The hydratase activity of malonate semialdehyde decarboxylase: Mechanistic and evolutionary implications

Article abstract of DOI:10.1021/ja044304n

Malonate semialdehyde decarboxylase (MSAD) is a member of the tautomerase superfamily, a group of structurally homologous proteins that have a characteristic β-α-β-fold and a catalytic amino-terminal proline. In addition to its physiological decarboxylase activity, the conversion of malonate semialdehyde to acetaldehyde and carbon dioxide, the enzyme has now been found to display a promiscuous hydratase activity, converting 2-oxo-3-pentynoate to acetopyruvate, with a k_cat/K_m value of 6.0 × 10² M^-1 s^-1. Pro-1 and Arg-75 are critical for both activities, and the pK_a of Pro-1 was determined to be _～9.2 by a direct ¹⁵N NMR titration. These observations implicate a decarboxylation mechanism in which Pro-1 polarizes the carbonyl oxygen of substrate by hydrogen bonding and/or an electrostatic interaction. Arg-75 may position the carboxylate group into a favorable orientation for decarboxylation. Both the hydratase activity and the pK_a value of Pro-1 are shared with trans-3-chloroacrylic acid dehalogenase, another tautomerase superfamily member that precedes MSAD in a bacterial degradation pathway for trans-1,3-dichloropropene. Hence, MSAD and CaaD could have evolved by divergent evolution from a common ancestral protein, retaining the necessary catalytic components for the conjugate addition of water. Copyright

Full text of DOI:10.1021/ja044304n

Published on Web 11/12/2004
The Hydratase Activity of Malonate Semialdehyde Decarboxylase:
Mechanistic and Evolutionary Implications
Gerrit J. Poelarends, Hector Serrano, William H. Johnson, Jr., David W. Hoffman, and
Christian P. Whitman*
DiVision of Medicinal Chemistry, College of Pharmacy, and Department of Chemistry and Biochemistry,
Institute for Cellular and Molecular Biology, The UniVersity of Texas, Austin, Texas 78712
Received September 18, 2004; E-mail: whitman@mail.utexas.edu
Scheme 1
Malonate semialdehyde decarboxylase (MSAD) from Pseudomo-
nas paVonaceae 170 catalyzes the metal ion-independent decar-
boxylation of 2 (Scheme 1), a catabolite generated by the bacterial
degradation of the nematocide trans-1,3-dichloropropene.1 Searches
of the protein databases did not uncover a relationship between
MSAD and known decarboxylases but instead suggested that the
enzyme might be a member of a new family within the tautomerase
superfamily.1 The members of this superfamily are structurally
homologous proteins that share a characteristic â-R-â-fold as well
as a catalytic amino-terminal proline.2 Sequence analysis, site-
directed mutagenesis, and chemical modification studies implicated
Pro-1 and Arg-75 as critical active-site residues in MSAD,1 thereby
supporting its association with the tautomerase superfamily and
suggesting two mechanisms for decarboxylation. In one mechanism
(Scheme 2A), Pro-1 is a charged species that polarizes the C-3
carbonyl group of 2 by hydrogen bonding and/or an electrostatic
interaction. In a second mechanism (Scheme 2B), Pro-1 is nucleo-
philic and reacts with the C-3 carbonyl group to form a Schiff base.
In both mechanisms, Arg-75 may position the carboxylate group
in an orientation that favors decarboxylation. A distinguishing
feature between these mechanisms is the ionization state of the
amino group of Pro-1.
Scheme 2
Scheme 3
Previous work has shown that the reactions of 4-oxalocrotonate
tautomerase (4-OT) and trans-3-chloroacrylic acid dehalogenase
(CaaD), the best characterized members of the 4-OT family in the
tautomerase superfamily, with 2-oxo-3-pentynoate (4, Scheme 3)
reflect both the ionization state of Pro-1 and the environment of
the active site.2-4 4-OT is irreversibly inactivated by 4 due to the
covalent modification of Pro-1, whereas CaaD converts 4 to
acetopyruvate (6, Scheme 3). The dissimilar reactions mirror
differences between the two active sites. Pro-1 of 4-OT has a pKa
of ∼6.4.5 Hence, at neutral pH, Pro-1 functions as a nucleophile
and attacks C-4 of 4 in a Michael-type reaction.6 In contrast, the
pKa of âPro-1 in CaaD is ∼9.2.7 Thus, a Michael-type reaction
between the amino group of proline and 4 is not favored, and in
the active site of CaaD, which is designed to carry out a hydration
reaction,8 4 is processed to 6.
In view of these observations, the reaction of MSAD with 4 was
examined. Incubation of 4 with MSAD resulted in a decrease in
the absorbance at 234 nm, corresponding to 4, accompanied by
the appearance of a new absorbance peak at 294 nm, which
corresponds to acetopyruvate (6).9 In addition to the characteristic
λmax of 294 nm, the identity of 6 in the incubation mixture was
confirmed by 1H NMR spectroscopy.10 MSAD was not inactivated
by 4 in the course of these experiments. The results clearly show
that MSAD has a hydratase activity, from which it can be inferred
that Pro-1 is cationic and not functioning as a nucleophile.11
The kinetic parameters for the hydratase activity were determined
next (Table 1).12 A comparison of the kcat/Km values shows that
Table 1. Kinetic Parameters for MSAD and CaaD
1
1
enzyme
sub
kcat (s-
)
Km (mM)
kcat/Km (M-1 s-
)
MSAD
MSAD
P1A14
R75A
CaaD
21
4
4
-
-
2.2 × 107
6.0 × 102
-
5.8 ( 0.4
9.6 ( 0.9
<0.05
<0.05
0.7 ( 0.1
-
-
4
-
44b
0.11 ( 0.01
6.4 × 103
MSAD is 3.7 × 104-fold less efficient in the conversion of 4 to 6
than it is in the conversion of 2 to 3, its physiological activity.13 In
comparison with the CaaD-catalyzed hydration of 4,4b the value of
kcat for MSAD is ∼8.3-fold higher than that measured for CaaD,
while the Km value is ∼87-fold higher. The net effect is a 10.7-
fold difference in the kcat/Km value. Mutagenesis of either Pro-1 or
Arg-75 to an alanine abolishes the hydratase activity of MSAD
(Table 1), thereby confirming both the importance of these residues
to the activity as well as the active-site nature of the activity.
To obtain direct evidence for the ionization state of Pro-1, the
pKa of the amino group was measured by a pH titration of the
uniformly 15N-labeled enzyme using 15N NMR spectroscopy.15 The
15N-chemical shift of Pro-1 is resolved from the other 15N
resonances of MSAD over the pH range 4.8-10.1, and monitoring
this resonance as a function of pH yields a pKa value of 9.2 ( 0.2
for the free enzyme (Figure 1).
The measured pKa value is consistent with the observed MSAD-
catalyzed hydration of 4 and indicates that Pro-1 is primarily a
charged species in MSAD. The combined results argue strongly
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J. AM. CHEM. SOC. 2004, 126, 15658-15659
10.1021/ja044304n CCC: $27.50 © 2004 American Chemical Society

C O M M U N I C A T I O N S
(6) 4-OT, an isomerase found in a degradation pathway for aromatic
hydrocarbons in P. putida mt-2, utilizes Pro-1 as a general base for the
conversion of 2-oxo-4E-hexenedioate to 2-oxo-3E-hexenedioate through
2-hydroxy-2,4E-hexadienedioate: Whitman, C. P.; Aird, B. A.; Gillespie,
W. R.; Stolowich, N. J. J. Am. Chem. Soc. 1991, 113, 3154-3162.
(7) Azurmendi, H. F.; Wang, S. C.; Massiah, M. A.; Poelarends, G. J.;
Whitman, C. P.; Mildvan, A. S. Biochemistry 2004, 43, 4082-4091.
(8) CaaD utilizes Pro-1 of the â-subunit as a general acid catalyst in the
conversion of 1 to 2 (Scheme 1):7 de Jong, R. M.; Brugman, W.;
Poelarends, G. J.; Whitman, C. P.; Dijkstra, B. W. J. Biol. Chem. 2004,
279, 11546-11552.
(9) The incubation mixture containing MSAD and 4 was monitored by UV
spectroscopy as described.4b An aliquot of MSAD (6 µL of a 16.7 mg/
mL solution) was diluted into 20 mM Na2HPO4 buffer (to a final volume
of 1 mL, pH 9.0) and incubated for 1 min at 26 °C. The assay was initiated
by the addition of a small quantity (1.5 µL) of 4 from a 60 mM stock
solution. Spectra were recorded every 4 min for a total of 32 min, and a
final spectrum was taken 90 min after the initiation of the reaction at
which time the reaction had neared completion. An aliquot (10 µL) of
the mixture was removed, diluted into 20 mM Na2HPO4 buffer (1 mL,
pH 9.0), and assayed for residual MSAD activity. No significant loss of
activity was observed.
Figure 1. The pH titration curve displaying the 15N-chemical shift of the
amino group of Pro-1 versus pH.
(10) The mixture contained 100 mM Na2HPO4 buffer (0.6 mL, pH ∼9.2) and
4 (4 mg, 0.04 mmol) dissolved in DMSO-d6 (30 µL). The pH of the
solution was adjusted to 7.6 by the addition of aliquots of NaOH (aq).
An aliquot of MSAD (100 µL of a 19.5 mg/mL solution in 20 mM NaH2-
PO4 buffer, pH 7.3) was added to the reaction mixture and a 1H NMR
spectrum was recorded 16 h later. The major signals present in the
for a decarboxylation mechanism involving the polarization of the
C-3 carbonyl group of 2 by hydrogen bonding and/or an electrostatic
interaction (Scheme 2A), analogous to the mechanism proposed
for methylmalonyl CoA decarboxylase.16
Interestingly, the pKa of Pro-1 as well as the ability to carry out
a hydration reaction are two features shared with CaaD, an
evolutionarily related enzyme that catalyzes the preceding step (i.e.,
1 to 2 in Scheme 1) in the same catabolic pathway. Thus, the
hydratase activity of MSAD is an adventitious one and an example
of catalytic promiscuity in MSAD.17 Similar to the mechanism
proposed for the CaaD-catalyzed conversion of 4 to 6,4b MSAD
might initiate the reaction by catalyzing the Michael addition of
water to the triple bond of 4 to form an allenic species. Rearrange-
ment of this species produces 5, which readily ketonizes to form
6. Arg-75 could facilitate the addition of water across the triple
bond by interacting with the 2-carbonyl group and polarizing this
group. Pro-1 then delivers a proton to the C-3 position of the allenic
species to complete the addition of water. In CaaD, the water
molecule is activated for attack by RGlu-52. Sequence analysis did
not identify a corresponding residue in MSAD, but a recent crystal
structure suggests that Asp-37 may play a similar role.18
MSAD is a fairly proficient hydratase using 4. Although this
activity has no known consequences for the organism’s metabolism,
its presence in both CaaD and MSAD coupled with the identical
pKa values of Pro-1, suggests that the two enzymes divergently
evolved from a common ancestor, conserving elements of the
catalytic machinery necessary for the conjugate addition of water.19
The results of this study further support a role for the catalytic and
binding promiscuity of the â-R-â-fold, the key structural com-
ponent of tautomerase superfamily members, in the diversification
of enzyme function within the tautomerase superfamily.
spectrum corresponded to 4, 6, the hydrate of 6, and the enol of 6.4b
A
similar reaction mixture without MSAD, incubated for 16 h, showed no
spectral evidence for 6 or derivatives. In aqueous solution, 6 is in
equilibrium with the hydrate and the enol: Guthrie, J. P. J. Am. Chem.
Soc. 1972, 94, 7020-7024.
(11) Although ∼39% of the enzyme exists with Pro-1 in the neutral state at
pH 9.0, enzyme inactivation is not observed under the experimental
conditions.
(12) The hydration of 4 by MSAD was monitored by following the formation
of 6 at 294 nm (ꢀ ) 7000 M-1 cm-1) in 20 mM Na2HPO4 buffer (pH
9.0) at 22 °C. An aliquot of MSAD (141 µL of a 8.5 mg/mL solution)
was diluted into buffer (20 mL) and incubated for 1 h. Subsequently, a 1
mL-portion of the diluted enzyme was transferred to a cuvette and assayed
by the addition of a small quantity of 4 from a 10 mM, 100 mM, or 1 M
stock solution. The stock solutions were made by dissolving the appropriate
amount of 4 in 100 mM Na2HPO4 buffer, pH 9.0. The pH of the stock
solutions was adjusted to about 8.5. The concentrations of 4 used in the
kinetic assay ranged from 0.05 to 12 mM.
(13) Only a kcat/Km value is reported because saturation with 2 is not possible.1
(14) The MSAD mutants were prepared as described1.
(15) 15N NMR spectra were collected at 23 °C on a Varian Inova spectrometer
operating at a proton frequency of 500.3 MHz. Titrations were performed
using samples (containing 10% D2O) which were ∼3 mM in subunits of
uniformly 15N-labeled wild-type MSAD in 10 mM NaH2PO4 buffer by
extensive exchange of the buffer for the one with the desired pH. One
dimensional 15N spectra were collected at 11 pH values, about evenly
spaced, between 4.83 and 10.0. Spectra were acquired using a broad-
band probe configured for 15N direct detection, without 1H decoupling. A
70° pulse was used for the 15N nuclei, with a sweep width of 20278 Hz,
an acquisition time of 0.4 s, and recycle time of 1 s between scans;
typically 32 000 scans were signal-averaged for a total acquisition time
of 9 h per spectrum. 15N chemical shifts were referenced by multiplying
the 0 ppm 1H frequency by 0.101329118: Wishart, D. S.; Bigam, C. G.;
Yao, J.; Abildgaard, F.; Dyson, H. J.; Oldfield, E.; Markley, J. L.; Sykes,
B. D. J. Biomol. NMR 1995, 6, 135-140. 1H chemical shifts were
referenced to 2,2-dimethyl-2-silapentane-5-sulfonate at 0 ppm. The enzyme
retained nearly full activity at the end of each experiment. The pKa of
Pro-1 was determined by fitting the data obtained from the NMR titration
curve to an equation for a single pKa found in the Grafit Program
(Erithacus Software Ltd., Horley, UK), and using a lower pH limit of
51.2 ppm and a higher pH limit of 41.4 ppm.
Acknowledgment. This research was supported by the National
Institutes of Health Grant GM-65324 and the Robert A. Welch
Foundation (F-1334 and F-1353). We thank Steve D. Sorey
(Department of Chemistry, The University of Texas at Austin) for
(16) Benning, M. M.; Haller, T.; Gerlt, J. A.; Holden, H. M. Biochemistry
2000, 39, 4630-4639.
(17) (a) Jensen, R. A. Annu. ReV. Microbiol. 1976, 30, 409-425. (b) Palmer,
D. R. J.; Garrett, J. B.; Sharma, V.; Meganathan, R.; Babbitt, P. C.; Gerlt,
J. A. Biochemistry 1999, 38, 4252-4258. (c) Copley, S. D. Trends
Biochem. Sci. 2000, 25, 261-265. (d) O’Brien, P. J.; Herschlag, D. Chem.
Biol. 1999, 6, R91-R105. (e) James, L. C.; Tawfik, D. S. Protein Sci.
2001, 10, 2600-2607.
1
his assistance in acquiring the H NMR spectra.
References
(18) Almrud, J. J.; Poelarends, G. J.; Serrano, H.: Johnson, W. H., Jr.; Hackert,
(1) Poelarends, G. J.; Johnson, W. H., Jr.; Murzin, A. G.; Whitman, C. P. J.
Biol. Chem. 2003, 278, 48674-48683.
M. L.; Whitman, C. P. 2004, unpublished results.
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C. P. Arch. Biochem. Biophys. 2002, 402, 1-13.
(19) MSAD displays CaaD activity, converting 1 to 2. The activity was
determined by 1H NMR identification of 2 and the colorimetric detection
of chloride release, using previously described protocols.4 The rate is ∼2
× 105-fold slower than that reported for CaaD.4a The presence of a
contaminating protein is unlikely but cannot be excluded. Nonenzymatic
decay of 2 prevents a determination of whether CaaD has low-level MSAD
activity.
(3) Johnson, W. H., Jr.; Czerwinski, R. M.; Fitzgerald, M. C.; Whitman, C.
P. Biochemistry 1997, 36, 15724-15732.
(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.;
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