Stereochemical Consequences of Vinylpyruvate Hydratase-Catalyzed Reactions

Article abstract of DOI:10.1021/acs.biochem.6b00552

A stereochemical analysis has been carried out on two vinylpyruvate hydratases (VPH), which convert 2-hydroxy-2,4-pentadienoate to 2-keto-4S-hydroxypentanoate in meta-fission pathways. Bacterial strains with this pathway can use aromatic compounds as sole sources of energy and carbon. The analysis was carried out using the 5-methyl and 5-chloro derivatives of 2-hydroxy-2,4-pentadienoate with the enzymes from Pseudomonas putida mt-2 (Pp) and Leptothrix cholodnii SP-6 (Lc). In both organisms, VPH is in a complex with the preceding enzyme in the pathway, 4-oxalocrotonate decarboxylase (4-OD). In D₂O, a deuteron is incorporated stereospecifically at the C-3 and C-5 positions of product by both Pp and Lc enzymes. Accordingly, the complexes generate (3S,5S)-3,5-[di-D]-2-keto-4S-hydroxyhexanoate and (3S,5R)-3,5-[di-D]-2-keto-4R-hydroxy-5-chloropentanoate (4R and 5R due to a priority numbering change). The substitution at C-5 (CH₃ or Cl) or the source of the enzyme (Pp or Lc) does not change the stereochemical outcome. One mechanism that can account for the results is the ketonization of the 5-substituted dienol to the α,β-unsaturated ketone (placing a deuteron at C-5 in D₂O), followed by the conjugate addition of water (placing a deuteron at C-3). The stereochemical outcome for VPH (from Pp and Lc) is the same as that reported for a related enzyme, 2-oxo-hept-4-ene-1,7-dioate hydratase, from Escherichia coli C. The combined observations suggest similar mechanisms for these three enzymes that could possibly be common to this group of enzymes.

Full text of DOI:10.1021/acs.biochem.6b00552

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Article
The Stereochemical Consequences of
Vinylpyruvate Hydratase-catalyzed Reactions
William H. Johnson, Tyler M.M. Stack, Stephanie M Taylor, Elizabeth A Burks, and Christian P. Whitman
Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b00552 • Publication Date (Web): 30 Jun 2016
Downloaded from http://pubs.acs.org on July 9, 2016
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Biochemistry
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The Stereochemical Consequences of Vinylpyruvate Hydrataseꢀcatalyzed Reactions
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REVISED June 29, 2016
William H. Johnson, Jr.‡, Tyler M.M. Stack§, Stephanie M. Taylor§, Elizabeth A. Burks‡, and
Christian P. Whitman‡,*
Division of Chemical Biology and Medicinal Chemistry, College of Pharmacy, and Department
of Molecular Biosciences, University of Texas, Austin, TX 78712
‡Division of Chemical Biology and Medicinal Chemistry
§Department of Molecular Biosciences
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ABSTRACT
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A stereochemical analysis has been carried out on two vinylpyruvate hydratases (VPH),
which convert 2ꢀhydroxyꢀ2,4ꢀpentadienoate to 2ꢀketoꢀ4Sꢀhydroxypentanoate in metaꢀfission
pathways. Bacterial strains with this pathway can use aromatic compounds as sole sources of
energy and carbon. The analysis was carried out using the 5ꢀmethyl and 5ꢀchloro derivatives of
2ꢀhydroxyꢀ2,4ꢀpentadienoate with the enzymes from Pseudomonas putida mtꢀ2 (Pp) and
Leptothrix cholodnii SPꢀ6 (Lc). In both organisms, VPH is in a complex with the preceding
enzyme in the pathway, 4ꢀoxalocrotonate decarboxylase (4ꢀOD). In D2O, a deuteron is
incorporated stereospecifically at the Cꢀ3 and Cꢀ5 positions of product by both Pp and Lc
enzymes. Accordingly, the complexes generate (3S,5S)ꢀ3,5ꢀ[diꢀD]ꢀ2ꢀketoꢀ4Sꢀhydroxyhexanoate
and (3S,5R)ꢀ3,5ꢀ[diꢀD]ꢀ2ꢀketoꢀ4Rꢀhydroxyꢀ5ꢀchloropentanoate (4R and 5R due to a priority
numbering change). The substitution at Cꢀ5 (CH3 or Cl) or the source of the enzyme (Pp or Lc)
does not change the stereochemical outcome. One mechanism that can account for the results is
the ketonization of the 5ꢀsubstituted dienol to the α,βꢀunsaturated ketone (placing a deuteron at
Cꢀ5 in D2O), followed by the conjugate addition of water (placing a deuteron at Cꢀ3). The
stereochemical outcome for VPH (from Pp and Lc) is the same as that reported for a related
enzyme, 2ꢀoxoꢀheptꢀ4ꢀeneꢀ1,7ꢀdioate hydratase, from Escherichia coli C. The combined
observations suggest similar mechanisms for these three enzymes that could possibly be common
to this group of enzymes.
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Bacterial metaꢀfission pathways convert monocyclic aromatic compounds to useful cellular
intermediates via catechol intermediates.1 Catechol (1, Scheme 1) or a substituted derivative
undergoes a signature metaꢀfission ring opening reaction, shown in Scheme 1, to generate a
compound that is processed through a series of reactions to 2ꢀhydroxyꢀ2,4ꢀpentadienoate (2,
Scheme 1), or a derivative.2,3 A metalꢀdependent (Mn2+ or Mg2+) hydratase then converts 2 to 2ꢀ
ketoꢀ4Sꢀhydroxypentanoate (4), most reasonably by the conjugate addition of water to the
intermediate, 2ꢀketoꢀ3ꢀpentenoate (3).2ꢀ6 Subsequently, the product undergoes an enzymeꢀ
catalyzed retroꢀaldol cleavage to yield pyruvate and acetaldehyde. The aldolase is tightly
associated with a dehydrogenase that uses NAD+ and CoASH to produce acetylꢀCoA from
acetaldehyde.7,8 In this manner, pyruvate and acetylꢀCoA are generated from aromatic
compounds.
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Several hydratases have been identified, purified, and studied in some detail.2ꢀ6,9,10 One,
designated vinylpyruvate hydratase (EC 4.2.1.80; VPH1) from Pseudomonas putida mtꢀ2, is part
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of an inducible pathway encoded by the TOL plasmid pWW0 that enables selected bacterial
strains to use toluene, mꢀ and pꢀxylene, 3ꢀethyltoluene, and 1,2,4ꢀtrimethylbenzene as their sole
sources of carbon and energy.2,3 VPH functions in a complex with the preceding enzyme in the
pathway, 4ꢀoxalocrotonate decarboxylase (4ꢀOD), and the 4ꢀOD/VPH complex converts 2ꢀketoꢀ
3Eꢀhexenedioate (5, Scheme 2) to 4. A stereochemical analysis of the reaction (using a complex
where VPH is nearly inactive) suggests that 4ꢀOD converts 5 to 2 and that VPH, in turn, converts
2 to 4.9
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Although VPH and other hydratases have been studied periodically over the last 35 years,
our understanding of their mechanisms is far from complete.2ꢀ6,9ꢀ12 The accumulated evidence
implicates the mechanism shown in Scheme 1 where 2 initially undergoes an enzymeꢀcatalyzed
ketonization to produce 3, which is then attacked by water at Cꢀ4. Protonation at Cꢀ3 completes
the conjugate addition of water. The experimental evidence argues against a Schiff base
mechanism,4 and suggests the involvement of a metalꢀion activated water molecule.4,5,10ꢀ12
Although the mechanism is reasonable, incubation of the trans isomer of 3 with the enzyme does
not afford product.4,5,11 The chemical incompetence of transꢀ3 raises three possibilities: the cisꢀ
isomer of 3 is the intermediate, various factors hamper the dissociation of the enzymeꢀbound
intermediate and its reꢀassociation with the enzyme once free in solution, or the mechanism is
not correct.4
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In this work, we report a stereochemical analysis of two VPHꢀcatalyzed reactions using 5ꢀ
methylꢀ and 5ꢀchloroꢀ2ꢀhydroxyꢀ2,4ꢀpentadienoate (4Zꢀ6 and 4Zꢀ7, respectively in Scheme 3),
and the enzymes from P. putida mtꢀ2 and Leptothrix cholodnii SPꢀ6 (designated Pp and Lc,
respectively). The analysis showed that the reactions produce the 4S isomers of 4 (in accord
with previous work) and 8, and the 4R isomer of 9 (due to a priority numbering change). When
the same reactions are carried out in D2O, products 8 and 9, are stereospecifically labeled at Cꢀ3
and Cꢀ5 with deuterium. Both deuterons are incorporated on the opposite face to that of the Cꢀ4
hydroxy group. The Pp and Lc enzymes show the same stereochemical result indicating that the
presence of the substituent does not change the mechanism. These results are consistent with the
enzymeꢀcatalyzed ketonization of the dienol (i.e., 6 or 7) to place a deuterium at Cꢀ5 for 8 and 9,
respectively, followed by the anti addition of water to incorporate deuterium at Cꢀ3 of the α,βꢀ
unsaturated ketone (with the transꢀconfiguration). If the α,βꢀunsaturated ketone has the cisꢀ
configuration, then ketonization of the dienol would be followed by the syn addition of water.
Nonetheless, both scenarios implicate the α,βꢀunsaturated ketone (i.e., 3 in Scheme 1) in the
VPH reaction mechanism.
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EXPERIMENTAL PROCEDURES
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Materials. Chemicals, biochemicals, buffers, solvents, and the components for LuriaꢀBertani
(LB) media were obtained from sources reported elsewhere.13 The syntheses of 2ꢀhydroxyꢀ2,4ꢀ
pentadienoate (2) is reported elsewhere.9 The 5ꢀmethyl derivative (6) was generated from 5ꢀ
(methyl)ꢀ2ꢀhydroxymuconate14 following the procedure used to produce 2.9 The Phenyl
Sepharose 6 Fast Flow, DEAEꢀSepharose resins, and the prepacked PDꢀ10 Sephadex Gꢀ25
columns were obtained from GE Healthcare (Piscataway, NJ). The EconoꢀColumn
chromatography columns and Freeze ’N Squeeze units were obtained from BioꢀRad
Laboratories, Inc. (Hercules, CA). Enzymes and reagents used for molecular biology procedures
were obtained from New England Biolabs, Inc. (Ipswich, MA). 5ꢀ(carboxymethyl)ꢀ2ꢀ
Hydroxymuconate isomerase (CHMI),15 4ꢀoxalocrotonate tautomerase (4ꢀOT)16,17, 4ꢀOD/VPH
and 4ꢀOD/E106QVPH from P. putida mtꢀ2 were purified by procedures reported elsewhere with
some modifications.9 The 4ꢀOD/VPH complex from L. cholodnii SPꢀ6 was purified similarly to
the P. putida mtꢀ2 complex.9 Activities were determined using previously described assays.9,15ꢀ17
Bacterial Strains, Plasmids, and Growth Conditions. Escherichia coli strains BL21ꢀ
Gold(DE3), C41(DE3), and the DH5α cells were obtained from Agilent Technologies (Santa
Clara, CA), Lucigen (Middleton, WI), and Invitrogen (Carlsbad, CA), respectively. L. cholodnii
SPꢀ6 was a gift from Dr. David Emerson (Bigelow Laboratory for Ocean Sciences, East
Boothbay, ME). The isolation of genomic DNA from L. cholodnii SPꢀ6 cells was carried out by
a procedure described elsewhere.18 The pET vectors were obtained from Novagen (Madison,
WI). Plasmids were isolated using the Sigma GenElute Plasmid Miniprep Kit, following the
manufacturer’s directions.
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General Methods. Techniques for restriction enzyme digestion, ligation, transformation, and
other standard molecular biology manipulations were based on methods described elsewhere19.
All PCR reactions were carried out in a GeneAmp 2700 thermocycler (Applied Biosystems,
Foster City, CA). Oligonucleotide primers were synthesized by SigmaꢀAldrich. DNA
sequencing was performed at the DNA core facility of the Institute of Cellular and Molecular
Biology (ICMB) at the University of Texas at Austin. Mass spectral data were obtained on an
LCQ electrospray ionꢀtrap mass spectrometer (Thermo, San Jose, CA) in the ICMB Protein and
Metabolite core facility. The samples were prepared as described previously.20 Kinetic data
were obtained at 24°C on an Agilent 8453 diodeꢀarray spectrophotometer. Nonlinear regression
data analysis was performed using Grafit (Erithacus Software Ltd., Staines, U.K.) obtained from
SigmaꢀAldrich. Protein concentrations were determined by the Waddell method.21 4ꢀOT was
analyzed using tricine sodium dodecyl sulfateꢀpolyacrylamide gel electrophoresis (SDSꢀPAGE)
on 15% gels.22 All other proteins were analyzed using Trisꢀglycine SDSꢀPAGE on 12% gels.23
Gels were run on a BioꢀRad MiniꢀProtean II gel electrophoresis apparatus. Nuclear magnetic
resonance (NMR) spectra were recorded on a Varian Unity+ 300 MHz or a Varian DirectDrive
600 MHz spectrometer (Palo Alto, CA), as noted. NMR signals were analyzed using the
software program SpinWorks 3.1.6 (Copyright © 2009 Kirk Marat, University of Manitoba).
Synthesis of 5-(chloro)-2-Hydroxy-2,4-hexadiene-1,6-dioate (12). The protocol for the
synthesis of 12 (Scheme 4) was based on the one used to generate 2ꢀhydroxymuconate (10)
except ethyl crotonate was replaced with ethyl 2ꢀchlorocrotonate24. The ethylꢀ2ꢀchlorocrotonate
was generated as follows. Over a 1 h period, chlorine gas was streamed over a solution of ethyl
crotonate (18.4 g, 161 mmol) dissolved in CCl4 (100 mL) and DMF (2 drops). The reaction was
carried out in a stoppered 500 mL round bottom flask. The solution was stirred at room
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temperature overnight. Solvent was then removed and the residue distilled. Fractions boiling at
176ꢀ182°C were collected to yield 15.9 g of product. 1H NMR analysis showed that it was
mostly ethyl 2,3ꢀdichlorobutyrate with a trace of ethyl 2ꢀchlorocrotonate. In order to convert the
remaining ethyl 2,3ꢀdichlorobutyrate to ethyl 2ꢀchlorocrotonate, the product mixture was
dissolved in acetone (150 mL) in a 500 mL round bottom flask. Potassium carbonate (20 g) was
added and the solution was allowed to gently reflux for 3 days. After cooling, the solid was
filtered off and rinsed with ethyl acetate. The solvent was removed in vacuo and the residue
purified by flash column chromatography (8:1 hexane/ethyl acetate). Fractions containing the
desired product were combined and evaporated to dryness. The residue was used without further
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purification. H NMR analysis showed an E/Z mixture of ethyl 2ꢀchlorocrotonate, where the Eꢀ
isomer predominates. Sodium metal (0.8 g, 1 eq) was allowed to react completely with ethanol
(50 mL) and then 300 mL of toluene was added. The resulting solution was distilled (to remove
water and ethanol) under an argon atmosphere until the temperature climbed above 80°C. The
sodium ethoxide solution was then cooled in an ice bath and diethyl oxalate (4.9 g, 33.6 mmol),
followed by ethyl 2ꢀchlorocrotonate (5 g, 33.7 mmol) was added. This mixture was equilibrated
to room temperature and stirred for 4 days. The bright yellow solid that precipitated was filtered,
washed with hexane, and airꢀdried to yield 4.23 g. The solid (3.84 g, 11 mmol) was suspended in
water (100 mL) and cooled in an ice bath. A 1 M solution of sodium hydroxide (32 mL, 32
mmol) was added dropwise over 30 min, and the reaction mixture was allowed to equilibrate to
room temperature overnight. The dark yellow solution was cooled in an ice bath and
concentrated phosphoric acid was added dropwise to adjust the pH to ~1.8. The solid that
precipitated was filtered, air dried, and then crystallized from ethyl acetate to yield 1.91 g of 4Zꢀ
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12. H NMR (CD3OD, 300 MHz) δ 6.50 (1H, d, J = 11.7 Hz), 7.94 (1H, d, J = 11.7 Hz); C
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NMR (CD3OD, 75 MHz) δ 105.6, 124.3, 132.7, 149.8, 165.9, 166.4.
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Enzymatic Synthesis of 5-(chloro)-2-Hydroxy-2,4Z-pentadienoate (7) using 4-
OD/E106QVPH (from P. putia mt-2). The generation and isolation of 7 (Scheme 4) was based
on the one used to generate 2 with the following modifications.9 Accordingly, 4Zꢀ12 (1.0 mmol)
was suspended in 100 mM Na2HPO4 buffer (25 mL) that contained 3 mM MgCl2. The pH was
adjusted with 1 M NaOH to 7.2. To this solution, 5ꢀ(carboxymethyl)ꢀ2ꢀhydroxymuconate
isomerase, (250 µL of a 3.4 mg/mL solution) was added and allowed to react for 5 min before
adding 4OD/E106QVPH complex from P. putida mtꢀ2 (2.5 mL of 1.2 mg/mL solution). The
reaction mixture was monitored by UV, and when the peak at 304 nm no longer changed (~20
min), it was quenched by adjusting the pH to ~1.8ꢀ2.0 with 8.5% phosphoric acid. Extraction
with ethyl acetate and further purification steps were carried out as reported previously9. (4Z)ꢀ7:
1H NMR (DMSOꢀd6, 600 MHz) δ 6.31 (1H, H3, dd, J = 11.2 Hz, 1.1 Hz), 6.34 (1H, H5, dd, J =
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7.1 Hz, 1.1 Hz), 6.75 (1H, H4, dd, J = 7.1 Hz, 11.3 Hz); C NMR (CD3OD, 75 MHz) δ 105.6,
120.6, 124.7, 144.8, 167.2. In order to verify that the 4Z-isomer of 7 was generated, compound
12 was subjected to thermal decarboxylation, as described elsewhere.25 Thermal decarboxylation
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yields a mixture of isomers.25 (4E)ꢀ7: H NMR (DMSOꢀd6, 600 MHz) δ 6.13 (1H, H3, dd, J =
11.1 Hz), 6.68 (1H, H5, dd, J = 13.3 Hz), 6.75 (1H, H4, dd, J = 11.1 Hz, 13.3 Hz).
Cloning of L. cholodnii SP-6 4-OT. A 50 µL PCR reaction was set up that contained
genomic DNA (480 ng), dNTPs (0.2 mM), Vent polymerase (0.5 unit), 10× buffer (when diluted
to 1× contributes 2 mM MgSO4), and forward (5′ꢀTAGTAGTAGCATATGCCCTTTGCCCAGꢀ
3′) and reverse (5′ꢀGATGATGATCTCGAGTCATTATTAGCGTCCGAGꢀ3′) primers (0.4 ꢀM
each), where the restriction enzyme sites are underlined (NdeI and XhoI). The PCR amplification
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protocol consisted of an initial 5ꢀmin denaturation cycle at 94° C, followed by 29 cycles of 94 °C
for 1 min, 55 °C for 2 min, and 72 °C for 3 min, a 10ꢀmin elongation cycle at 72 °C, and ending
with a hold at 4 °C. The reaction produced a DNA piece of the correct size that was eluted from
a 1% agarose gel and processed through a Freeze ’N Squeeze unit. The liquid that was recovered
was used as the template DNA for subsequent amplification in which four reactions (50 µL each)
were set up. Each reaction contained 1 µL of the template DNA along with the components
listed above. The PCR amplification protocol was identical to the one above except it began with
a 2ꢀmin denaturation cycle. The reaction product was recovered from an agarose gel using a
Freeze ’N Squeeze unit followed by ethanol precipitation of the DNA. The PCR product was
inserted between the NdeI and XhoI restriction sites of a modified pET24a vector (the BglII
restriction site was removed) following procedures described elsewhere.19 The ligation product
was used to transform E. coli DH5α cells by electroporation using a BioꢀRad MicroPulser
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Electroporation unit. The transformation reaction mixture was plated on LB/Kn (30 ꢀg/mL)
plates and the cells were grown overnight at 37° C. Following PCR screening, a positive colony
was identified and used for the generation of additional plasmid for sequencing and protein
expression.
Construction of the L. cholodnii SP-6 4-OD/VPH Expression Vector. The 4OD/VPH
complex from L. cholodnii SPꢀ6 was cloned from genomic DNA, using the following primers.
For the 4ꢀOD gene, 5′ꢀTAGTAGTAGCATATGGCACTGAATCGCACCGꢀ3′ was used as the
forward primer and 5′ꢀGATGATGATAAGCTTTCATTATTAGACGAAACGCATCGACACꢀ3′
was used as the reverse primer (where the NdeI and HindIII restriction sites are underlined). For
the VPH gene, 5′ꢀTAGTAGTAGCATATGGACGCACTCACCATCCꢀ3′ was used as the
forward primer and 5′ꢀGATGATGATGGATCCTCATTATTAAATAAACCGCACCGACGꢀ3′
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was used as the reverse primer (where the NdeI and BamHI restriction sites are underlined). For
the initial PCR of each gene from genomic DNA, three reactions (50 µL each) were prepared
where each reaction contained genomic DNA (0.5 µg), the accompanying 10× buffer (5 µL), 10
mM dNTPs (1 µL), 2.5 µL bovine serum albumin (10 mg/mL), Vent DNA polymerase (0.5
units), the forward and reverse primers (2 µL each), and varying amounts of MgSO4 in distilled
H2O. Reactions were subjected to the PCR protocol described for the cloning of the Lc 4ꢀOT
gene. PCR reactions were eluted on 1% agarose gels. Bands of the correct size were excised,
isolated using the Freeze ’N Squeeze units, and the product was used as template DNA in future
reactions, following the same protocol. The PCR product and pET24a were then treated with the
appropriate restriction enzymes (NdeI and HindIII for the 4ꢀOD construct and NdeI and BamHI
for the VPH construct), ligated, and processed as described in the previous section to construct
separate plasmids containing each gene (pET24aꢀ4OD and pET24aꢀVPH).
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In order to insert the 4ꢀOD gene after VPH in the pET24aꢀVPH vector, an additional forward
primer, 5′ꢀTAGTAGTAGGGATCCAGAAGGAGATATACATGGCACTGAATCGCACCGꢀ3′,
was used (where the BamHI restriction site is underlined). Accordingly, the PCR was repeated
as described above using this primer (and the reverse primer used to synthesize the 4ꢀOD gene)
and pET24aꢀ4OD as the PCR template. The PCR amplification protocol consisted of an initial
2ꢀmin denaturation cycle at 94° C, followed by 29 cycles of 94 °C for 1 min, 55 °C for 1 min,
and 72 °C for 3 min, a 10ꢀmin elongation cycle at 72 °C, and ending with a hold at 4 °C. The
pET24a vector containing the VPH gene and the PCR product containing the 4ꢀOD sequence
with the BamHI restriction site as well as a ribosome binding site and TATA box were treated
with HindIII and BamHI and processed as described above to construct a plasmid containing
both genes.
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Generation and Isolation of 3-Hydroxybutyrate (15), 3-Hydroxypentanoate (16), and 3-
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Hydroxy-γ-butyrolactone (17). The VPH products (i.e., 4, 8, and 9, Scheme 4) were generated
8
9
from 2ꢀhydroxymuconate (10, Scheme 4), 5ꢀmethylꢀ2ꢀhydroxymuconate (11), and 5ꢀchloroꢀ2ꢀ
hydroxymuconate (12) using 4ꢀOT (Pp 4ꢀOT: 6.7 mg/mL; Lc 4ꢀOT: 1 mg/mL) and 4ꢀOD/VPH
(Pp 4ꢀOD/VPH: 2.1 mg/mL; Lc 4ꢀOD/VPH: 0.8 mg/mL). Each substrate (~32 mg of 10, 11, or
12) was processed to its VPH product (4, 8, or 9, respectively).
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For each reaction, the substrate was dissolved in 100 mM Na2HPO4 buffer, pH 9 (~4.8 mL). The
pH was adjusted to ~7.0 with 2 M NaOH (~10 ꢀL) and an aliquot (10 ꢀL) of the Pp or Lc 4ꢀOT
was added. The solutions were then distributed evenly into 8 test tubes (~0.6 mL each) and
aliquots of 4ꢀOD/VPH (50 ꢀL of Pp or Lc in 10 mM NaH2PO4 buffer containing 5 mM MgCl2)
were added to the individual tubes. Aliquots were removed from representative tubes at ~10ꢀmin
intervals and the UV absorbance was measured. The reactions were judged to be complete (~30ꢀ
90 min) by the absence of significant UV absorbance between 220ꢀ300 nm. Hydrogen peroxide
(~60 µL of a 30% solution) was added to each reaction mixture and the resulting mixture was
allowed to incubate at room temperature for 1 h.2,4 A solution of catalase (10 mg/mL) was made
in 20 mM NaH2PO4 buffer, and 5ꢀ10 µL of this solution was added to each reaction.2,4 After
bubbling ceased (indicating H2O2 was no longer present), the reaction mixtures were combined
and the pH was adjusted to 1.8 with 8.5% H3PO4 (for 3ꢀhydroxybutyrate, 15, and 3ꢀ
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hydroxypentanoate, 16, in Scheme 5). These solutions were saturated with NaCl and extracted
with ethyl acetate (3× ~25 mL). The ethyl acetate layers were combined and dried over
anhydrous Na2SO4. After evaporation to dryness, the residue was dissolved in a small amount of
ethyl acetate and purified by flash chromatography eluting with ethyl acetate. Yields were ~7.8
and 1.8 mg for 15 and 16, respectively. For the generation of 3ꢀhydroxyꢀγꢀbutyrolactone (17),
the pH was not adjusted. The combined reaction mixtures were saturated with NaCl and
extracted with ethyl acetate (3× ~25 mL). The residue was dissolved in a small amount of ethyl
acetate and purified by flash chromatography (4:1 ethyl acetate: hexanes) to generate 4.2ꢀ5.5 mg
of 17. 3ꢀHydroxybutyric acid (15) 1H NMR (CD3OH, 600 MHz) δ 1.19 (3H, d, J = 6.3 Hz), 2.37
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(1H, dd, J = 5.6 Hz, 15.3 Hz), 2.41 (1H, dd, J = 7.4 Hz, 15.3 Hz), 4.14 (1H, m); C NMR
(CD3OH, 125 MHz) δ 23.3, 44.7, 65.6, 175.6. 3ꢀHydroxypentanoic acid (16) 1H NMR (CD3OH,
600 MHz) δ 0.95 (3H, t), 1.50 (2H, m), 2.35 (1H, dd, J = 8.2 Hz, 15.3 Hz), 2.44 (1H, dd, J = 4.7
13
Hz, 15.3 Hz), 3.89 (1H, m); C NMR (CD3OH, 125 MHz) δ 10.3, 31.0, 42.9, 70.9, 175.8. 3ꢀ
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Hydroxyꢀγꢀbutyrolactone (17) H NMR (CD3OH, 600 MHz) δ 2.35 (1H, m), 2.80 (1H, dd, J =
13
5.9 Hz, 17.7 Hz), 4.20 (1H, m), 4.41 (1H, dd, J = 4.3 Hz, 10.0 Hz), 4.54 (1H, m); C NMR
(CD3OH, 125 MHz) δ 38.6, 68.4, 77.8, 179.1.
Assignment of the Stereochemistry at C-3 of 3-Hydroxybutyrate (15), 3-Hydroxypentanoate
(16), and 3-Hydroxy-γ-butyrolactone (17). Samples were dissolved separately in methanol (15
and 17) or chloroform (16) and the optical rotations were measured using a Perkin Elmer 241
MC Polarimeter. Several readings were taken (~10) and averaged to produce the rotation that
was used to calculate the reported specific rotations (Table 1). A specific rotation was also
determined for the commercially available Sꢀisomer of 15.26 The specific rotations for 16 and 17
were compared to those reported in the literature.27,28
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Assignment of the Stereochemistry at C-3 and C-5 of 8 and 9 by 1H NMR Spectroscopy. The
Pp and Lc 4ꢀOD/VPH were exchanged into 100 mM Na2HPO4 buffer, pH 7.2, or 100 mM
Na2DPO4 buffer, pH 7.2, where each buffer contained 5 mM MgCl2. To an NMR tube was added
600 µL of 100 mM Na2HPO4 buffer or 100 mM Na2DPO4 buffer (both at pH ~ 9) along with 30
µL of a solution of 6 (Scheme 3 to produce 8) (16.8 mg) dissolved in DMSOꢀd6 (126 µL). The
addition of substrate adjusted the pH to ~7. An initial 1H NMR spectrum of the reaction mixture
was recorded before the addition of enzyme. After an aliquot of enzyme (25 µL Pp or Lc 4ꢀ
OD/VPH) was added, spectra were acquired every 3 min until the reaction was complete (~20
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min) for the reactions run in 100 mM Na2HPO4 buffer (H2O). This same series of H NMR
experiments was repeated using 30 µL of a solution of 7 (12.3 mg in 92 µL DMSOꢀd6) to
1
produce 9 (Scheme 3). For the H NMR experiments carried out in Na2DPO4 buffer (D2O), a
single 1H NMR spectrum was recorded for both 8 and 9 in order to minimize the exchange at Cꢀ3
1
and Cꢀ5. 8: H NMR (NaH2PO4, 600 MHz) δ 0.72 (3H, t), 1.33 (2H, m), 2.66 (1H, dd, J = 8.4
Hz, 16.6 Hz), 2.75 (1H, dd, J = 4.2 Hz, 16.6 Hz), 3.87 (1H, m). 3,5ꢀdi[D]8: 1H NMR (NaD2PO4,
600 MHz) δ 0.71 (3H, d, J = 7.4 Hz), 1.30 (1H, m, J = 7.4ꢀ7.6 Hz), 2.64 (0.9H, d, J = 8.4 Hz),
1
3.87 (1H, t). 9: H NMR (NaH2PO4, 600 MHz) δ 2.84 (1H, dd, J = 7.8 Hz, 17.4 Hz), 2.89 (1H,
dd, J = 4.8 Hz, 17.4 Hz), 3.45 (1H, dd, J = 5.8 Hz, 11.6 Hz), 3.53 (1H, dd, J = 3.9 Hz, 11.6 Hz),
1
4.21 (1H, m). 3,5ꢀdi[D]9: H NMR (NaD2PO4, 600 MHz) δ 2.82 (0.3H, d, J = 8.2 Hz), 3.44
(0.8H, d, J = 5.7 Hz), 4.19 (1H, m).
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RESULTS
Assignment of C-4 Stereochemistry of 3-Hydroxybutyrate (15), 3-Hydroxypentanoate (16),
and 3-Hydroxy- -butyrolactone (17). The stereochemistry at the C4 hydroxyl of 2ꢀketoꢀ4ꢀ
γ
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hydroxypentanoate (4), 5ꢀmethylꢀ2ꢀketoꢀ4ꢀhydroxypentanoate (8), and 5ꢀchloroꢀ2ꢀketoꢀ4ꢀ
hydroxypentanoate (9) was determined by their respective enzymatic generation from 10, 11, and
12 (Scheme 4) and their subsequent chemical degradation to 3ꢀhydroxybutyrate (15), 3ꢀ
hydroxypentanoate (16), and 3ꢀhydroxyꢀγꢀbutyrolactone (17) (Schemes 5A and B). The chemical
degradation is based on previous protocols and involves the oxidative decarboxylation of the
VPH products (using H2O2) and purification.2,4,29 In the course of these reactions, the
degradation product of 9 (Scheme 5B) cyclizes to lactone 17 with the loss of the chloride, but
retains the stereochemistry at Cꢀ4 (of 9).
The optical rotations of the three samples (generated using the Pp and Lc enzymes) are
shown in Table 1 along with those of the authentic (and configurationally known) compounds.
In all cases, the optical rotations are positive. The S isomer of 15 and 16 has a positive rotation
and the R isomer of 17 has a positive rotation. This indicates that the Pp and Lc enzymes
generate 2ꢀketoꢀ4Sꢀhydroxypentanoate (4) and 2ꢀketoꢀ4Sꢀhydroxyhexanoate (8), and that neither
the methyl group nor the enzyme alters the stereochemistry. The stereochemical outcome
reported here for 4 agrees with previous work.2,4,29 The positive rotation for 17 indicates that the
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R isomer of 17 has been generated and that both enzymes produce 5ꢀchloroꢀ2ꢀketoꢀ4Rꢀ
hydroxypentanoate (9).
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1H NMR Spectroscopic Analysis of the Products Generated by Pp and Lc 4-OD/VPH Using 6
and 7 in D2O. The preceding analysis shows that the Pp and Lc 4ꢀOD/VPHs introduce a chiral
carbon at C4 of 4, 8, and 9. Hence, the protons at C3 of 4, 8, and 9 are diastereotopic as are the
protons at C5 of 8 and 9. (The C5 methyl group of 4 is achiral.) These protons will present a
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complex set of signals in a H NMR spectrum when the reactions are carried out in H2O. The
stereoselective or stereospecific incorporation of deuterium will simplify the set of signals when
the same reactions are carried out in D2O.
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A partial H NMR spectrum corresponding to the fully protio 8 is shown in Figure 1A. The
signals corresponding to the diastereotopic protons at C3 and C5 are indicated. Each
diastereotopic proton at C3 appears as a doublet of doublets (centered at 2.66 ppm) with the
downfield one centered at 2.75 ppm and the upfield one centered at 2.64 ppm. When the Pp
reaction is carried out in D2O, there is a loss of the downfield doublet of doublets and the
collapse of the upfield signal into a broadened doublet at 2.64 ppm (Figure 1B). The Lc reaction
gives the same result (Figure 1C). (The doublet at 2.78 ppm, indicated by the asterisk in Figures
1B and 1C, corresponds to the C3 methylene group of 2ꢀoxoꢀ4ꢀhexenoate.)
The two diastereotopic protons at C5 appear as a complex multiplet centered at 1.33 ppm
(Figure 1A) due to coupling with the C4 proton and the additional coupling with the C6 methyl
group (along with the germinal coupling of the C5 protons). When the Pp reaction is carried out
in D2O, the signal simplifies into a broadened tripletꢀmultiplet at (1.30 ppm). Again, the Lc
reaction gives the same result (Figure 1C). (The doublet at 1.43 ppm, indicated by the asterisk
Figures 1B and 1C, corresponds to the C5 methyl group of 2ꢀoxoꢀ4ꢀhexenoate.)
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The same set of experiments was carried out using the Pp and Lc 4ꢀOD/VPH with 7. A
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partial H NMR spectrum corresponding to the fully protio 9 is shown in Figure 2A where the
7
8
9
diastereotopic protons at C3 and C5 are indicated. Each diastereotopic proton at C3 appears as a
doublet of doublets (centered at 2.87 ppm). When the Pp reaction is carried out in D2O, there is a
loss of the downfield doublet of doublet and a collapse of the upfield doublet of doublets into a
broadened doublet centered at 2.82 ppm (Figure 2B). The Lc reaction in D2O gives the same
result (Figure 2C), indicating that the halogen or the enzymes does not change the
stereochemical outcome.
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In this case, the signals corresponding to the diastereotopic protons at C5 (of 9) are shifted
downfield. Each diastereotopic proton at C5 gives rise to a doublet of doublets (centered at 3.49
ppm) where the downfield signal is centered at 3.53 ppm and the upfield one is centered at 3.45
ppm (Figure 2A). When the Pp reaction is carried out in D2O, there is a loss of the downfield
doublet of doublet and a collapse of the upfield doublet of doublets into a broadened doublet
centered at 3.44 (Figure 2B). The Lc reaction in D2O gives the same result (Figure 2C),
indicating that the halogen does not change the stereochemical outcome.
Assignment of the Stereochemistry at C-3 and C-5 of 8 and 9. The stereochemistry at C3 of
1
3,5ꢀdi[D]8 and 3,5ꢀdi[D]9 can be assigned based on the coupling constants in the H NMR
spectra (Figures 1B,C and 2B,C). We have previously shown the stereoselective incorporation of
1
a deuteron at Cꢀ3 of 4, and assigned the stereochemistry by chemical degradation and H NMR
analysis of the resulting product.9,29 These analyses showed that the 4ꢀOD/VPH complex
produces the (3S,4S)ꢀ[3ꢀD]4 in D2O. A deuteron is also incorporated at Cꢀ5, but Cꢀ5 remains
achiral. In the present analysis, we confirmed that the complex produces the 3S isomer of 3ꢀ[D]8
and 3ꢀ[D]9. In both products, the vicinal coupling constant (8.4 and 8.2 Hz, respectively)
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indicates an anti relationship between the remaining Cꢀ3 proton and the Cꢀ4 proton (due to the
conformation stabilized by a hydrogen bond between the Cꢀ4 hydroxyl group and Cꢀ2 carbonyl
oxygen11, as shown in Scheme 6).30 For Cꢀ5 in 8 and 9, the vicinal coupling constant (7.4 and
5.7 Hz, respectively) indicates an anti relationship between the remaining Cꢀ5 proton and the Cꢀ4
proton (assuming both molecules have a conformation stabilized by a hydrogen bond between
the Cꢀ4 hydroxyl group and Cꢀ2 carbonyl oxygen11, as shown in Scheme 6).30 In this case, the
complex produces the 5S isomer of [5ꢀD]8 and the 5R isomer of [5ꢀD]9 (due to a change in
priority). Hence, the 4ꢀOD/VPH complex from P. putida mtꢀ2 and L. cholodnii produced
(3S,4S,5S)ꢀ3,5ꢀ[diꢀD]8 and (3S,4R,5R)ꢀ3,5ꢀ[diꢀD]9.
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DISCUSSION
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In addition to VPH from the TOL plasmid (pWW0), two other hydratases have been studied
in detail.5,11,12 The first, designated MhpD, catalyzes the same reaction in a strain of E. coli (E.
coli str. Kꢀ12 substr. W3110) that degrades phenylpropionate (18, Scheme 7) via a metaꢀfission
pathway.5 MhpD does not exist as a complex with the preceding enzyme in the pathway, which
is MhpC, a hydrolase that converts 2ꢀhydroxyꢀ6ꢀketononaꢀ2,4ꢀdiene 1,9ꢀdioate (19) to 2 and
succinate. It has 42% sequence identity and 58% similarity with VPH (including the
conservation of several regions of the sequence).5
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The second enzyme, 2ꢀoxoꢀheptꢀ4ꢀeneꢀ1,7ꢀdioate hydratase (designated HpcG or OHED
hydratase), converts 2ꢀoxoꢀheptꢀ4Zꢀeneꢀ1,7ꢀdioate (20, Scheme 8) to 2ꢀoxoꢀ4Sꢀhydroxyꢀheptaꢀ
1,7ꢀdioate (21) in the homoprotocatechuate (HPC) pathway in E. coli C.11,12 The HPC pathway
parallels the reactions of the catechol metaꢀfission pathway (Scheme 1), but the intermediates
ꢀ
have a carboxymethyl group (i.e., ꢀCH2CO2 ) at Cꢀ5 (of 2 and 4 in Scheme 1, for example). The
pathway provides a degradative route for phenylalanine and tyrosine.11,12 OHED hydratase
functions as a “freeꢀstanding” enzyme that is not part of a complex.
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Studies of VPH have been complicated by the observation that the enzyme exists as a
complex with 4ꢀOD, the preceding enzyme in the pathway.3,4,9 Both 4ꢀOD and VPH from P.
putida mtꢀ2 are largely insoluble when expressed separately.3,9 Nonetheless, a number of
insights were obtained by studies of the complex (metal ion dependence where Mn2+ is preferred
over Mg2+, lack of a Schiff base or lactone intermediate, and the complex produces 4Sꢀ4) and the
complex where each enzyme is inactivated by mutagenesis of key residues identified by
sequence analysis. Studies of the 4ꢀOD/E106QVPH complex (which is nearly devoid of
hydratase activity) yielded two critical insights: Gluꢀ106 is essential for catalysis by VPH, and 2
is the product of 4ꢀOD and the likely substrate for VPH.9
These findings are largely consistent with those observed for MhpD and HpcG.5,11,12 One
apparent variation is that 20 appears to be the substrate for the OHED hydratase reaction, and not
2ꢀhydroxyꢀ2,4Zꢀheptadieneꢀ1,7ꢀdioate (22), the 5ꢀcarboxymethyl analog of 2. This conclusion is
based on the observation that the previous enzyme in the pathway, 5ꢀ(carboxymethyl)ꢀ2ꢀoxoꢀ3ꢀ
hexeneꢀ1,6ꢀdioate decarboxylase (COHED), produces 3ꢀ[D]20 when the reaction is carried out in
D2O.25 (The COHEDꢀcatalyzed reaction produces the 4Zꢀisomer of 20, and the 4ꢀODꢀcatalyzed
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reaction produces the 4Z isomers of 6 and 7 from 13 or 14, respectively.) However, 20 and 22
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are in rapid equilibrium in aqueous buffer so a direct determination is not possible.
A
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stereochemical analysis of the OHED hydrataseꢀcatalyzed reaction (in D2O) showed that the
final product of the reaction is (3S,4S,5S)ꢀ3,5ꢀ[diꢀD]21.11 [In the initial report, the stereochemical
assignment was inadvertently switched at Cꢀ3 of (4S)ꢀ21].11 The analysis clearly established that
deuterons were incorporated at C3 and C5 in a stereospecific manner when the reaction was
carried out in D2O. Any proposed mechanism would have to account for the involvement of
both centers.
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We (and others) have suggested a ketonization/hydration mechanism (for VPH) or an
isomerization/hydration mechanism (for OHED hydratase). In these scenarios, water would be
added by the enzyme to an α,βꢀunsaturated ketone (3 in Scheme 1).5,11,12 In the VPHꢀcatalyzed
reaction, ketonization of 2 in D2O would label Cꢀ5 (of 3) with deuterium. The addition of water
(D2O) would then label Cꢀ3 (of product, 4) with deuterium. Various groups have proposed that
the water would be activated by the metal ion. 4,5,10ꢀ12
The strongest support for this mechanism comes from the crystallographic analysis of OHED
hydratase carried out by Izumi et al.12 Based on this analysis, the mechanism shown in Figure 3
was formulated. The authors suggested that the metal ion (Mg2+) is coordinated by the Cꢀ2
carbonyl group and the Cꢀ1 carboxylate group (of 20) based on modeling the substrate into the
crystal structure of the enzymeꢀoxalate complex.12 (Oxalate is a competitive inhibitor of the
enzyme.5) This coordination and a hydrogen bond to Lysꢀ61 favor the enol form (22) of the
substrate (20). A nearby water molecule (hydrogen bonded to Gluꢀ106) is responsible for
proton/deuteron exchange at Cꢀ3 (in D2O). The enol form then undergoes ketonization to form
the α,βꢀunsaturated ketone (e.g., 23, Scheme 9) with incorporation of a deuteron at Cꢀ5. This
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deuteron comes from a nearby water molecule held in place by the backbone carbonyl group of
Alaꢀ166 and the side chain carboxylate group of Aspꢀ79. The loss of the proton from this water
molecule is proposed to activate the water so it can add to Cꢀ4 (according to Izumi et al).12
Hence, the deuteron at Cꢀ5 (in D2O) and the hydroxyl group at Cꢀ4 come from the same side (in
the active site). The crystal structure further indicates that the water molecule is not one of those
coordinated by the Mg2+, which argues against metal activated water molecule. Finally, the
reaction is completed by the return of the deuteron at Cꢀ3 (from the water molecule hydrogen
bonded to Gluꢀ106). In this case, the addition of the hydroxyl group at Cꢀ4 and the deuteron at
Cꢀ3 are on opposite sides (suggesting an anti addition).
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Izumi et al. also provided an explanation for the observation that the α,βꢀunsaturated ketone
(in this case, 3Eꢀ23) is not processed by the enzyme when introduced exogenously.12 The
ketonization of 22 to 23 places a proton at Cꢀ5 (deuteron in D2O). Because this step activates the
water molecule for attack at Cꢀ4, the water molecule would be in a much less reactive state to
add to 23 when presented with 23.12 Although 23 might bind to the enzyme, it would not be
processed.12 (The 3Z isomer of 23 puts the two carboxylate groups in an unfavorable
conformation so that it is not a likely intermediate.11,12
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The two VPHꢀcatalyzed reactions show the same stereochemical outcome as that of the
OHEDꢀcatalyzed reaction. (When product 21 is released into solution, it adopts the more stable
conformation as shown in Scheme 10.11) The same mechanism proposed by Izumi et al.12 could
be operative although the ketonization of 2 to 3 might not be a discrete step, but simply a
delocalization of the electrons across the dienolate system. In fact, the mechanism might be a
common one to this group of enzymes.
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Many more similar hydratases have been identified in the course of various sequencing
projects. Among these hydratases is one that is proposed to process halogenated substrates in a
metaꢀfission pathway in Comamonas sp. strain CNBꢀ1.31,32 Comamonas sp. strain CNBꢀ1 utilizes
4ꢀchloronitrobenzene as a sole carbon source.31,32 The proposed degradative pathway parallels
the canonical metaꢀfission pathway where the intermediates corresponding to 10, 5, 2, and 4 are
depicted with a halogen substituent at the X position (Scheme 11).
The presence of the halogen raises questions about its effect on binding and reactivity and
whether the corresponding enzymes from C. sp. strain CNBꢀ1 have evolved the capability to
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process the halogenated intermediates selectively over the nonꢀhalogenated ones.6,33 However,
the strain is not available. Although L. cholodnii SP-6 is not known for its ability to degrade
halogenated aromatic compounds,34 4ꢀOT, 4ꢀOD, and VPH from L. cholodnii SP-6 share high
pairwise sequence identity and similarity with the reported sequences for the C. strain CNBꢀ1 (4ꢀ
OT: 78%, 87%; 4ꢀOD: 82%, 90%; VPH: 85%, 92%; identity and similarity, respectively). In
contrast, the P. putida enzymes show lower sequence identity and similarity with the Lc enzymes
(4ꢀOT: 45%, 71%; 4ꢀOD: 64%, 82%; VPH: 67%, 78%; identity and similarity, respectively).
These similarities suggest that the Lc enzymes might be representative models for the
unavailable C. sp. strain CNBꢀ1 enzymes.
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The effects of halogen substituents have been studied more extensively in polychlorinated
biphenyl (PCB)ꢀdegrading organisms.33 The studies were prompted by the observation that the
bacteria can only partially degrade PCBs. Several roadblocks were uncovered, but they all have
a steric basis. For example, one enzyme hydrolyzed a series of 3ꢀsubstituted substrates in the
order H > F > Cl > CH3.33 The larger the substituent, the less likely it was to be hydrolyzed.
Large substituents also block the formation of a key intermediate and led to nonꢀproductive
binding modes.33
Our study using 6 (where the 5ꢀCH3 might present a steric hindrance) and 7 (where the 5ꢀCl
might present a steric and/or electronic impairment) as substrates for VPH indicates that there are
no differences in the overall stereochemical outcomes. Although kinetic parameters have not
been reported, this finding suggests that there are no major differences in the arrangement of the
active site groups involved in the ketonization/hydration mechanism. In the course of this study,
we found that the 5ꢀhalogenatedꢀ2ꢀhydroxymuconates (e.g., 10 in Scheme 11 where R = Cl, Br,
and F) behave very differently in solution than 2ꢀhydroxymuconate (10 where R = H). There
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were also subtle differences in the processing of these compounds by the Pp and Lc enzymes,
with consequences for activity. These results will be reported in the near future.
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AUTHOR INFORMATION
Corresponding Author
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*Eꢀmail: whitman@austin.utexas.edu. Telephone: (512) 471ꢀ6198. Fax (512) 232ꢀ2606.
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Funding
This research was supported by the National Institutes of Health Grants (GMꢀ41239) and the
Robert A. Welch Foundation Grant (Fꢀ1334).
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGEMENTS
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The protein mass spectrometry analysis was conducted in the Institute for Cellular and
Molecular Biology Protein and Metabolite Analysis Facility at the University of Texas at Austin.
We thank Steve D. Sorey (Department of Chemistry, University of Texas at Austin) for his
expert assistance in the acquisition of the 1H NMR spectra.
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ABBREVIATIONS
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DMF, dimethyl formamide; DMSOꢀd6, dimethyl sulfoxideꢀd6; Lc, Leptothrix cholodnii SPꢀ6;
LB, LuriaꢀBertani; MhpD, 2ꢀhydroxypentadienoic acid hydratase; 4ꢀOD, 4ꢀoxalocrotonate
decarboxylase; 4ꢀOT, 4ꢀoxalocrotonate tautomerase; PCB, polychlorinated biphenyl; PCR,
polymerase chain reaction; Pp, Pseudomonas putida mtꢀ2; SDSꢀPAGE, sodium dodecyl sulfateꢀ
polyacrylamide gel electrophoresis; VPH, vinylpyruvate hydratase
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REFERENCES
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Manjasetty, B., Powlowski, J., and Vrielink, A. (2003) Crystal structure of a bifunctional
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