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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.
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
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ABSTRACT |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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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.
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INTRODUCTION |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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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.
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MATERIALS AND METHODS |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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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.
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 N2. The residue was dissolved in methanol and blown for 30 min with hydrogen using PtO2 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 min1. 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.
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RESULTS AND DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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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.
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/-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.
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 s1;
Km, 26.3 µM; and
kcat/Km, 35.23 × 105 M1 s1. 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.
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-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.
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ACKNOWLEDGMENTS |
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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.
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FOOTNOTES |
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* 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.
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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