Characterization of a newly identified mycobacterial tautomerase with promiscuous dehalogenase and hydratase activities reveals a functional link to a recently diverged cis -3-chloroacrylic acid dehalogenase

Article abstract of DOI:10.1021/bi200071k

The enzyme cis-3-chloroacrylic acid dehalogenase (cis-CaaD) is found in a bacterial pathway that degrades a synthetic nematocide, cis-1,3-dichloropropene, introduced in the 20th century. The previously determined crystal structure of cis-CaaD and its prom

Full text of DOI:10.1021/bi200071k

ARTICLE
pubs.acs.org/biochemistry
Characterization of a Newly Identified Mycobacterial Tautomerase
with Promiscuous Dehalogenase and Hydratase Activities Reveals
a Functional Link to a Recently Diverged cis-3-Chloroacrylic Acid
Dehalogenase
Bert-Jan Baas, Ellen Zandvoort, Anna A. Wasiel, Wim J. Quax, and Gerrit J. Poelarends*
Department of Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1,
9713 AV Groningen, The Netherlands
S Supporting Information
b
ABSTRACT: The enzyme cis-3-chloroacrylic acid dehalogen-
ase (cis-CaaD) is found in a bacterial pathway that degrades a
synthetic nematocide, cis-1,3-dichloropropene, introduced in
the 20th century. The previously determined crystal structure of
cis-CaaD and its promiscuous phenylpyruvate tautomerase
(PPT) activity link this dehalogenase to the tautomerase super-
family, a group of homologous proteins that are characterized by
a catalytic amino-terminal proline and a β-R-β structural fold.
The low-level PPT activity of cis-CaaD, which may be a vestige
of the function of its progenitor, prompted us to search the
databases for a homologue of cis-CaaD that was annotated as a
putative tautomerase and test both its PPT and cis-CaaD activity. We identified a mycobacterial cis-CaaD homologue (designated
MsCCH2) that shares key sequence and active site features with cis-CaaD. Kinetic and 1H NMR spectroscopic studies show that
MsCCH2 functions as an efficient PPT and exhibits low-level promiscuous dehalogenase activity, processing both cis- and trans-3-
chloroacrylic acid. To further probe the active site of MsCCH2, the enzyme was incubated with 2-oxo-3-pentynoate (2-OP). At pH
8.5, MsCCH2 is inactivated by 2-OP due to the covalent modification of Pro-1, suggesting that Pro-1 functions as a nucleophile at
pH 8.5 and attacks 2-OP in a Michael-type reaction. At pH 6.5, however, MsCCH2 exhibits hydratase activity and converts 2-OP to
acetopyruvate, which implies that Pro-1 is cationic at pH 6.5 and not functioning as a nucleophile. At pH 7.5, the hydratase and
inactivation reactions occur simultaneously. From these results, it can be inferred that Pro-1 of MsCCH2 has a pKa value that lies in
between that of a typical tautomerase (pKa of Pro-1 ∼ 6) and that of cis-CaaD (pKa of Pro-1 ∼ 9). The shared activities and
structural features, coupled with the intermediate pKa of Pro-1, suggest that MsCCH2 could be characteristic of an evolutionary
intermediate along the past route for the divergence of cis-CaaD from an unknown superfamily tautomerase. This makes MsCCH2
an ideal candidate for laboratory evolution of its promiscuous dehalogenase activity, which could identify additional features
necessary for a fully active cis-CaaD. Such results will provide insight into pathways that could lead to the rapid divergent evolution of
an efficient cis-CaaD enzyme.
he bacterial enzymes trans-3-chloroacrylic acid dehalogenase
(CaaD) and cis-3-chloroacrylic acid dehalogenase (cis-CaaD)
CaaD is a heterohexamer consisting of three R-subunits (75
amino acid residues each) and three β-subunits (70 amino acid
residues each).1 Similarities in sequence and tertiary and qua-
ternary structures connect CaaD to members of the 4-oxalocro-
tonate tautomerase (4-OT) family within the tautomerase
superfamily.1,9,15 4-OT is part of a bacterial degradation pathway
for aromatic hydrocarbons such as toluene (5) and converts
2-hydroxy-2,4-hexadienedioate (6) to 2-oxo-3-hexenedioate (7)
(Scheme 2).15ꢀ19 The evolutionary link between CaaD and
members of the 4-OT family is strengthened by the observations
that 4-OT from Pseudomonas putida mt-2 and YwhB, a 4-OT
T
catalyze the hydrolytic dehalogenation of the trans- and cis-isomers of
3-chloroacrylate (2 and 3, respectively) to yield malonate semialde-
hyde (4) and HCl (Scheme 1).1ꢀ3 These reactions represent key
steps in the degradation of the synthetic nematocide 1,3-dichloro-
propene (1), which was introduced into the environment in the 20th
century.4,5 Both dehalogenases belong to the tautomerase superfamily
and as such share a β-R-β structural fold and a catalytic amino-
terminal proline.6ꢀ8 However, they are not members of the same
family and likely have evolved independently from respective family
progenitors.3,8 This is quite surprising given the fact that CaaD and cis-
CaaD use similar catalytic mechanisms to process the different
isomers of 3-chloroacrylate.9ꢀ14 Despite their most recent emergence,
the evolutionary origins of CaaD and cis-CaaD remain elusive.
Received: January 17, 2011
Revised:
March 2, 2011
Published: March 03, 2011
r
2011 American Chemical Society
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|

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Scheme 1
Scheme 2
Scheme 4
Scheme 3
that Pro-1 of MsCCH2hasa pKa value thatliesinbetweenthatofa
typical tautomerase (pKa of Pro-1 ∼ 6) and that of cis-CaaD (pKa
of Pro-1 ∼ 9). These results suggest that MsCCH2 could be
characteristic of an evolutionary intermediate along the past route
for the divergence of cis-CaaD from an unknown homotrimeric
tautomerase.
’ MATERIALS AND METHODS
homologue from Bacillus subtilis, show low-level CaaD activity.20
This suggests that a 4-OT-like enzyme could have been recruited
to serve as a CaaD because it fortuitously had a low but useful
level of dehalogenase activity.7,20 Indeed, CaaD exhibits vestigial
tautomerase activity and, like 4-OT and YwhB, converts pheny-
lenolpyruvate (8) to phenylpyruvate (9) (Scheme 3).21
Materials. The sources of the chemicals, biochemicals, com-
ponents of buffers and media, PCR purification, gel extraction,
and Miniprep kits, Ni-NTA sepharose, prepacked PD-10 Sepha-
dex G-25 columns, and the oligonucleotides, enzymes, and
reagents used in the molecular biology procedures are reported
elsewhere.25 Phenylpyruvate, (p-hydroxyphenyl)-pyruvate, trans-
3-chloroacrylic acid, and cis-3-chloroacrylic acid were purchased
from Sigma-Aldrich Chemical Co. (St. Louis, MO). 2-Oxo-3-
pentynoate was a kind gift of Professor C. P. Whitman
(University of Texas at Austin, TX).
General Methods. BLASTP searches of the National Center
for Biotechnology Information (NCBI) databases were per-
formed using the cis-CaaD amino acid sequence (GenBank:
AAR00932.1) as the query sequence. Amino acid sequences
were aligned using a version of the CLUSTALW multiple-
sequence alignment routines available in the computational tools
at the EMBL-EBI Web site. Techniques for restriction enzyme
digestions, ligation, transformation, and other standard molecu-
lar biology manipulations were based on methods described
elsewhere26 or as suggested by the manufacturer. The PCR was
carried out in a DNA thermal cycler (model GS-1) obtained from
Biolegio (Nijmegen, The Netherlands). DNA sequencing was
performed by ServiceXS (Leiden, The Netherlands) or Macro-
gen (Seoul, Korea). Protein was analyzed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDSꢀPAGE) on
gels containing 10% polyacrylamide. The gels were stained with
Coomassie brilliant blue. Protein concentrations were deter-
mined by the method of Waddell.27 Kinetic data were obtained
on a V-650 or V-660 spectrophotometer from Jasco (IJsselstein,
The Netherlands). The kinetic data were fitted by nonlinear
cis-CaaD, a trimer composed of three identical monomers
(149 amino acid residues each), is the title enzyme of a separate
family within the tautomerase superfamily.3 This dehalogenase
also exhibits vestigial tautomerase activity, albeit at a low level,
converting 8 to 9.21 Its closest family member described to date,
Cg10062 from Corynebacterium glutamicum, is considerably
homologous in sequence (34% identical and 53% similar) to
cis-CaaD, and a pairwise alignment suggests that all key catalytic
residues of cis-CaaD are conserved in Cg10062.22 As expected,
Cg10062 possesses dehalogenase activity, converting both 2 and
3 with a strong preference for 3.22 However, the enzyme exhibits
no significant tautomerase activity using 8.a Hence, a functional
link of shared promiscuous activities between cis-CaaD and a
tautomerase from the same family has not yet been identified.
As promiscuous activities could provide important clues regard-
ing the progenitor of a newly diverged family member,7,23,24 we
searched the sequence databases for a homologue of cis-CaaD that
was tentatively annotated as a putative tautomerase and tested
both its phenylenolpyruvate tautomerase (PPT) and cis-CaaD
activity. Herein, we present the characterization of a cis-CaaD
homologue (designated MsCCH2) from Mycobacterium smegma-
tis strain MC2 155 that functions as an efficient tautomerase,
having a kcat/Km valueof 9.8ꢁ 103 Mꢀ1 sꢀ1 for 8 andhaslow-level
promiscuous cis-CaaD and CaaD activity. Hydratase assays and
labeling studies using 2-oxo-3-pentynoate (12, Scheme 4) indicate
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regression data analysis using the Grafit program (Erithacus,
Software Ltd., Horley, U.K.) obtained from Sigma Chemical Co.
1H NMR spectra were recorded on a Varian Inova 500 (500
MHz) spectrometer using a pulse sequence for selective pre-
saturation of the water signal. Chemical shifts for protons are
reported in parts per million scale (δ scale) downfield from
tetramethylsilane and are referenced to protium (H2O: δ =
4.67). The native molecular mass of purified MsCCH2 was
determined by Superdex 75 gel filtration using a fast protein
liquid chromatography system according to the instructions
provided with the HiLoad 16/60 Superdex 75 prep grade column
(Pharmacia). The standard molecular mass markers were albu-
min (67 kDa), ovalbumin (43 kDa), chymotrypsinogen A
(25 kDa), and ribonuclease A (13.7 kDa). Electrospray ioniza-
tion mass spectrometry (ESI-MS) and MS/MS spectra were
recorded using an LCQ electrospray mass spectrometer
(Applied Biosystems, Foster City, CA). Matrix-assisted laser
desorption/ionization-time of flight (MALDI-TOF) and TOF/
TOF mass spectra were recorded using a 4700 Proteomics
analyzer (Applied Biosystems). The enzymes CaaD and 4-OT
were purified according to a previously published procedure.28
Construction of Expression Vectors for MsCCH2 and the
MsCCH2-P1A Mutant, and Expression and Purification of
the Two His-Tagged Proteins. The experimental procedures
used for the construction of expression vectors for MsCCH2
wild-type and the MsCCH2-P1A mutant, and the overproduc-
tion and purification of these two proteins are provided in the
Supporting Information.
Enzyme Assays. The tautomerization activities of MsCCH2
and the P1A mutant were measured by monitoring the ketoniza-
tion of 8 to 9 and 10 to 11 in 10 mM Na2HPO4 buffer (pH 7.3) at
22 °C. Stock solutions of 8 and 10 were generated by dissolving
the appropriate amount of phenylpyruvic acid or (p-hydroxy-
phenyl)pyruvic acid in absolute ethanol. The crystalline free acid
of phenylpyruvic acid or (p-hydroxyphenyl)pyruvic acid is exclu-
sively the enol form. The ketonization of 8 to 9 was monitored by
following the decrease in absorbance at either 310 nm (ε = 1400
Mꢀ1 cmꢀ1) or 315 nm (ε = 300 Mꢀ1 cmꢀ1) using substrate
concentrations ranging from 0.05 to 8 mM. The ketonization of 10
to 11 was monitored by following the decrease in absorbance at
325 nm (ε = 1800 Mꢀ1 cmꢀ1) using substrate concentrations
ranging from 0.1 to 1.8 mM. An appropriate quantity of enzyme
(from a 5 mg/mL stock solution in 10 mM Na2HPO4 buffer, pH
7.3) was diluted into the sodium phosphate buffer (1 mL in a
cuvette), and the assay was initiated by the addition of a small
aliquot (5ꢀ20 μL) of substrate (either 8 or 10) from a stock
solution. At all substrate concentrations, the nonenzymatic rate
was subtracted from the enzymatic rate of ketonization.
were recorded according to protocols reported elsewhere.2,3,25
The modifications to these protocols are provided in the
Supporting Information.
UV Spectroscopic Analysis of the Reactions of CaaD, 4-OT,
and MsCCH2 with 2-Oxo-3-Pentynoate (12). UV spectra
monitoring the CaaD, 4-OT, and MsCCH2 reactions with 12
were recorded as follows. An appropriate amount of enzyme (45
μg of CaaD, 0.5 mg of 4-OT or 0.5 mg of MsCCH2) was
incubated with 12 (400 μM) in 1 mL of 10 mM Na2HPO4 buffer
at 22 °C, with the final pH of the incubation mixture ranging
from 6.5 to 8.5. A fresh stock solution of 12 (40 mM) was made
up in 10 mM Na2HPO4 buffer, and the pH was adjusted to 7.3.
The hydration of 12 (λmax = 234 nm) was monitored by following
the formation of 13 at 294 nm.2 The covalent modification of 4-OT
or MsCCH2 by 12 was monitored by following the formation of 14
(for 4-OT, λmax = 340 nm; for MsCCH2, λmax = 324 nm).
To determine the mass of (unmodified and modified)
MsCCH2 after its incubation with 12 in 10 mM Na2HPO4
buffer at pH 6.5, 7.3, 7.5, or 8.5, a sample was withdrawn from the
incubation mixture, diluted 10-fold in 5 mM NH4HCO2 buffer
(pH 7.3), and directly analyzed by ESI-MS.
Mass Spectral Analysis of the Product of the MsCCH2-
Catalyzed Hydration of 2-Oxo-3-Pentynoate (12). The ex-
perimental procedure used for the identification of 13 as the
product of the MsCCH2-catalyzed hydration of 12 is provided in
the Supporting Information.
Mass Spectral Analysis of Modified MsCCH2 and Peptide
Mapping. The MsCCH2 sample was made up as follows. The
enzyme (1 mg) was incubated with 12 (400 μM) in 1 mL of
10 mM NaH2PO4 buffer (pH 7.3) for 3 h at 22 °C. The
formation of the covalent adduct (14) was monitored spectro-
photometrically at 324 nm. An aliquot of a 500 mM stock
solution of NaBH4 in water was added to the sample to give a
final NaBH4 concentration of 25 mM, after which the mixture
was incubated overnight at 22 °C. This protocol was repeated
(three times) until the absorbance at 324 nm was completely lost.
Subsequently, the buffer was exchanged against 5 mM
NH4HCO2 buffer (pH 7.3) using a prepacked PD-10 Sephadex
G-25 gelfiltration column. An aliquot of this protein sample was
directly analyzed by ESI-MS.
For the peptide mapping studies, a quantity (50 μg) of the
MsCCH2 sample was vacuum-dried. The protein pellet was
dissolved in 10 μL of 10 M guanidine-HCl and incubated for 2 h
at 37 °C. Subsequently, the sample was diluted 10-fold by the
addition of 90 μL of 100 mM NH4HCO3 buffer (pH 8.0) and
incubated for 48 h at 37 °C with protease Glu-C (0.5 μL from a
10 mg/mL stock solution in water). The sample was analyzed by
MALDI-TOF MS without further purification. Selected ions of
modified and umodified peptide fragments were subjected to
TOF/TOF MS analysis.
The dehalogenation activities of MsCCH2 and the P1A
mutant were measured by following the dechlorination of 2
and 3 at 22 °C in 50 mM Tris-SO4 buffer (pH 8.0) using a
colorimetric assay.1,29 An appropriate amount of enzyme (1 mg)
was incubated with the desired concentration of 2 or 3 in 3 mL of
the Tris-SO4 buffer. The concentrations of substrate in the assay
ranged from 20 to 350 mM. Stock solutions (400 mM) of 2 and 3
were made up in 50 mM Tris-SO4 buffer. The pH of the stock
solution was adjusted to 8.0. Chloride concentrations were
measured colorimetrically at different time intervals.
’ RESULTS
Identification of cis-CaaD Homologues. A sequence simi-
larity search in the NCBI microbial database was performed with
the BLASTP program using the cis-CaaD amino acid sequence as
the query. This search yielded several bacterial proteins that
shared significant sequence identity with cis-CaaD. The top hits
included two sequences from M. smegmatis strain MC2 155,
designated MsCCH1 and MsCCH2, as well as the previously
characterized Cg10062 protein from C. glutamicum (Figure 1).22
1H NMR Spectroscopic Analysis of the Reaction of
1
MsCCH2 with 2, 3 or 10. H NMR spectra monitoring the
MsCCH2-catalyzed conversion of trans-3-chloroacrylate (2), cis-
3-chloroacrylate (3), or (p-hydroxyphenyl)enolpyruvate (10)
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Figure 1. Amino acid sequence alignment of cis-CaaD from coryneform bacterium strain FG41 3 and three selected cis-CaaD homologues. The NCBI
reference sequences for the selected proteins are as follows: Cg10062 from Corynebacterium glutamicum: NP_599314.1; MsCCH1 from Mycobacterium
smegmatis strain MC2 155: YP_887666.1; MsCCH2 from Mycobacterium smegmatis strain MC2 155: YP_885986.1. Identical residues are shaded in
black. The six active site residues of cis-CaaD that have been implicated as critical residues for its activity3,12 are indicated by an asterisk.
The mycobacterial protein MsCCH2 is annotated as a putative
tautomerase and was selected for further study.
Table 1. Kinetic Parameters for cis-CaaD, MsCCH2, and
MsCCH2-P1A Using Phenylenolpyruvate (8) and (p-Hydro-
xyphenyl)enolpyruvate (10)
The mature MsCCH2 protein is predicted to be 138 amino
acids in length, assuming excision of the initiating methionine
residue. The genomic context of the gene encoding MsCCH2
does not provide any clues about the biological function of this
protein in M. smegmatis. The sequence of MsCCH2 is 28%
identical and 39% similar to that of cis-CaaD. Despite the low
sequence identity, four of the six active site residues that have
been implicated as critical residues for cis-CaaD activity (Pro-1,
His-28, Arg-70, and Glu-114) are conserved in MsCCH2 (Pro-1,
His-28, Arg-68, and Glu-112) (Figure 1).3,12 The other two
residues, Arg-73 and Tyr-103, are not conserved and replaced by
His-71 and Ile-101 in MsCCH2. To obtain insight into the
functional properties of this cis-CaaD homologue, MsCCH2 was
overproduced, purified, and subjected to kinetic and mechanistic
characterization (as decribed below).
Expression and Purification of MsCCH2. The gene coding
for MsCCH2 was amplified from genomic DNA of M. smegmatis
strain MC2 155 and cloned into the expression vector pET20b-
(þ), resulting in the construct pMsCCH2. The MsCCH2
encoding gene in pMsCCH2 is under transcriptional control of
the T7 promoter, and the enzyme was produced constitutively in
E. coli BL21(DE3) as a C-terminal hexahistidine fusion protein.
The recombinant enzyme was purified by a one-step Ni-Sephar-
ose chromatography protocol, which typically provides ∼15 mg
of homogeneous enzyme per liter of culture. The purified
MsCCH2 was analyzed by ESI-MS and gel filtration chromatog-
raphy. Analysis of MsCCH2 by ESI-MS showed one major peak
corresponding to a mass of 16093 ( 1 Da. A comparison of this
value to the calculated subunit mass (16 223 Da) indicates that
the initiating methionine is removed during posttranslational
processing, which results in a protein with an N-terminal proline.
The native molecular mass of MsCCH2 was estimated by gel
filtration chromatography to be ∼48 kDa, which suggests that
the native enzyme is a homotrimeric protein.
enzyme
substrate kcat (sꢀ1
)
Km (μM) kcat/Km (Mꢀ1 sꢀ1
)
MsCCH2a
MsCCH2a
P1A-MsCCH2a
P1A-MsCCH2a 10
cis-CaaDb
a These steady-state kinetic parameters were measured in 10 mM
Na2HPO4 buffer (pH 7.3) at 22 °C. b These kinetic data were obtained
from Poelarends et al.21 Errors are standard deviations.
8
34 ( 4
3.2 ( 0.3
3500 ( 300
890 ( 90
9.8 ꢁ 103
3.5 ꢁ 103
3.3 ꢁ 103
2.5 ꢁ 102
1.8 ꢁ 103
10
8
0.8 ( 0.02 250 ( 30
0.2 ( 0.02 790 ( 150
0.2 ( 0.03 110 ( 30
8
MsCCH2 catalyzes the tautomerization of 8. The results show that
MsCCH2 converts 8 to 9 and that the activity (in terms of kcat) is
170-fold higher than that observed for cis-CaaD (Table 1).
A comparison of the kinetic parameters further shows that the Km
value for MsCCH2 is significantly higher (32-fold) than that
measured for cis-CaaD. Together, this results in a 5.4-fold higher
kcat/Km. Additionally, MsCCH2 also catalyzes the conversion of
(p-hydroxyphenyl)enolpyruvate (10) to (p-hydroxyphenyl)pyruvate
(11) (Scheme 3) with a kcat = 3.2 sꢀ1 and a Km of 0.89 mM, resulting
in a kcat/Km of 3.5 ꢁ 103 Mꢀ1 sꢀ1 (Table 1). The formation of 11 in
1
the reaction of MsCCH2 with 10 was confirmed by H NMR
spectroscopy (see Supporting Information).
Dehalogenase Activity of MsCCH2. Having established that
MsCCH2 exhibits high-level tautomerase activity, we next
investigated whether the enzyme exhibits promiscuous dehalo-
genase activity. Accordingly, MsCCH2 was incubated with cis-3-
chloroacrylic acid (3) and trans-3-chloroacrylic acid (2)
1
(Scheme 1), and the reactions were monitored by H NMR
spectroscopy. After incubation of 3 with MsCCH2 in 100 mM
phosphate buffer (pH 9.2) for 14 days at 22 °C, the intensity of the
two signals correspondingto 3 (6.25 and 6.17ppm) decreased and
four new signals appeared. Two signals (9.53 and 2.10) corre-
spond to acetaldehyde, whereas the other two signals (5.11 and
1.18 ppm) correspond to its hydrate. Integration of the signals
indicates that ∼10% of 3 has been converted to acetaldehyde (and
its hydrate). In contrast to cis-CaaD, MsCCH2 also processes
Tautomerase Activity of MsCCH2. It has previously been
determined that cis-CaaD exhibits promiscuous low-level tauto-
merase activity, converting phenylenolpyruvate (8) to phenylpyr-
uvate (9) (Scheme 3),21 which may be a vestige of the function of
its progenitor. This observation prompted us to examine whether
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Table 2. Kinetic Parameters for cis-CaaD, Cg10062, and
MsCCH2 Using cis-3-Chloroacrylate (3) and trans-3-Chlor-
oacrylate (2)
enzyme substrate
kcat (sꢀ1
)
Km (mM) kcat/Km (Mꢀ1 sꢀ1
)
MsCCH2a
MsCCH2a
Cg10062c
Cg10062c
cis-CaaDc
3
2
3
2
3
NDb
ND
1.6 ꢁ 10ꢀ3
4.0 ꢁ 10ꢀ3
10
(4.0 ( 0.1) ꢁ 10ꢀ4 96 ( 6
1.6 ( 0.3
156 ( 42
(2.0 ( 1) ꢁ 10ꢀ3 54 ( 40
4.0 ꢁ 10ꢀ2
4.6 ( 0.3
0.152 ( 0.02
3.0 ꢁ 104
a The steady-state kinetic parameters were measured in 50 mM Tris-SO4
buffer (pH 8.0) at 22 °C. b Not determined. These kinetic data were
c
obtained from Poelarends et al.22 Errors are standard deviations.
2:∼13% of 2 hasbeen converted toacetaldehyde(and its hydrate)
after 14 days at 22 °C.
The MsCCH2-catalyzed conversions of 2 and 3 were also
followed in 100 mM phosphate buffer at pH 6.5. Integration of
the signals indicates that ∼6% of 2 and ∼3% of 3 has been
converted to acetaldehyde (and its hydrate) after 2 weeks at
22 °C. No product formation was detected for control reactions
without enzyme (2 or 3 incubated in 100 mM phosphate buffer,
either pH 6.5 or pH 9.2, for 14 days at 22 °C), ruling out a
nonenzymatic dehalogenation.
A previously described colorimetric assay, which monitors
halide release, was used to measure kinetic parameters (Table 2).1,29
MsCCH2 catalyzes the dehalogenation of 2with a kcat =4ꢁ 10ꢀ4 sꢀ1
and a Km of 96 mM, resulting in a kcat/Km of ∼4 ꢁ 10ꢀ3 Mꢀ1 sꢀ1.
Saturation with 3 was not achieved for MsCCH2 and therefore
only the kcat/Km value was determined. A comparison of this
value to that measured for the dehalogenation of 2 shows that
MsCCH2 is ∼2.5-fold more efficient in catalyzing the dehalo-
genation of 2 than 3. In comparison to the cis-CaaD-catalyzed
dehalogenation of 3, MsCCH2 shows a 7.5 ꢁ 106-fold lower
kcat/Km. Hence, MsCCH2 exhibits low-level dehalogenase activ-
ity, accepting both isomers of 3-chloroacrylate as substrates with
a slight preference for the trans-isomer.
Reaction of MsCCH2 with 2-Oxo-3-Pentynoate. Previous
work has shown that the tautomerase 4-OT is irreversibly
inactivated by 2-oxo-3-pentynoate (12) due to the covalent
modification of Pro-1,30 whereas the dehalogenases CaaD and
cis-CaaD convert 12 to acetopyruvate (13) (Scheme 4).2,3 These
dissimilar reactions reflect differences in the ionization state and
reactivity of Pro-1 in the tautomerase versus dehalogenase active
sites. In order to probe the active site of MsCCH2, the reaction of
this enzyme with 12 was examined and compared to the well-
characterized 4-OT and CaaD reactions with 12.
As expected, incubation of 12 with CaaD (at pH 7.3) resulted
in a decrease in the absorbance at 234 nm, corresponding to 12,
accompanied by the appearance of one new absorbance peak at
294 nm, which corresponds to 13 (Figure 2A).2 Incubation of 12
with 4-OT (at pH 7.3) resulted in the rapid inactivation of 4-OT
and the appearance of one new absorbance peak at 340 nm,
which likely corresponds to the enamine species 14 (Scheme 4)
(Figure 2B).30 Surprisingly, the conversion of 12 by MsCCH2
(at pH 7.3) resulted in the appearance of two new absorbance
peaks, one at 294 nm and the other at 324 nm (Figure 2C). From
comparison to the product absorbance peaks formed in the
reactions of CaaD and 4-OT with 12, it can be inferred that the
peak observed at 294 nm corresponds to acetopyruvate (13),
whereas the peak observed at 324 nm likely corresponds to an
Figure 2. UV spectra monitoring the dissimilar reactions of CaaD,
4-OT, and MsCCH2 with 2-oxo-3-pentynoate (12, 400 μM) in
10 mM phosphate buffer at pH 7.3. (A) UV spectra monitoring the
CaaD-catalyzed conversion of 12 (λmax = 234 nm) to acetopyruvate
(13, λmax = 294 nm).2 Spectra were recorded every 2 min. (B) UV
spectrum of 4-OT inactivated by 12. The absorbance at 234 nm
corresponds to unreacted 12, while the absorbance peak at 340 nm
likely corresponds to enamine 14 (Scheme 4), which results from the
reaction between 12 and Pro-1 of 4-OT.30 (C) UV spectra monitoring
the MsCCH2-catalyzed conversion of 12 (λmax = 234 nm). The
product peak at 294 nm corresponds to 13, whereas the peak observed
at 324 nm likely corresponds to enamine 14, which results from the
reaction between 12 and Pro-1 of MsCCH2. Spectra were recorded
every 40 min.
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Table 3. Identification of Pro-1 as the Sole Site of Labeling by
12 Using Glu-C Digestion and MALDI-TOF-MS and MALDI-
TOF/TOF-MS Analyses
calculated mass observed mass
peptide fragment
(Da)a
(Da)
PVYTVTMSRGTLNGE
C5H8O3-PVYTVTMSRGTLNGE
PVYTVTMSRGTLNGETKAALAAE
PVYTVTMSRGTLN
PV
1624.8
1740.8
2380.2
1420.7
197.1
1625.5
1741.6
2381.8b
1421.9c
197.1c
C5H8O3-
2496.2
2497.8b
PVYTVTMSRGTLNGETKAALAAE
C5H8O3-PVYTVTMSRGTLN
C5H8O3-PV
1536.7
313.1
1537.9c
313.2c
a These values are calculated using the average molecular mass. b This
c
value corresponds to the total mass of the parent ion. The reported
mass corresponds to the b-ion.
enamine species (14), which could result from the reaction
between 12 and Pro-1 of MsCCH2. Importantly, the formation
of 13 and covalently modified MsCCH2 (presumably 14) in the
reaction of 12 with MsCCH2 was confirmed by ESI-MS and
MS/MS analyses (see Supporting Information).
Identification of the Modified Active Site Residue by Mass
Spectrometry. In order to identify the site of modification,
MsCCH2 was treated with 12 at pH 7.3, reduced with NaBH4,b
and analyzed by ESI-MS. The reconstruct of the ESI mass
spectrum revealed two major peaks, one corresponding to the
mass of unmodified MsCCH2 (16094 Da) and the other to the
mass expected for the enzyme modified by a single molecule of 12
and reduced by NaBH4 (16209 Da). This MsCCH2 sample was
then digested with endoproteinase Glu-C,31 and the resulting
peptide mixture was analyzed by MALDI-TOF-MS. A comparison
of the peaks of the modified MsCCH2 component to those of the
unmodified MsCCH2 component in the peptide mixture revealed
a single modification by a species having a mass of 116 Da on the
fragments Pro-1 to Glu-15 and Pro-1 to Glu-23 (Table 3). Analysis
of the remaining peaks showed no modification of other frag-
ments. These data indicate that a single site on the enzyme has
been modified and that the site of modification is localized within
the amino-terminal fragment Pro-1 to Glu-15.
To determine the actual site of the single modification, the
unmodified and modified Pro-1 to Glu-15 and Pro-1 to Glu-23
peptides were subjected to MALDI-TOF/TOF-MS analysis.
Because the two Pro-1 to Glu-23 peptides gave a more complete
fragmentation pattern than the two Pro-1 to Glu-15 peptides, the
masses of the fragment ions observed in the TOF/TOF spectra
of the modified and unmodified Pro-1 to Glu-23 peptides were
further analyzed. The spectrum of the precursor ion correspond-
ing to the unmodified Pro-1 to Glu-23 peptide displayed
characteristic b-ions resulting from the peptide fragments
PVYTVTMSRGTLN and PV. MALDI-TOF/TOF-MS analysis
of the precursor ion corresponding to the modified Pro-1 to Glu-
23 peptide revealed an increase in mass of 116 Da for these b-ions
(Table 3). Because one of these fragment ions is generated by the
dipeptide Pro-1 to Val-2, we conclude that the active site Pro-1
residue is the sole site of modification by 12.
Figure 3. UV spectra monitoring the reaction of MsCCH2 with 2-oxo-
3-pentynoate (12, 400 μM) in 10 mM phosphate buffer at three different
pH values. (A) Spectra monitoring the reaction at pH 6.5. Spectra were
recorded every 40 min. (B) Spectra monitoring the reaction at pH 7.5.
Spectra were recorded every 40 min. (C) Spectra monitoring the
reaction at pH 8.5. Spectra were recorded every 5 min. The decrease
in absorbance at 234 nm corresponds to the loss of 12, whereas the
increases in absorbance at 294 and 324 nm correspond to the formation
of 13 and 14, respectively.
three different pH values (Figures 3 and 4). Incubation of
MsCCH2 with 12 at pH 6.5 resulted in the formation of 13,
but no absorbance peak at 324 nm (indicative of covalently
modified MsCCH2; that is, the presumed enamine species 14)
Effect of pH on Product Formation in Incubations of
MsCCH2 with 2-Oxo-3-Pentynoate. To probe the ionization
state of Pro-1, the reaction of MsCCH2 with 12 was examined at
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at pH 6.5, converting 12 to 13, which implies that Pro-1 is
cationic at pH 6.5 and not functioning as nucleophile. However,
incubation of 12 with MsCCH2 at pH 8.5 did not result in
formation of 13 but only resulted in the formation of covalently
modified MsCCH2, as indicated by the absorbance peak at
324 nm (Figure 3C). As expected, analysis of this incubation
mixture by ESI-MS showed one major peak corresponding to the
mass (16206) of modified MsCCH2 (Figure 4C). This suggests
that at pH 8.5, Pro-1 has the correct protonation state to be able
to act as a nucleophile and attacks 12 to form a covalently
modified enzyme (14). Consistent with the preceding experi-
ment at pH 7.3, incubation of 12 with MsCCH2 at pH 7.5
resulted in the formation of both 13 and covalently modified
enzyme (Figures 3B and 4B). Assuming a pKa of Pro-1 of about
7.5, ∼50% of the enzyme would be in the correct protonation
state to react with 12 to give 13, and ∼50% of the enzyme would
be in the correct protonation state to attack 12 to give 14.
Kinetic Properties of the P1A Mutant of MsCCH2. The
results above show that incubation of MsCCH2 with 12 (at pH 8.5)
leads to the modification of Pro-1 and the concomitant loss of
hydratase activity. To further assess the importance of Pro-1 for the
activities of MsCCH2, the P1A mutant was constructed, over-
produced in E. coli BL21(DE3) as a C-terminal hexahistidine fusion
protein, and purified to homogeneity using the metal affinity
chromatography protocol developed for wild-type MsCCH2. ESI-
MS analysis revealed a mass of 16 067 Da (calc. 16 066 Da for the
enzyme without the initiating methionine) for the P1A mutant,
indicating that the initiating methionine is removed during post-
translational processing. The kinetic properties of the P1A mutant
were analyzed using 2, 3, 8, 10, and 12 as substrates and compared
to those measured for the wild-type enzyme.
For 8, the P1A mutant showed a 41-fold decrease in kcat and a
14-fold decrease in Km, resulting in a ∼3-fold decrease in kcat/Km
(Table 1). For 10, the P1A mutant showed a 16-fold decrease in
kcat, whereas the Km was not significantly affected. Hence, this
resulted in a 16-fold decrease in kcat/Km. The P1A mutant had no
detectable dehalogenase activity toward 2 and 3 using the
colorimetric assay, and no detectable activity toward 12 (at pH
7.3) using the UV spectroscopic assay (i.e., the reaction mixture
showed no product absorbance peaks). The CaaD and cis-CaaD
1
activities of the P1A mutant were also assessed by H NMR
spectroscopy after a lengthy incubation period (14 days at 22 °C)
in 100 mM phosphate buffer (pH 9.2). However, the reaction
mixtures showed no product. These findings are not surprising in
view of the low-level dehalogenase activities of the wild-type
enzyme. Taken together, the results suggest that Pro-1 is critical
for the tautomerase, dehalogenase, and hydratase activities of
MsCCH2.
Figure 4. ESI-MS analysis of the MsCCH2 protein in the three
incubation mixtures described in Figure 3. (A) ESI-MS analysis of
MsCCH2 after incubation with 12 at pH 6.5; see Figure 3A. (B) ESI-MS
analysis of MsCCH2 after incubation with 12 at pH 7.5; see Figure 3B.
(C) ESI-MS analysis of MsCCH2 after incubation with 12 at pH 8.5; see
Figure 3C. The major peaks in the spectra correspond to either
unmodified MsCCH2 (16093 Da) or MsCCH2 modified by a single
molecule of 12 (16206 Da). The additional minor peaks at the right of
each major peak result from (one or two) sodium adducts.
’ DISCUSSION
The mycobacterial enzyme (MsCCH2) characterized in this
study proficiently tautomerizes phenylenolpyruvate (8) to phe-
nylpyruvate (9) and (p-hydroxyphenyl)enolpyruvate (10) to (p-
hydroxyphenyl)pyruvate (11) (Scheme 3). MsCCH2 catalyzes
the conversion of 8 to 9 with a kcat/Km of 9.8 ꢁ 103 Mꢀ1 sꢀ1
,
which is somewhat lower (∼40-fold) than the values measured
for the same conversion catalyzed by the tautomerases 4-OT
(kcat/Km = 3.7 ꢁ 105 Mꢀ1 s
)
ꢀ1 32 and YwhB (kcat/Km = 4.2 ꢁ 105
was observed (Figure 3A). Indeed, analysis of this incubation
mixture by ESI-MS showed one major peak corresponding to the
mass (16093) of unmodified MsCCH2 (Figure 4A). These
observations suggest that MsCCH2 exhibits hydratase activity
Mꢀ1 ꢀ1).19 The lower catalytic efficiency of the MsCCH2-
s
catalyzed reaction results mainly from a higher Km value, which
suggests suboptimal binding of 8 in the active site of MsCCH2.
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Scheme 5
A comparison of the kinetic parameters for the MsCCH2 (kcat
=
,
alanine completely abolished the dehalogenase activity of
MsCCH2, indicating that dehalogenation, like tautomerization,
is an active site process that involves Pro-1.
3.2 ( 0.3 sꢀ1, Km = 890 ( 90 μM) and YwhB (kcat = 4.1 ( 0.4 sꢀ1
Km = 160 ( 22 μM)19 catalyzed conversion of 10 to 11 shows
similar catalytic efficiencies for both enzymes. It is important to
emphasize that we have also tested compound 6, the native
substrate of 4-OT (Scheme 2),18,19 as potential substrate for
MsCCH2, but no detectable 1,5-ketoꢀenol tautomerase activity
was observed. These results indicate that MsCCH2 functions as
an effective 1,3-ketoꢀenol tautomerase, converting 8 to 9 and 10
to 11, given that the physiological substrate for this tautomerase
has yet to be identified.
Although MsCCH2 has a low-level dehalogenase activity, the
rates of dehalogenation are still significant in comparison with
the reported nonenzymatic rate of ∼2.2 ꢁ 10ꢀ12 sꢀ1 at 25 °C
and pH 7.36 Using this value for the spontaneous, uncatalyzed
dehalogenation of 2 and the kcat value for the MsCCH2-catalyzed
dehalogenation of 2 (Table 2), it can be estimated that MsCCH2
affords a ∼2 ꢁ 108-fold rate enhancement. Although the
individual kcat value for the MsCCH2-catalyzed dehalogenation
of 3 could not be determined, a similar 108-fold rate enhance-
ment can be assumed. For comparison, cis-CaaD, which is
thought to have evolved for the purpose of degrading 3,3,6 affords
a ∼2 ꢁ 1012-fold rate enhancement for the conversion of 3 to 4.
Onthe basis of experimentalstudies,3,11ꢀ13 a mechanism for the
cis-CaaD-catalyzed conversion of3 to 4 was proposed (Scheme 5).
The first step in catalysis is the nucleophilic attack of an activated
water molecule on C-3 of 3 to form an enediolate intermediate.
The enzyme presumably uses the side chains of two residues, Glu-
114 and Tyr-103, to activate the nucleophilic water molecule. The
side chains of His-28, Arg-70, and Arg-73 interact with the C-1
carboxylate group of 3. These interactions position and polarize
the substrate to facilitate the attack of water and stabilize the
resulting enediolate species. Tautomerization of the enediolate
intermediate may be assisted by Pro-1, which is thought to place a
proton at the C-2 atom, and generates an unstable chlorohydrin
intermediate (Scheme 5a). Collapse of the proposed chlorohydrin
to afford 4 and HCl could be an enzymatic or a nonenzymatic
process. Alternatively, the enediolate species can undergo an
elimination reaction to release the chlorine as chloride ion,
followed by tautomerization of the enol intermediate and proton-
ation at C-2 by Pro-1, yielding 4 (Scheme 5b).
Similarities in sequence and quaternary structure firmly link
MsCCH2 to the cis-CaaD family, which is one of the five known
families in the tautomerase superfamily.3,12 With the previously
3
studied family members being cis-CaaD
and Cg10062,22
MsCCH2 is the first known family member that exhibits a
pronounced tautomerase activity. All known tautomerase super-
family members are characterized by a catalytic amino-terminal
proline.6,7 The importance of the conserved Pro-1 in MsCCH2 for
its tautomerase activity was demonstrated by mutating this residue
to an alanine. For both substrates (8 and 10), the major effect of
this mutation is observed in kcat (Table 1). Hence, the function of
Pro-1 is predominantly catalytic. In the well-studied tautomerases
4-OT and YwhB, the catalytic Pro-1 functions as the general base
and is responsible for proton transfer.19,33ꢀ35 It is therefore
reasonable to suggest that Pro-1 in MsCCH2 also functions as
the general base and may abstract the C-2 enol proton (of 8 or 10)
for delivery to the C-3 position (yielding 9 or 11).
MsCCH2 also exhibits dehalogenase activity using trans-3-
chloroacrylate (2) and cis-3-chloroacrylate (3) as substrates
(Scheme 1), but this activity is much lower (106-fold) compared
to its tautomerase activity (as assessed by kcat/Km values). Hence,
the tautomerase activity is the primary activity of MsCCH2. A
comparison of the kinetic parameters for the conversion of 3 to 4
shows that the catalytic efficiency (kcat/Km) of MsCCH2 is 104-
and 107-foldlower thanthatof Cg10062and cis-CaaD (Table 2).22
Unlike cis-CaaD, MsCCH2 also catalyzes the conversion of 2 to 4
but with a catalytic efficiency that is 10-fold lower than that
measured for Cg10062 (Table 2). Replacement of Pro-1 by an
In the context of this proposed cis-CaaD mechanism, the much
lower dehalogenase activity of MsCCH2 (compared to cis-CaaD)
could potentially be explained by active site differences. Sequence
comparisons suggested that four of the six active site residues that
have been implicated as critical residues for cis-CaaD activity
(Pro-1, His-28, Arg-70, and Glu-114) are conserved in MsCCH2
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(Pro-1, His-28, Arg-68, and Glu-112) (Figure 1).3,12 However,
two active site residues, Arg-73 and Tyr-103, are replaced by His-
71 and Ile-101 in MsCCH2. The replacement of Arg-73 in cis-
CaaD by His-71 in MsCCH2 may result in less efficient binding
and polarization of 3, thereby affecting water addition. The
replacement of Tyr-103 in cis-CaaD by Ile-101 in MsCCH2 may
even be more significant because it eliminates one of the presumed
water-activating residues. These active site differences may reflect
the different reaction specificities of MsCCH2 and cis-CaaD, and
suggest that MsCCH2 could be only a few mutations away from
being a highly specific and efficient cis-CaaD. Hence, mutation of
His-71 to an arginine accompanied by the replacement of Ile-101
with a tyrosine might increase the dehalogenase activity and
decrease the tautomerase activity of MsCCH2. The consequences
of these mutations are currently being examined.
Another potential explanation for the lower dehalogenase
activity and increased tautomerase activity of MsCCH2 could
be a lowered pKa of its Pro-1 residue when compared to the pKa
of Pro-1 (∼9.3) in cis-CaaD.11c Support for this view comes from
studies with 2-oxo-3-pentynoate (12, Scheme 4). Previous work
has shown that the reactions of 4-OT and CaaD, the best
characterized members of the 4-OT family in the tautomerase
superfamily, with 12 reflect both the ionization state of Pro-1
(neutral versus cationic) and the environment of the active site.2,30
Whereas 4-OT is irreversibly inactivated by 12 due to the covalent
modification of Pro-1, CaaD converts 12 to acetopyruvate (13)
(Scheme 4). These dissimilar reactions reflect differences between
the two active sites. Pro-1 of 4-OT has a pKa of ∼6.4, enabling it to
function as a general base catalyst in its physiological tautomerase
activity (conversion of 6 to 7, Scheme 2).33,34 Hence, at neutral
pH, Pro-1 functions as a nucleophile and attacks C-4 of 12 in a
Michael-type reaction, presumably yielding 14 (Scheme 4).30 In
contrast, the pKa of Pro-1 in CaaD is ∼9.2, enabling it to function
as a general acid catalyst in its physiological dehalogenase activity
(conversion of 2 to 4, Scheme 1).10 Thus, a Michael-type reaction
between the amino group of proline and 12 is not favored, and in
the active site of CaaD, which has evolved to carry out water
addition, 12 is processed to 13.2 Like CaaD, cis-CaaD and
Cg10062 process 12 to 13, instead of being inactivated by 12,
which implies that their Pro-1 residues have a pKa comparable to
that determined for Pro-1 of CaaD.3,22
In view of these observations, the reaction of MsCCH2 with
12 was examined at different pH values. At pH 8.5, MsCCH2 is
inactivated by 12 due to the covalent modification of Pro-1. This
suggests that Pro-1 is neutral at pH 8.5 and functions as a
nucleophile, attacking C-4 of 12 in a Michael-type reaction. The
identification of 15 by mass spectrometry experiments, coupled
with the observed λmax of 324 nm for the reaction product,
strongly suggests that the reaction results in the formation of 14.
Indeed, reduction with NaBH4 results in the complete loss of the
absorbance at 324 nm, consistent with the rearrangement of 14
into the corresponding imine, which is reduced by NaBH4 to give
15. At pH 6.5, however, MsCCH2 exhibits hydratase activity and
converts 12 to 13. This implies that Pro-1 is cationic at pH 6.5
and not functioning as a nucleophile. At pH 7.3ꢀ7.5, MsCCH2
converts 12 to both 13 and 14, indicating that the hydratase and
inactivation reactions occur simultaneously. These observations,
coupled with the results of control reactions using 12 and 4-OT
or CaaD at pH 7.3, suggest that Pro-1 of MsCCH2 has a pKa
value that lies in between that of a typical tautomerase like 4-OT
(pKa of Pro-1 ∼ 6.4) and that of the dehalogenases CaaD and cis-
CaaD (pKa of Pro-1 ∼ 9.2). This makes MsCCH2 the first
known tautomerase superfamily member with such an inter-
mediate pKa value for its catalytic Pro-1 residue. This intermedi-
ate pKa value, estimated to be ∼7.5, may enable Pro-1 to serve (at
cellular pH) as a general base catalyst in its primary tautomerase
activity and as a general acid catalyst in its promiscuous dehalo-
genase activity.
Given that Pro-1 in CaaD and cis-CaaD has a pKa of ∼9.2,10,11
enabling it to function as a general acid catalyst in the physio-
logical dehalogenase activity, an interesting route for the labora-
tory evolution of MsCCH2 into a more efficient dehalogenase
can be proposed. The introduction of one or more hydrophilic
residues could make the active site of MsCCH2 less hydropho-
bic, thereby making the active site more amenable for water
addition as well asraising the pKa of Pro-1. Thiswould increase the
concentration of enzyme with Pro-1 in the correct protonation
state to function as a general acid catalyst. The appropriate
experiments to investigate this intriguing possibility are underway.
In conclusion, the identification of MsCCH2 as tautomerase
with promiscuous dehalogenase and hydratase activities now
establishes a functional link between the recently diverged
Figure 5. Proposed evolutionary pathway for the divergence of an ancestral tautomerase, which may have been a specialist, to cis-CaaD, a specialist
dehalogenase that is selective for the cis-isomer of 3-chloroacrylate.3 The family members MsCCH2 (this study) and Cg1006222 could be characteristic
of evolutionary intermediates 1 and 2, respectively. In the course of divergence, the pKa of the essential Pro-1 residue likely increased from ∼6 (in the
ancestral tautomerase) to ∼7.5 (in the generalist intermediate 1) to ∼9 (in the specialist dehalogenase), allowing this residue to efficiently serve as a
general acid in the newly evolved dehalogenase activity. For clarity, and because the three-dimensional structures of MsCCH2 and Cg10062 are not
reported yet, the β-R-β building block carrying the N-terminal proline is shown.
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cis-CaaD and a tautomerase of the same family. Hence, the major
phenylpyruvate tautomerase activity of MsCCH2 comprises the
promiscuous activity of cis-CaaD, and the primary dehalogenase
activity of cis-CaaD comprises the promiscuous activity of
MsCCH2. In addition, both enzymes possess significant hydra-
tase activity. A reasonable route for the divergence of cis-CaaD
from an unknown tautomerase is presented in Figure 5. The
ancestral tautomerase may have been a specialist tautomerase
with a low pKa value for its catalytic Pro-1 residue. Mutations in
the specialist may have raised the pKa of its Pro-1 residue to about
7.5. This may have introduced a low-level promiscuous dehalo-
genase activity in the enzyme, without significantly decreasing
the original tautomerase activity. This thus yielded, in effect, a
generalist intermediate (intermediate 1 in Figure 5) exhibiting
both tautomerase and dehalogenase activity. MsCCH2 could be
characteristic of this generalist intermediate and may therefore
resemble the progenitor of cis-CaaD, using its promiscuous
dehalogenase activity as an essential starting point. Further
mutation and selection for an increase of the promiscuous
dehalogenase activity may have resulted in intermediate 2 and
finally in the specialist dehalogenase, cis-CaaD. Cg10062, which
has pronounced dehalogenase activity, but lacks isomer
specificity,22 could be characteristic of intermediate 2. In the
course of divergence, the pKa of the essential Pro-1 residue likely
increased from ∼6 (in the ancestral tautomerase) to ∼7.5 (in the
generalist intermediate 1) to ∼9 (in intermediate 2 and the
specialist dehalogenase), allowing this residue to efficiently serve
as a general acid in the newly evolved dehalogenase activity.
Although this route is highly speculative, laboratory evolution of
the promiscuous dehalogenase activity of MsCCH2 could pro-
vide important insight into pathways that could lead to the rapid
divergent evolution of an efficient cis-CaaD enzyme.
Christian P. Whitman (University of Texas at Austin, TX) for the
kind gift of 2-oxo-3-pentynoate. We thank Annie van Dam and
Margot Jeronimus-Stratingh (University of Groningen) for their
expert assistance in acquiring the MS spectra.
’ ABBREVIATIONS USED
CaaD, trans-3-chloroacrylic acid dehalogenase; cis-CaaD, cis-3-
chloroacrylic acid dehalogenase; EBI, European Bioinformatics
Institute; EMBL, European Molecular Biology Laboratory; ESI-
MS, electrospray ionization mass spectrometry; MALDI-TOF,
matrix assisted laser desorption/ionization time-of-flight;
MsCCH, Mycobacterium smegmatis cis-CaaD homologue; NCBI,
National Center for Biotechnology Information; NMR, nuclear
magnetic resonance; 4-OT, 4-oxalocrotonate tautomerase; PCR,
polymerase chain reaction; PPT, phenyl(enol)pyruvate tauto-
merase; SDSꢀPAGE, sodium dodecyl sulfateꢀpolyacrylamide
gel electrophoresis.
’ ADDITIONAL NOTE
a G. J. Poelarends and C. P. Whitman, unpublished results.
b Without this reduction step, the bound species is lost under the
conditions of the MALDI-TOF MS experiments. Reduction with
NaBH4 results in the complete loss of the absorbance at 324 nm,
consistent with the rearrangement of the presumed enamine
species (14, Scheme 4) into the corresponding imine, which is
reduced by NaBH4.
c The pKa of the catalytic Pro-1 in CaaD has been determined to
be 9.2 by direct titration using 15N NMR spectroscopy.10 A direct
titration of Pro-1 in cis-CaaD has not been conducted, but a
pHꢀrate profile of the cis-CaaD reaction implicated an acid
catalyst with a pKa of ∼9.3.11 It is assumed that this pKa value
corresponds to that of Pro-1 on the basis of the expected
mechanistic parallels with CaaD.
’ ASSOCIATED CONTENT
S
’ REFERENCES
Supporting Information. The experimental procedures
b
used for the construction, expression, overproduction, and
purification of His-tagged MsCCH2 wild-type and MsCCH2-
P1A mutant, and the H NMR spectroscopic analysis of the
reaction of MsCCH2 with 2 and 3. The experimental procedures
used for and results of the 1H NMR spectroscopic analysis of the
reaction of MsCCH2 with 10 and the mass spectral analysis of
the products (13 and 14) of the MsCCH2-catalyzed reaction
with 12. This material is available free of charge via the Internet at
http://pubs.acs.org.
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1
’ AUTHOR INFORMATION
Corresponding Author
*Tel: þ31-50-3633354; fax: þ31-50-3633000; e-mail: g.j.poelarends@
rug.nl.
Funding Sources
This research was financially supported by VIDI Grant 700.56.421
(to G.J.P.) from the Division of Chemical Sciences of The
Netherlands Organisation for Scientific Research (NWO-CW).
’ ACKNOWLEDGMENT
(7) Poelarends, G. J., Puthan Veetil, V., and Whitman, C. P. (2008)
The chemical versatility of the β-R-β fold: catalytic promiscuity and
divergent evolution in the tautomerase superfamily. Cell. Mol. Life Sci.
65, 3606–3618.
We thank Professor William R. Jacobs, Jr. (Howard Hughes
Medical Institute, Chevy Chase, MD) for the kind gift of genomic
DNA of Mycobacterium smegmatis strain MC2 155 and Professor
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dx.doi.org/10.1021/bi200071k |Biochemistry 2011, 50, 2889–2899