The 4-Oxalocrotonate Tautomerase- and YwhB-Catalyzed Hydration of 3E-Haloacrylates: Implications for the Evolution of New Enzymatic Activities

Article abstract of DOI:10.1021/ja0370948

4-Oxalocrotonate tautomerase (4-OT) catalyzes the conversion of 2-oxo-4E-hexenedioate to 2-oxo-3E-hexenedioate through the intermediate, 2-hydroxy-2,4E-hexadienedioate. 4-OT and a homologue found in Bacillus subtilis (designated YwhB) share sequence identity and two key catalytic groups, Pro-1 and Arg-11, with the two subunits comprising trans-3-chloroacrylic acid dehalogenase (CaaD). 4-OT and YwhB have now been found to display a low-level hydratase activity, resulting in the dehalogenation of 3E-haloacrylates. The enzymes are highly selective for the (E)-isomer, and Pro-1 is critical for the activity while an arginine is likely required. Two mechanisms are proposed in which Pro-1 functions as a general base or a general acid catalyst and, along with the arginine, facilitates the Michael addition of water. Both mechanisms suggest an intriguing route for the evolution of the CaaD activity. One or more mutations could decrease the hydrophobic environment of the active site, which would make it more favorable for a hydrolytic reaction, thereby raising the pK_a of Pro-1 and increasing the concentration of enzyme in the reactive form. Copyright

Full text of DOI:10.1021/ja0370948

Published on Web 11/01/2003
The 4-Oxalocrotonate Tautomerase- and YwhB-Catalyzed Hydration of
3E-Haloacrylates: Implications for the Evolution of New Enzymatic Activities
Susan C. Wang, William H. Johnson, Jr., and Christian P. Whitman*
DiVision of Medicinal Chemistry, College of Pharmacy, The UniVersity of Texas, Austin, Texas 78712-1074
Received July 6, 2003; E-mail: whitman@mail.utexas.edu
Scheme 1
Nature may exploit the catalytic promiscuity observed in some
enzymes in order to create new enzymes.1 A catalytically promiscu-
ous enzyme has one or more low-level activities in addition to its
primary, physiological activity.2 Duplication of the gene, followed
by a series of mutations to amplify the desired activity, generates
Scheme 2
a new enzyme now having the progenitor’s low-level activity as
the primary activity. We report herein the observation of low-level
hydration activities in the bacterial isomerase, 4-oxalocrotonate
tautomerase (4-OT) from Pseudomonas putida mt-2 and a 4-OT
homologue, YwhB, found in Bacillus subtilis.3 In addition to
providing very clear examples of catalytically promiscuous en-
zymes, these observations support the proposed evolutionary link
was required to convert ∼30% of (E)-4 to a mixture of 8 and 9.
Both enzymes will process (E)-5: ∼92% of (E)-5 is converted to
between these enzymes and the recently characterized trans-3-
8 and 9 after 209 h by 4-OT and ∼93% of (E)-5 is converted to a
chloroacrylic acid dehalogenase (CaaD)4 and show that 4-OT- and
mixture of 8 and 9 after 616 h by YwhB.
YwhB-like sequences have diverse catalytic capabilities and may
have served as templates for the creation of new enzymatic
activities.
A control experiment demonstrates that the hydration of (E)-4
is an enzyme-catalyzed process. An H NMR spectrum of (E)-4
1
incubated in Na2HPO4 buffer (pH 6.8) for 192 h (8 days) showed
that only (E)-4 was present, ruling out a nonenzymatic hydration.
The nonenzymatic hydration of (E)-4 requires much harsher
conditions: only ∼10% of the chloride is removed after 24 h in
0.5 M aqueous NaOH at 60 °C.11
The results show that the incubation of (E)-4 and (E)-5 with
4-OT or YwhB generates 8, which is readily hydrated to yield 9.
A likely scenario for the formation of 8 from these compounds
involves the initial enzymatic hydration of the 3E-haloacrylates to
produce an unstable halohydrin species (6, Scheme 2), which
decomposes to malonate semialdehyde (7).4b Nonenzymatic decar-
boxylation of 7 yields 8. Compound 7 is not sufficiently stable to
accumulate in quantities detectable by 1H NMR spectroscopy during
the lengthy incubation periods.
The preparations of 4-OT and YwhB used in these experiments
were highly purified, but it remained possible that a contaminating
enzyme could be responsible for the observed activity. To eliminate
this concern, two control experiments were performed. First, a
partially purified protein sample from cells harboring an “empty”
pET-24a(+) vector (i.e., the genes encoding 4-OT or YwhB are
absent) was examined.12 Neither (E)-4 nor (E)-5 was converted to
product after 10.5 days using 2.6 mg of protein. In addition,
incubation of synthetically prepared 4-OT (likely to be free of
contaminating cellular enzymes) with (E)-4 for 208 h led to the
same product mixture in comparable amounts.
The isomer specificities of 4-OT and YwhB were also investi-
gated. An 1H NMR spectrum of a reaction mixture containing (Z)-4
and 4-OT showed that ∼3-4% of the Z-isomer was converted to
8 and 9 after lengthy incubation (139 h using 0.64 mg). In contrast,
YwhB showed no detectable activity with (Z)-4 after prolonged
incubation (209 h using 0.51 mg). The enzymes show a clear
preference for the E-isomer, which is consistent with the structural
resemblance of (E)-4 to the acrylate portion of 4E-1 and 2.
Moreover, this observation implicates the active sites of the two
enzymes in the low-level activity.13
4-OT, found in a pathway that degrades aromatic hydrocarbons,
catalyzes the conversion of 2-oxo-4E-hexenedioate (1; Scheme 1)
to 2-oxo-3E-hexenedioate (3) through 2-hydroxy-2,4E-hexadiene-
dioate (2).5 Among the key catalytic residues are Pro-1, which
abstracts the C-3 proton of 1, and Arg-11, which participates in
both substrate binding (at C-6) and in catalysis.6 4-OT is the title
enzyme of the 4-OT family, whose structurally homologous
members are constructed from short monomers (61-81 amino
acids), which conserve a signature â-R-â structural motif as well
as Pro-1.7 YwhB shares 36% pairwise sequence identity with 4-OT
and retains both Pro-1 and Arg-11.
Sequence analysis first suggested that the heterohexameric CaaD,
responsible for the hydrolytic dehalogenation of 3E-chloro- and
3E-bromoacrylate [(E)-4 and (E)-5, respectively; Scheme 2], might
be a 4-OT family member.4a The pairwise sequence identities
observed between YwhB and the R-subunit of CaaD (35%) and
4-OT and the â-subunit (25%) are low, but mutagenesis indicated
that â-Pro-1 and R-Arg-11 are critical for activity, further
substantiating a relationship between CaaD and the 4-OT family.4a
In view of these observations, we examined whether 4-OT and
YwhB catalyzed the hydration of 3E-haloacrylates, resulting in their
dehalogenation. Accordingly, each protein was incubated with (E)-
1
4, and the reactions were monitored by H NMR spectroscopy.8
The 1H NMR spectrum of (E)-4 in Na2HPO4 buffer (pH 6.8) shows
two doublets (6.09 and 6.89 ppm), which correspond to the C-2
and C-3 protons, respectively. After incubation of (E)-4 with 4-OT
(0.6 mg) for 136 h, the intensity of these two signals diminishes
and four new signals appear. Two signals (2.04 and 9.48 ppm)
correspond to acetaldehyde (8), whereas the other two signals (1.13
and 5.05 ppm) correspond to its hydrate (9).4a,9 Integration of the
signals indicates that ∼74% of (E)-4 has been converted to a
mixture of 8 and 9. Incubation of YwhB with (E)-4 results in the
same products, although the reaction is not as efficient.10 Although
less YwhB (0.3 mg) was used, a longer incubation period (171 h)
9
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J. AM. CHEM. SOC. 2003, 125, 14282-14283
10.1021/ja0370948 CCC: $25.00 © 2003 American Chemical Society

C O M M U N I C A T I O N S
Table 1. Kinetic Parameters for 4-OT, YwhB, and Mutants
enzyme sub.
cat, (s-1 kcat/Km, (M-1 s-1
4-OT
26c
decrease the hydrophobic environment of 4-OT’s active site, making
it more hydrophilic. The resulting active site would now be more
amenable to a hydrolytic reaction, thereby raising the pKa of Pro-1
and increasing the concentration of enzyme in the reactive form.
This possibility is being explored.
k
)
)
3500 ( 500
(2.0 ( 0.3) × 107
(2.6 ( 0.4) × 10-2
(2.0 ( 0.3) × 10-4
(1.6 ( 0.1) × 10-1
(2.8 ( 0.1) × 104
(4.4 ( 1.0) × 10-2
(1.9 ( 0.2) × 10-3
(1.2 ( 0.1) × 105
4-OT
(E)-4
(E)-4
(E)-4
2
(8.3 ( 0.5) × 10-4
P1A-4-OT
R11A-4-OT
YwhB
-
-
Acknowledgment. We thank Dr. Michael C. Fitzgerald (De-
partment of Chemistry, Duke University) for synthetic 4-OT. This
research was supported by the National Institutes of Health Grant
GM-41239 and the Robert A. Welch Foundation (F-1334). S.C.W.
is a Fellow of the American Foundation for Pharmaceutical
Education.
26 ( 1.4
YwhB
R11A-YwhB
CaaD
(E)-4
(E)-4
(E)-44b
-
-
3.8 ( 0.1
Having established a low-level CaaD activity for both enzymes,
kinetic parameters were determined (Table 1).14 Saturation with
(E)-4 was not achieved for YwhB. A comparison of the kcat/Km
values shows that 4-OT and YwhB are 4.6 × 106-fold and 2.7 ×
106-fold, respectively, less efficient than CaaD.15 For 4-OT, binding
(Km) and turnover (kcat) are adversely and comparably impacted
such that a 103-fold decrease in Km coupled with a 103-fold increase
in kcat will produce CaaD activity levels. With regard to the
conversion of 2 to 3, the CaaD activities of 4-OT and YwhB are
7.7 × 108-fold and 6.4 × 105-fold less efficient, respectively.
To gauge the importance of Pro-1 and Arg-11 to the activity,
kcat/Km values were also determined for three mutants, as saturation
could not be achieved with these mutants (Table 1).16 The kcat/Km
value for the P1A-4-OT mutant is 108-fold less than that of the
wild type, whereas the kcat/Km value for the R11A-4-OT mutant is
slightly greater (6-fold). The kcat/Km value for the R11A-YwhB
mutant is 23-fold less than that of YwhB, whereas there is no
detectable activity for the P1A-YwhB mutant. These results provide
further evidence indicating that 4-OT and YwhB are responsible
for the observed activities and that Pro-1 is critical for activity in
both enzymes, whereas Arg-11 is only essential for the CaaD
activity of YwhB.
Two mechanisms might explain the observed hydratase activity.
In both mechanisms, a positively charged residue (e.g., Arg-11)
may interact with the C-1 carboxylate group of (E)-4 or (E)-5 and
draw electron density away from the C-3 position to form an enolic
intermediate. The partial positive charge at C-3 is now susceptible
to a Michael addition of water. 4-OT has two active-site arginines,
Arg-11 and Arg-39, whereas YwhB has only Arg-11. Thus,
mutation of Arg-11 in 4-OT might not lead to a loss of activity
because (E)-4 may interact with Arg-39. In one mechanism, Pro-1
may function as a general base and activate the water molecule for
addition to C-3. Subsequently, Pro-1, now functioning as a general
acid, would deliver the proton to C-2 to complete the addition of
water. In a second mechanism, water may add to C-3 as a result of
the partial positive charge, and Pro-1 might act as a general acid
catalyst and deliver a proton to the C-2 position of (E)-4 or (E)-5
upon ketonization of the enediolate intermediate.
References
(1) (a) Jensen, R. A. Annu. ReV. Microbiol. 1976, 30, 409-425. (b) Hughes,
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(4) (a) Poelarends, G. J.; Saunier, R.; Janssen, D. B. J. Bacteriol. 2001, 183,
4269-4277. (b) Wang, S. C.; Person, M. D.; Johnson, W. H., Jr.;
Whitman, C. P. Biochemistry 2003, 42, 8762-8773.
(5) Whitman, C. P.; Aird, B. A.; Gillespie, W. R.; Stolowich, N. J. J. Am.
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(6) (a) Stivers, J. T.; Abeygunawardana, C.; Mildvan, A. S.; Hajipour, G.;
Whitman, C. P. Biochemistry 1996, 35, 814-823. (b) Czerwinski, R. M.;
Johnson, W. H., Jr.; Whitman, C. P.; Harris, T. K.; Abeygunawardana,
C.; Mildvan, A. S. Biochemistry 1997, 36, 14551-14560. (c) Harris, T.
K.; Czerwinski, R. M.; Johnson, W. H., Jr.; Legler, P. M.; Abeygunawar-
dana, C.; Massiah, M. A.; Stivers, J. T.; Whitman, C. P.; Mildvan, A. S.
Biochemistry 1999, 38, 12343-12357.
(7) Almrud, J. J.; Kern, A. D.; Wang, S. C.; Czerwinski, R. M.; Johnson, W.
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(8) The 1H NMR spectra were recorded in 100% H2O on a Varian Unity
INOVA-500 spectrometer.4b The NMR tube contained 100 mM Na2HPO4
buffer (0.6 mL, pH 9.2) and (E)-4 (4 mg, 0.04 mmol) dissolved in DMSO-
d6 (30 µL). Adding (E)-4 adjusted the pH to 6.8. Subsequently, a quantity
of enzyme (in 20 mM NaH2PO4 buffer, pH 7.3) was added to the NMR
tube. The purification of 4-OT has been reported.6b The enzymes do not
lose significant activity in the time frame of these experiments.
(9) Gil, A. M.; Duarte, I. F.; Delgadillo, I.; Colquhoun, I. J.; Casuscelli, F.;
Humpfer, E.; Spraul, M. J. Agric. Food Chem. 2000, 48, 1524-1536.
(10) The purification and characterization of YwhB has been reported. YwhB
converts 2 to 1 as well as 2 to 3 but does not catalyze the latter reaction
as efficiently as 4-OT (Table 1). Wang, S. C.; Johnson, W. H., Jr.;
Czerwinski, R. M.; Whitman, C. P. Biochemistry, in press.
(11) Braddon, S. A.; Dence, C. W. Tappi 1968, 51, 249-256.
(12) An aliquot of pET-24a(+) (0.5 µL) was transformed into electrocompetent
Escherichia coli strain BL21-Gold(DE3)pLysS cells. Cells carrying the
pET-24a(+) plasmid were grown and induced for protein expression as
described.6b Cells were lysed by sonication, the cell lysate was centrifuged,
and the resulting supernatant was made 2 M in (NH4)2SO4. After being
stirred for 1 h (4 °C), the solution was centrifuged, and the supernatant
was transferred to a Millipore Ultrafree 15 (MWCO 5000 Da) centrifugal
concentrator and exchanged into 20 mM NaH2PO4 buffer, pH 7.3, to give
a final volume of 0.6 mL.
(13) The low-level cis-CaaD activity of 4-OT suggests that a 4-OT-like
sequence could have been the template for the evolution of a known cis-
CaaD: van Hylckama Vlieg, J. E. T.; Janssen, D. B. Biodegradation 1992,
2, 139-150. cis-CaaD has recently been identified as a tautomerase
superfamily member. Poelarends, G. J.; Serrano, H.; Person, M. D.;
Johnson, W. H., Jr.; Murzin, A. G.; Whitman, C. P. Biochemistry, in press.
(14) Assay mixtures (1-mL) contained 100 mM K2HPO4 buffer, pH 7.8, NAD+
(0.27 mg), aldehyde dehydrogenase (0.7 mg), â-mercaptoethanol (4 µg),
substrate (4-OT: 0.94-79.4 mM; YwhB: 23.5-70.4 mM), and enzyme
(4-OT: 0.5 mg; YwhB: 0.05 mg). Assays were performed at 23 °C
following the conversion of NAD+ to NADH.
The proposed role for Pro-1 in the latter mechanism is suggested
by recent findings implicating the â-Pro-1 as a general acid catalyst
in the CaaD-catalyzed reaction.17 The â-Pro-1 of CaaD has a pKa
of ∼9.2, enabling it to function as a general acid catalyst.17 In
contrast, the catalytic Pro-1 in 4-OT has a pKa of ∼6.4 due to its
presence in the hydrophobic active site.6a This lowered pKa value
enables it to function as a general base catalyst in its physiological
activity at cellular pH. Under the conditions of the kinetic
experiments (pH 7.8), very little 4-OT is present with Pro-1 in the
correct protonation state to function as a general acid catalyst. Both
mechanisms offer an interesting evolutionary route for the produc-
tion of a more efficient dehalogenase. One or more mutations could
(15) The kcat for 4-OT using (E)-5 is (3.2 ( 0.4) × 10-3 s-1, and the kcat/Km
is 0.52 ( 0.12 M-1 s-1, which are 3.8-fold and 2-fold greater than those
measured for 4-OT using (E)-4.
(16) The preparation of the 4-OT mutants is described elsewhere.6b,c YwhB
mutants were constructed by overlap extension PCR (Ho, S. N.; Hunt, H.
D.; Horton, R. M.; Pullen, J. K.; Pease, L. R. Gene 1989, 77, 51-59)
using the protocol described for the P1A- and R11A-4-OT mutants with
the YwhB gene as template.6b,c
(17) Azurmendi, H. F.; Wang, S. C.; Massiah, M. A.; Whitman, C. P.; Mildvan,
A. S. Biochemistry 2003, 42, 8619; Chem. Abstr. 2003, 42, 113.
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