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Applied and Environmental Microbiology, June 2000, p. 2668-2672, Vol. 66, No. 6
0099-2240/00/$04.00+0
trans-o-Hydroxybenzylidenepyruvate
Hydratase-Aldolase as a Biocatalyst
Richard W.
Eaton*
Gulf Ecology Division, National Health and
Environmental Effects Research Laboratory, U.S. Environmental
Protection Agency, Gulf Breeze, Florida 32561
Received 7 February 2000/Accepted 31 March 2000
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ABSTRACT |
The hydratase-aldolase-catalyzed conversion of
trans-o-hydroxybenzylidenepyruvate to salicylaldehyde and
pyruvate is an intermediate reaction in the conversion of naphthalene
to salicylate by bacteria. Here, a variety of aromatic aldehydes and
some nonaromatic aldehydes together with pyruvate have been shown to be
substrates for aldol condensations catalyzed by this enzyme in extracts
of the recombinant strain Escherichia coli JM109(pRE701).
Some of the products of these reactions were also compared as
substrates in the opposite (hydration-aldol cleavage) reaction.
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TEXT |
Naphthalene and more complex
fused-ring polycyclic aromatic compounds are metabolized through
pathways that often include the formation and cleavage of a
4-substituted 2-ketobut-3-enoate intermediate. In the naphthalene
catabolic pathway, this intermediate is
trans-o-hydroxybenzylidenepyruvate (o-tHBPA),
which is converted by a hydratase-aldolase to salicylaldehyde and
pyruvate (3). The reaction catalyzed by the
hydratase-aldolase is reversible, and the enzyme may therefore be used
as a biocatalyst to form novel products by aldol condensation.
Previously, we proposed a (forward) reaction mechanism for the enzyme
in which removal of the proton from the ortho hydroxyl group
of o-tHBPA initiates rearrangements leading to hydration prior to aldol cleavage (Fig. 1A)
(3). This proposal was based on the observation that
benzylidenepyruvate was not a substrate for tHBPA
hydratase-aldolase, yet could be formed by the enzyme by condensation
of benzaldehyde and pyruvate. In the synthetic (reverse) direction, the
phenolic hydroxyl group apparently was not required, since the product
of aldol condensation (Fig. 1, compound I) could have been formed by
spontaneous dehydration of a 4-phenyl-4-hydroxy-2-ketobutyrate (Fig. 1,
compound IV) due to the greater stability of the highly conjugated
substituted benzylidenepyruvate.
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FIG. 1.
Possible involvement of aromatic hydroxyl groups in the
transformation of tHBPAs by tHBPA
hydratase-aldolase. (A) Previously proposed mechanism for the
metabolism of o-tHBPA (compound I), in which the initiating
step in hydration of the substrate is removal of the hydroxyl proton by
the enzyme leading to the formation of a quinonemethide-stabilized
carbanion intermediate (II). (B) Possible formation of a
quinonemethide-stabilized carbanion intermediate (VIII) during the
transformation of p-tHBPA (VII). (C) m-tHBPA (IX)
cannot form a quinonemethide intermediate similar to II and VIII.
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Over 30 additional aldehydes have been examined here as substrates for
tHBPA hydratase-aldolase. Several of these aldehydes were
used to generate products which were characterized and used as
substrates in the forward (hydration-aldol cleavage) reaction. The
results indicate that the enzyme accepts a broad range of aldehydes and
4-substituted 2-keto-but-3-enoates as substrates, that dehydration of
4-substituted 4-hydroxy-2-ketobutyrates is probably enzyme catalyzed,
and that the previously proposed enzyme reaction mechanism is not correct.
Source of o-tHBPA hydratase-aldolase.
The
bacterial strain Escherichia coli JM109, which carries the
tHBPA hydratase-aldolase-encoding plasmid pRE701, was
described previously (2, 3). The strain was grown and
induced with isopropyl-
-D-thiogalactoside, and crude
extracts were prepared and used without purification as described
previously (3).
Screening.
Screening of chemicals as substrates for the
hydratase-aldolase was carried out in a Perkin-Elmer Lambda 6 double-beam spectrophotometer (3). Both spectrophotometer
cuvettes contained 10 mM sodium pyruvate and 50 mM K-Na phosphate
buffer (pH 7) in 1 ml. The sample cuvette also contained 0.05 to 0.1 mM
aldehyde. A spectrum was recorded prior to addition of 3 to 10 µl of
extract (up to 200 µg of protein) and then at various times afterward
in order to document spectral changes occurring as the reaction
progressed. Reaction mixtures contained a large excess of pyruvate to
force the equilibrium in the direction of condensation. A variety of aldehydes were examined. All of these were obtained from Aldrich Chemical Co., except for 2-thiophenecarboxaldehyde (Fluka) and benzaldehyde (Fisher). Chemicals were considered to be substrates for
tHBPA hydratase-aldolase-catalyzed condensation with
pyruvate if the incubations caused the disappearance of the substrate
spectrum accompanied by the appearance of the spectrum of the product. Those aromatic aldehydes that are substrates include benzaldehyde, 4-biphenylcarboxaldehyde, 2-carboxybenzaldehyde, 2-chlorobenzaldehyde, 2,3-dihydroxybenzaldehyde, 2-formylbenzenesulfonate, 2-furaldehyde, 3-furaldehyde, 2-hydroxybenzaldehyde (salicylaldehyde),
3-hydroxybenzaldehyde, 4-hydroxybenzaldehyde,
1-hydroxy-2-naphthaldehyde, 2-hydroxy-1-naphthaldehyde, 2-hydroxy-5-nitrobenzaldehyde, indole-3-carboxaldehyde,
4-isopropylbenzaldehyde, 2-methoxybenzaldehyde
(o-anisaldehyde), 3-methoxysalicylaldehyde (o-vanillin), 1-methylindole-3-carboxaldehyde,
1-naphthaldehyde, 2-naphthaldehyde, 2-nitrobenzaldehyde,
phenanthrene-9-carboxaldehyde, phthalaldehyde,
2-pyridinecarboxaldehyde, 3-pyridinecarboxaldehyde, 2-quinolinecarboxaldehyde, 3-quinolinecarboxaldehyde,
4-quinolinecarboxaldehyde, 2-thiophenecarboxaldehyde,
3-thiophenecarboxaldehyde, o-tolualdehyde, and
p-tolualdehyde. By using the same criteria, two nonaromatic aldehydes, cyclohexanecarboxaldehyde and crotonaldehyde, were also
determined to be substrates for tHBPA hydratase-aldolase. Acetophenone, 2'-hydroxyacetophenone, phenylacetaldehyde, and trans-cinnamaldehyde are not substrates.
Preparative-scale aldol condensation reactions.
Products of
certain condensation reactions were prepared on a scale sufficient to
allow purification so that they could be characterized and examined as
substrates in the cleavage reaction. A typical preparative reaction
mixture contained, in a 400-ml beaker, 250 ml of 10 mM phosphate buffer
(pH 6.8), 10 mM aldehyde, and 20 mM pyruvate. A dialysis bag containing
10 ml of JM109(pRE701) extract (200 to 300 mg of protein) was floated
in this solution. The mixture was slowly stirred with a magnetic
stir-bar overnight at room temperature (20 to 24°C). Progress of the
reaction was monitored by recording changes in the UV-visible spectra
of the diluted reaction mixture. When the reaction was judged to be
complete, the dialysis bag was discarded, and the reaction mixture was
freeze-dried. The residue was then redissolved in a minimum volume of
water, and products were separated from residual starting materials by chromatography on Sephadex G-25 with water as the solvent
(3). Elution of chemicals was monitored by recording the
UV-visible spectra of diluted fractions with an HP8452A diode-array
spectrophotometer; peak fractions were pooled and freeze-dried.
Identification of products.
Analysis of condensation products
used gas chromatography-mass spectrometry (GC-MS) of trimethylsilyl
(TMS) derivatives prepared by using
N,O-bis (trimethylsilyl)-trifluoroacetamide
(BSTFA) containing 1% trimethylchlorosilane (TMCS) according to the
manufacturer (Pierce). GC-MS analyses were carried out with a
Hewlett-Packard model 5988A mass spectrometer coupled to a
Hewlett-Packard model 5890 gas chromatograph as previously described
(3, 4). Proton and 13C nuclear magnetic
resonance (NMR) spectra in d6-dimethyl sulfoxide were obtained with a General Electric model QE Plus spectrometer at 300 and 75 MHz, respectively.
GC-MS gave, for each product (except that from
1-hydroxy-2-naphthaldehyde [see below]), two major chromatographic
peaks with
mass spectra that are very similar to each other (Table
1). In
all cases, the molecular ions and
fragmentation patterns are indicative
of 4-substituted
2-ketobut-3-enoates formed by condensation of
pyruvate with an aldehyde
followed by dehydration of the 4-substituted
2-keto-4-hydroxybutyrate
(aldol) product.
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TABLE 1.
GC-MS data for TMS derivatives of products formed by
aldol condensation of pyruvate with various aldehydes catalyzed by
extracts of E. coli JM109(pRE701)
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Based on previous GC-MS analyses of TMS derivatives of
o-tHBPA and its isomer, 2-hydroxychromene-2-carboxylate
(
3), the
earlier-eluting GC peak in each GC-MS analysis is
likely to be
the
cis isomer, while the later, more abundant
peak is likely
to be the more extended
trans isomer
(
3). When 4-substituted
2-keto-3-butenoates have been
analyzed by both GC-MS and NMR spectroscopy
(
3,
4
[and see below]), the sole compound evident in the
NMR spectrum prior
to derivatization has been the
trans isomer,
some of which
may be converted to the
cis isomer during derivatization
in
preparation for GC-MS or in the GC
injector.
Is dehydration spontaneous or enzyme catalyzed?
Since
dehydration yields a relatively stable product having a double bond in
conjugation with a keto group and the aromatic ring, it could, in some
cases, occur spontaneously, as previously proposed (3), or
it could be enzyme catalyzed. Support for the latter comes from a
comparison of the products of condensation of pyridine-2-carboxaldehyde
and pyruvate by tHBPA hydratase-aldolase and another enzyme,
2-keto-3-deoxy-6-phosphogalactonate aldolase (KDPGal aldolase)
(5). While KDPGal aldolase produced
(R)-4-hydroxy-2-keto-4-(2'-pyridyl)butyrate (5),
tHBPA hydratase-aldolase yielded the dehydration product 2-keto-4-(2'-pyridyl)-but-3-enoate. Dehydration of 4-hydroxy-2-keto 4-(2'-pyridyl)butyrate is, therefore, probably not spontaneous but is
catalyzed by tHBPA hydratase-aldolase. By analogy,
dehydration of other 4-substituted 4-hydroxy-2-ketobutyrate
condensation products may also be catalyzed by tHBPA
hydratase-aldolase.
Because of the relevance of the products of condensation of pyruvate
with 3-hydroxybenzaldehyde and 4-hydroxybenzaldehyde
for determining
the reaction mechanism of
tHBPA hydratase-aldolase,
those
products were further characterized by NMR spectroscopy
(Fig.
2). Both
13C and
1H NMR spectra are consistent with identification of the
products
as the
trans isomers of
m-HBPA
(
m-tHBPA) and
p-HBPA (
p-tHBPA),
respectively.
p-tHBPA has two pairs of identical aromatic
protons,
while
m-tHBPA has four different aromatic protons,
one of which
is not coupled to the other three. The two vinylic protons
of
both
m-tHBPA and
p-tHBPA have
trans
coupling constants (
J = 16.2
Hz).
Several of the 4-substituted 2-ketobut-3-enoates produced here were
examined as substrates for
tHBPA hydratase-aldolase (Table
2). Most were cleaved at rates that were
less than 2% of the
rate with
o-tHBPA. Previously, we
determined that two of these,
benzylidenepyruvate and
o-methoxybenzylidenepyruvate, are not
substrates
(
3); this led us to propose a reaction mechanism
in which
there is a requirement for an
ortho hydroxyl group for
hydration to occur (Fig.
1A). However, the fact that these chemicals
actually are substrates, although very poor ones (cleaved at about
0.5% of the rate with
o-tHBPA) indicates that there is not
a strict
requirement for an
ortho hydroxyl group to initiate
the reaction
and that the proposed mechanism is not correct. Although
not essential
for activity, enol-keto tautomerization of the aromatic
hydroxyl
to form a quinonemethide could provide some stability for
reaction
intermediates such as the proposed carbanion (Fig.
1A,
compound
II). If that were the case,
p-tHBPA, which could
tautomerize to
form a similar quinonemethide-stabilized carbanion (Fig.
1B, compound
VIII), should also be a good substrate, but it is not.
(
p-tHBPA
was cleaved at 1% the rate of
o-tHBPA.)
Surprisingly, the best
substrate analog examined here is
m-tHBPA (Table
2 and Fig.
3),
which was cleaved at 75% of the rate with
o-tHBPA. Since
tautomerization
ofthe
meta hydroxyl group of
m-tHBPA could not yield a carbanion-stabilizing
quinonemethide (Fig.
1C), the hydroxyls of
o-tHBPA and
m-tHBPA
are probably not involved in the reaction mechanism
but may have
a more peripheral role, such as in coordination of the
substrate
in the active site.
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FIG. 3.
Conversion of m-tHBPA to
m-hydroxybenzaldehyde and pyruvate by cell extracts of
E. coli JM109(pRE701) at 30°C. The sample and reference
curvettes contained 50 mM potassium-sodium phosphate buffer (pH 7.0) in
1-ml volumes. The sample curvette also contained 100 nmol of
m-tHBPA. Spectra were recorded before the addition of 5 µl
of extract containing 21 µg of protein to both curvettes and after
0.17, 2.5, 5, 7.5, 10, 12.5, and 15 min.
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In most of the condensation reactions carried out here, the
transformation products had UV-visible spectra with absorbance
maxima
at longer wavelengths than those of the spectra of the
aldehyde
substrates. This property would be expected for compounds
possessing an
increased number of conjugated double bonds, as
are produced by aldol
condensation followed by dehydration. Spectral
changes accompanying
condensations of 1-hydroxy-2-naphthaldehyde
and
2-hydroxy-1-naphthaldehyde with pyruvate were exceptions,
yet appeared
to be very similar to each other. In the condensation
of
1-hydroxy-2-naphthaldehyde with pyruvate, the initial spectrum
due to
1-hydroxy-2-naphthaldehyde, having maxima at 316 and 365
nm, changed to
a spectrum with maxima at 300, 313, 334, and 348
nm. The condensation
product was prepared on a large scale, purified,
and examined,
following trimethylsilylation, by GC-MS. The mass
spectrum of the major
derivative, which eluted in a broad peak
at 58.48 min, suggests that it
is the tri-TMS derivative of
2-keto-4-hydroxy-4-(1'-hydroxy-2'-naphthyl)-butyrate
with the following
m/z of major ion peaks (proposed composition,
percentage of
intensity): 476 (M
+, 65), 475 ([M
H]
+, 64), 459 (17), 432 (30), 359 ([M
TMS
CO
2]
+, 76), 331 ([M
TMS
CO
2 CO]
+, 13), 329 (13), 302 (20), 297 ([M
OTMS
OTMS
H]
+, 100), 207 ([M
OTMS
OTMS
OTMS
H
H]
+, 16), 181 (12), 151 (13), 73 (TMS
+, 56).
It appears that this condensation product is not dehydrated
by the
enzyme and survives purification without loss of
water.
The plasmid pRE701 carries a hybrid
lacZ-nahE gene encoding
a single peptide with a molecular weight of 36,559 (8 LacZ and
323 NahE
amino acids), which may form a homotrimer, as shown for
the related
hydratase-aldolase of the naphthalenesulfonate-degrading
strain
Pseudomonas vesicularis BN6 (
2,
6). This single
enzyme
catalyzes the coupled and reversible hydration and aldol
cleavage
of
o-tHBPA and a variety of substrate analogs. A
mechanism for
these reactions in which the aromatic ring is not
involved can
be proposed (Fig.
4). This
differs from a previously proposed
mechanism in that both hydration and
dehydration are obligately
enzyme catalyzed and tightly connected to
aldol cleavage and condensation.
One disappointing aspect of this
mechanism is that although the
enzyme is capable of forming and
breaking carbon-carbon bonds
of a potentially large number of
substrates, it is not useful
for making chiral products (4-substituted
4-hydroxy-2-ketobutyrates)
in the way that other aldolases such as
2-keto-3-deoxy-6-phosphogluconate
aldolase and
2-keto-3-deoxy-6-phosphogalactonate aldolase are
(
1,
5,
7,
8), since, once formed, most chiral intermediates
will be either
dehydrated or cleaved. However, the results obtained
with the
hydroxynaphthaldehydes suggest that coupling of hydratase
and aldolase
activities is not absolute and that it might be possible
to eliminate
either of them by genetic engineering of the enzyme
active site.
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FIG. 4.
Proposed mechanism for tHBPA
hydratase-aldolase. The typical hydration and aldol cleavage reactions
(9) do not require involvement of the aromatic substituent
as proposed previously (Fig. 1).
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ACKNOWLEDGMENTS |
I thank Wallace Gilliam of this laboratory for GC-MS analyses and
Jerome Gurst of the Chemistry Department, University of West Florida,
Pensacola, for NMR spectroscopy analyses.
Partial support for the purchase of the NMR spectrometer at the
University of West Florida was provided by grant USE-9050802 from the
National Science Foundation.
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FOOTNOTES |
*
Mailing address: Gulf Ecology Division, NHEERL, U.S.
Environmental Protection Agency, 1 Sabine Island Dr., Gulf Breeze, FL 32561. Phone: (850) 934-9345. Fax: (850) 934-9201. E-mail:
eaton.richard{at}epa.gov.
Contribution 1095 from the Gulf Ecology Division, NHEERL,
U.S. Environmental Protection Agency, Gulf Breeze, Fla.
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Applied and Environmental Microbiology, June 2000, p. 2668-2672, Vol. 66, No. 6
0099-2240/00/$04.00+0
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