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.
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.
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.
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-β-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.
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)
| Product elution time (% of total) |
m/z of major ion peaks (proposed composition, % of intensity) |
|---|---|
| m-HBPA | |
| 20.57 min (21) | 336 (M+, 2), 291 ([M − CH3 − CH3 − CH3]+, 8), 277 (3), 264 (3), 249 (4), 219 ([M − TMS − CO2]+, 66), 203 ([M − TMS − CO2 − O]+, 24), 185 (3), 147 (4), 135 (3), 115 (2), 102 (3), 75 (11), 73 (TMS+, 100) |
| 22.93 min (72) | 336 (M+, 4), 321 (2), 306 (2), 291 ([M − CH3 − CH3 − CH3]+, 10), 277 (4), 264 (3), 249 (6), 219 ([M − TMS − CO2]+, 82), 203 ([M − TMS − CO2 − O]+, 28), 161 (2), 147 (4), 135 (2), 115 (2), 102 (2), 75 (9), 73 (TMS+, 100) |
| p-HBPA | |
| 22.25 min (19) | 336 (M+, 2), 321 ([M − CH3]+, 5), 291 (1), 277 (2), 249 (2), 219 ([M − TMS − CO2]+, 100), 203 ([M − TMS − CO2 − O]+, 5), 191 ([M − TMS − CO2 − CO]+, 3), 161 (2), 153 (1.3), 147 (2), 135 (1), 115 (2), 102 (2), 75 (6), 73 (TMS+, 54) |
| 24.38 min (75) | 336 (M+, 2), 321 ([M − CH3]+, 4), 291 (1), 277 (3), 249 (2), 219 ([M − TMS − CO2]+, 100), 203 ([M − TMS − CO2 − O]+, 4.4), 191 ([M − TMS − CO2 − CO]+, 3), 175 (2), 153 (1), 147 (2), 135 (1), 115 (2), 102 (1), 75 (5), 73 (TMS+, 53) |
| Benzylidenepyruvate | |
| 15.68 min (13) | 248 (M+, 2), 233 (1), 218 (2), 203 ([M − CH3 − CH3 − CH3]+, 19), 189 (12), 176 (6), 161 (7), 145 (2), 131 ([M − TMS − CO2]+, 76), 115 ([M − TMS − CO2 − O]+, 16), 103 ([M − TMS − CO2 − CO]+, 30), 77 ([M − TMS − CO2 − CO − C2H2]+, 21), 73 (TMS+, 100) |
| 17.61 min (87) | 248 (M+, 3), 233 (2), 218 (2), 203 ([M − CH3 − CH3 − CH3]+, 20), 189 (14), 176 (5), 161 (6), 145 (3), 131 ([M − TMS − CO2]+, 90), 117 (4), 115 ([M − TMS − CO2 − O]+, 16), 103 ([M − TMS − CO2 − CO]+, 30), 77 ([M − TMS − CO2 − CO − C2H2]+, 20), 73 (TMS+, 100) |
| o-Methylbenzylidenepyruvate | |
| 16.15 min (32) | 262 (M+, 3), 247 (2), 232 (1), 217 ([M − CH3 − CH3 − CH3]+, 9), 203 (19), 190 (5), 175 (5), 172 (3), 159 (2), 145 ([M − TMS − CO2]+, 100), 129 ([M − TMS − CO2 − O]+, 15), 117 ([M − TMS − CO2 − CO]+, 15), 115 ([M − TMS − CO2 − O − CH3]+, 30), 102 (1.6), 91 ([M − TMS − CO2 − CO − C2H2]+, 10), 77 (1), 75 (17), 73 (TMS+, 97) |
| 19.06 min (54) | 262 (M+, 2), 247 (2), 232 (1), 217 ([M − CH3 − CH3 − CH3]+, 7), 203 (15), 190 (2), 175 (3), 172 (3), 159 (1), 145 ([M − TMS − CO2]+, 100), 129 ([M − TMS − CO2 − O]+, 14), 117 ([M − TMS − CO2 − CO]+, 16), 115 ([M − TMS − CO2 − O − CH3]+, 26), 102 (1), 91 ([M − TMS − CO2 − CO − C2H2]+, 9), 89 (2), 77 (1), 75 (13), 73 (TMS+, 84) |
| o-Chlorobenzylidenepyruvate | |
| 17.82 min (22) | 267 ([M − CH3]+, 1), 247 (1), 239 (2), 237 ([M − CH3 − CH3 − CH3]+, 3), 223 (5), 210 (2), 203 (9), 195 (2), 167 (7), 165 ([M − TMS − CO2]+, 24), 151 (2), 149 ([M − TMS − CO2 − O]+, 5), 139 (2), 137 ([M − TMS − CO2 − CO]+, 8), 130 (6), 117 (4), 116 (3), 102 ([M − TMS − CO2 − CO − Cl]+, 11), 101 (12), 95 (2), 93 (3), 75 (16), 73 (TMS+, 100) |
| 20.42 min (73) | 284 (M+, 0.2), 282 (M+, 0.6), 267 ([M − CH3]+, 1), 252 (1), 237 ([M − CH3 − CH3 − CH3]+, 5), 223 (7), 210 (2), 203 (12), 195 (2), 187 (1.5), 167 (10), 165 ([M − TMS − CO2]+, 29), 159 (2), 151 (2), 149 ([M − TMS − CO2 − O]+, 7), 139 (3), 137 ([M − TMS − CO2 − CO]+, 8), 130 (7), 117 (4), 116 (4), 102 ([M − TMS − CO2 − CO − Cl]+, 12), 101 (14), 95 (2), 93 (4), 75 (18), 73 (TMS+, 100) |
| o-Methoxybenzylidenepyruvate | |
| 19.05 min (34) | 278 (M+, 4), 263 ([M − CH3]+, 1), 233 (1), 219 (6), 191 (2), 176 (1), 161 ([M − TMS − CO2]+, 100), 157 (3), 146 ([M − TMS − CO2 − CH3]+, 5), 145 ([M − TMS − CO2 − O]+, 4), 131 (2), 118 (6), 115 (2), 105 (7), 103 (3), 89 (4), 79 (3), 77 (7), 75 (5), 73 (TMS+, 37) |
| 22.02 min (62) | 278 (M+, 4), 263 ([M − CH3]+, 1), 233 (1), 219 (6), 191 (2), 176 (1), 161 ([M − TMS − CO2]+, 100), 157 (3), 146 ([M − TMS − CO2 − CH3]+, 5), 145 ([M − TMS − CO2 − O]+, 4), 131 (2), 118 (6), 115 (2), 105 (7), 103 (3), 89 (4), 79 (3), 77 (7), 75 (5), 73 (TMS+, 37) |
| o-Carboxybenzylidenepyruvate | |
| 24.56 min (18) | 364 (M+, 1), 349 ([M − CH3]+, 12), 305 (1.5), 292 (2), 277 (5), 274 (3), 247 ([M − TMS − CO2]+, 5), 246 ([M − TMS − CO2 − H]+, 8), 219 ([M − TMS − CO2 − CO]+, 7), 203 ([M − TMS − TMS − O + H]+, 10), 190 (2), 187 (2), 173 (2), 147 ([M − TMS − TMS − CO2 − CO2 + H]+, 59), 133 (8), 131 (3), 129 (2), 105 (2), 102 ([M − TMS − TMS − CO2 − CO − CO2]+, 3), 75 (10), 73 (TMS+, 100) |
| 24.72 min (55) | 364 (M+, 0.03), 349 ([M − CH3]+, 1), 292 (4), 277 (1), 259 (1), 247 ([M − TMS − CO2]+, 10), 231 (1), 219 ([M − TMS − CO2 − CO]+, 9), 203 ([M − TMS − TMS − O + H]+, 13), 188 (1), 173 (1), 157 (1), 147 ([M − TMS − TMS − CO2 − CO2 + H]+, 17), 133 (2), 129 (2), 117 (1), 105 (1), 102 ([M − TMS − TMS − CO2 − CO − CO2]+, 5), 73 (TMS+, 100) |
| 2-Keto-4-(2′-pyridyl)-but-3-enoate | |
| 11.93 min (16) | 205 ([M − CH3 − CH3 − CH3 + H]+, 72), 190 (7), 176 ([M − TMS]+, 5), 162 (6), 132 ([M − TMS − CO2]+, 29), 117 (2), 104 ([M − TMS − CO2 − CO]+, 7), 78 ([M − TMS − CO2 − CO − C2H2]+, 8), 73 (TMS+, 100) |
| 18.34 min (77) | 234 ([M − CH3]+, 0.5), 205 ([M − CH3 − CH3 − CH3 + H]+, 23), 190 (14), 176 ([M − TMS]+, 8), 162 (14), 132 ([M − TMS − CO2]+, 42), 117 (2), 104 ([M − TMS − CO2 − CO]+, 11), 78 ([M − TMS − CO2 − CO − C2H2]+, 16), 73 (TMS+, 100) |
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).
FIG. 2.
13C and 1H NMR data for m-HBPA and p-HBPA in d6-dimethyl sulfoxide. d, doublet; t, triplet.
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.
TABLE 2.
Cleavage reactions catalyzed by o-tHBPA hydratase-aldolasea
| Reaction substrate/product | Wavelength measured (nm) | Difference in ɛ of substrate and product (M−1 cm−1) | Relative rateb |
|---|---|---|---|
| o-tHBPA/2-hydroxybenzaldehyde | 296 | 10,630 | 1.0 |
| m-tHBPA/3-hydroxybenzaldehyde | 297 | 15,800 | 0.75 |
| p-tHBPA/4-hydroxybenzaldehyde | 331 | 10,590 | 0.008 |
| o-Chlorobenzylidenepyruvate/2-chlorobenzaldehyde | 297 | 16,900 | 0.016 |
| o-Methylbenzylidenepyruvate/o-tolualdehyde | 303 | 16,600 | 0.003 |
| 2-Keto-4-(2′-pyridyl)-but-3-enoate/pyridine-2-carboxaldehyde | 300 | 13,300 | 0.0064 |
| 2-Keto-4-(3′-indolyl)-but-3-enoate/indole-3-carboxaldehyde | 367 | 21,600 | 0.0046 |
| o-Carboxybenzylidenepyruvate/2-carboxybenzaldehyde | 300 | 11,900 | 0.014 |
| Benzylidenepyruvate/benzaldehyde | 300 | 13,300 | 0.0052 |
| o-Methoxybenzylidenepyruvate/2-methoxybenzaldehyde | 296 | 11,400 | 0.0046 |
Assay mixtures contained, in 1-ml volumes, 0.1 mM substrate, 50 mM K-Na phosphate buffer (pH 7), and 1 to 20 μl of extract. Assay wavelengths, which were absorbance maxima of the substrates, and extinction coefficients (ɛ) used in assays were determined from qualitative assays for each transformation similar to that shown in Fig. 3 for the transformation of m-tHBPA and from the spectra of purified substrates and products. Extracts of E. coli JM109 lacking pRE701 had no activity toward these substrates.
Rates are relative to that for o-tHBPA, which varied from 1.6 to 3.6 μmol min−1 mg of protein−1 in different extracts.
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.
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 − CO2]+, 76), 331 ([M − TMS − CO2 − 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.
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).
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.
Footnotes
Contribution 1095 from the Gulf Ecology Division, NHEERL, U.S. Environmental Protection Agency, Gulf Breeze, Fla.
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