mn-dependent BphC. Microbiology
151: 4139-4151
[Abstract]
[Full Text]
-
Martinez-Perez, O., Moreno-Ruiz, E., Floriano, B., Santero, E.
(2004). Regulation of Tetralin Biodegradation and Identification of Genes Essential for Expression of thn Operons. J. Bacteriol.
186: 6101-6109
[Abstract]
[Full Text]
-
Habe, H., Chung, J.-S., Kato, H., Ayabe, Y., Kasuga, K., Yoshida, T., Nojiri, H., Yamane, H., Omori, T.
(2004). Characterization of the Upper Pathway Genes for Fluorene Metabolism in Terrabacter sp. Strain DBF63. J. Bacteriol.
186: 5938-5944
[Abstract]
[Full Text]
-
Moreno-Ruiz, E., Hernaez, M. J., Martinez-Perez, O., Santero, E.
(2003). Identification and Functional Characterization of Sphingomonas macrogolitabida Strain TFA Genes Involved in the First Two Steps of the Tetralin Catabolic Pathway. J. Bacteriol.
185: 2026-2030
[Abstract]
[Full Text]
-
Hernaez, M. J., Floriano, B., Rios, J. J., Santero, E.
(2002). Identification of a Hydratase and a Class II Aldolase Involved in Biodegradation of the Organic Solvent Tetralin. Appl. Environ. Microbiol.
68: 4841-4846
[Abstract]
[Full Text]
-
Diaz, E., Ferrandez, A., Prieto, M. A., Garcia, J. L.
(2001). Biodegradation of Aromatic Compounds by Escherichia coli. Microbiol. Mol. Biol. Rev.
65: 523-569
[Abstract]
[Full Text]
Top
Abstract
Introduction
