Kinetic and structural characterization of a heterohexamer 4-oxalocrotonate tautomerase from chloroflexus aurantiacus J-10-fl: Implications for functional and structural diversity in the tautomerase superfamily

Article abstract of DOI:10.1021/bi100502z

4-Oxalocrotonate tautomerase (4-OT) isozymes play prominent roles in the bacterial utilization of aromatic hydrocarbons as sole carbon sources. These enzymes catalyze the conversion of 2-hydroxy-2,4-hexadienedioate (or 2-hydroxymuconate) to 2-oxo-3-hexene

Full text of DOI:10.1021/bi100502z

5016 Biochemistry 2010, 49, 5016–5027
DOI: 10.1021/bi100502z
Kinetic and Structural Characterization of a Heterohexamer 4-Oxalocrotonate
Tautomerase from Chloroflexus aurantiacus J-10-fl: Implications for Functional and
Structural Diversity in the Tautomerase Superfamily†,‡
Elizabeth A. Burks,§ Christopher D. Fleming, Andrew D. Mesecar, Christian P. Whitman,*,§ and Scott D. Pegan*, ,^
§Division of Medicinal Chemistry, College of Pharmacy, The University of Texas, Austin, Texas 78712, Center for Pharmaceutical
Biotechnology (MC 870), College of Pharmacy, University of Illinois, 900 South Ashland Avenue, Chicago, Illinois 60607-7173, and
^Department of Chemistry and Biochemistry, University of Denver, Denver, Colorado 80208-0183
Received April 2, 2010; Revised Manuscript Received May 12, 2010
ABSTRACT: 4-Oxalocrotonate tautomerase (4-OT) isozymes play prominent roles in the bacterial utilization
of aromatic hydrocarbons as sole carbon sources. These enzymes catalyze the conversion of 2-hydroxy-2,4-
hexadienedioate (or 2-hydroxymuconate) to 2-oxo-3-hexenedioate, where Pro-1 functions as a general base
and shuttles a proton from the 2-hydroxyl group of the substrate to the C-5 position of the product. 4-OT, a
homohexamer from Pseudomonas putida mt-2, is the most extensively studied 4-OT isozyme and the founding
member of the tautomerase superfamily. A search of five thermophilic bacterial genomes identified a coded
amino acid sequence in each that had been annotated as a tautomerase-like protein but lacked Pro-1.
However, a nearby sequence has Pro-1, but the sequence is not annotated as a tautomerase-like protein. To
characterize this group of proteins, two genes from Chloroflexus aurantiacus J-10-fl were cloned, and the
corresponding proteins were expressed. Kinetic, biochemical, and X-ray structural analyses show that the two
expressed proteins form a functional heterohexamer 4-OT (hh4-OT), composed of three Rβ dimers. Like the
P. putida enzyme, hh4-OT requires the amino-terminal proline and two arginines for the conversion of
2-hydroxymuconate to the product, implicating an analogous mechanism. In contrast to 4-OT, hh4-OT does
not exhibit the low-level activity of another tautomerase superfamily member, the heterohexamer trans-3-
chloroacrylic acid dehalogenase (CaaD). Characterization of hh4-OT enables functional assignment of the
related enzymes, highlights the diverse ways the β-R-β building block can be assembled into an active
enzyme, and provides further insight into the molecular basis of the low-level CaaD activity in 4-OT.
4-Oxalocrotonate tautomerase (4-OT),1 initially cloned from
the TOL plasmid pWW0 in Pseudomonas putida mt-2, catalyzes
the conversion of 2-hydroxy-2,4-hexadienedioate, known more
commonly as 2-hydroxymuconate [1 (Scheme 1)] to 2-oxo-3-
hexenedioate (2) (1-5). The enzyme is part of the meta-fission
pathway, which is a catabolic pathway for aromatic hydrocar-
bons. Organisms having the TOL plasmid can process simple
aromatic hydrocarbons (e.g., benzene, toluene, m- and p-xylene,
3-ethyltoluene, and 1,2,4-trimethylbenzene) as their sole sources
of carbon and energy (3).
4-OT is a member of the tautomerase superfamily, which is a
group of structurally homologous proteins, characterized by a
β-R-β scaffold and a catalytic amino-terminal proline (6-11).
There are five known families of this superfamily represented by
the key members, 4-OT, 5-(carboxymethyl)-2-hydroxymuconate
isomerase (CHMI), macrophage migration inhibitory factor
(MIF) (7, 8), cis-3-chloroacrylic acid dehalogenase (cis-CaaD) (9),
and malonate semialdehyde decarboxylase (MSAD) (10). The
4-OT family includes trans-3-chloroacrylic acid dehalogenase
(CaaD). Superfamily members have been described as trimers
(CHMI, MIF, cis-CaaD, and MSAD), a heterohexamer (CaaD), a
homodimer (a 4-OT homologue designated YdcE) (11), and homo-
hexamers (4-OT and a 4-OT homologue designated YwhB) (7, 8).
These enzymes mediate tautomerization, dehalogenation, hydra-
tion, and decarboxylation reactions. Thus far, Pro-1 is a critical
residue for the activities of all of these enzymes.
†This research was supported by National Institutes of Health (NIH)
Grants GM-41239 (C.P.W.) and AI-60915 (S.D.P.), Robert A. Welch
Foundation Grant F-1334 (C.P.W.), and U.S. Department of Defense
Grant W81XWH0710445 USAMRAA (S.D.P.). Use of the Advanced
Photon Source is supported by the U.S. Department of Energy, Office of
Science, Office of Basic Energy Sciences, under Contract W-31-109-
Eng-38. The Analytical Instrumentation Facility Core (College of
Pharmacy, The University of Texas) is supported by NIH Center Grant
ES07784.
‡The atomic coordinates and structure factors have been deposited
with the Brookhaven Protein Data Bank (entry 3MB2).
*To whom correspondence should be addressed. C.P.W.: telephone,
(512) 471-6198; fax, (512) 232-2606; e-mail, whitman@mail.utexas.edu.
S.D.P.: telephone, (303) 871-2533; fax, (303) 871-2254; e-mail, scott.pegan@
du.edu.
1Abbreviations: Ap, ampicillin; BSA, bovine serum albumin; dNTPs,
deoxyribose nucleotide triphosphates; CHMI, 5-(carboxymethyl)-2-
hydroxymuconate isomerase; CaaD, trans-3-chloroacrylic acid dehalogen-
ase; cis-CaaD, cis-3-chloroacrylic acid dehalogenase; DSC, differential
scanning calorimetry; HEPES, N-(2-hydroxyethyl)piperazine-N0-2-ethane-
sulfonate; hh4-OT, heterohexamer 4-oxalocrotonate tautomerase; IPTG,
isopropyl β-D-thiogalactoside; Kn, kanamycin; LB, Luria-Bertani; MIF,
macrophage migration inhibitory factor; MSAD, malonate semialdehyde
decarboxylase; NCBI, National Center for Biotechnology Information;
NMR, nuclear magnetic resonance; 4-OT, 4-oxalocrotonate tautomerase;
PEG, polyethylene glycol; PPT, phenylpyruvate tautomerase; PCR, poly-
merase chain reaction; rmsd, root-mean-square deviation; SDS-PAGE,
sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
During the course of a search for other superfamily members,
the genomes of five recently sequenced thermophilic organisms
exhibited an intriguing peculiarity: each had a protein annotated
r
pubs.acs.org/Biochemistry
Published on Web 05/13/2010
2010 American Chemical Society

Article
Biochemistry, Vol. 49, No. 24, 2010 5017
Scheme 1
General Methods. Techniques for restriction enzyme diges-
tion, ligation, transformation, and other standard molecular
biology manipulations were based on methods described else-
where (15). Oligonucleotide primers were synthesized by Invitro-
gen. DNA sequencing was performed at the DNA core facility of
the Institute for Cellular and Molecular Biology at The Uni-
versity of Texas. Mass spectral data were obtained on an LCQ
electrospray ion-trap mass spectrometer (Thermo, San Jose, CA)
in the Analytical Instrumentation Facility Core in the College of
Pharmacy at The University of Texas. Samples were prepared as
described elsewhere (17). Kinetic data were obtained at 24 ꢀC on
an Agilent 8453 diode-array spectrophotometer. Nonlinear re-
gression data analysis was performed using Grafit (Erithacus
Software Ltd., Staines, U.K.) obtained from Sigma-Aldrich.
Plasmids were isolated from cell cultures using the QIAprep
Miniprep Kit (Qiagen, Valencia, CA). Protein concentrations
were determined by the method described by Waddell (18).
Protein was analyzed by tricine sodium dodecyl sulfate-poly-
acrylamide gel electrophoresis (SDS-PAGE) on 15% T/2% C
gels on a vertical gel electrophoresis apparatus obtained from
Idea Scientific (Minneapolis, MN) (19). BLAST and iterative
PSI-BLAST searches of the National Center for Biotechnology
Information (NCBI) databases were performed using the 4-OT
amino acid sequence from the TOL plasmid of P. putida mt-2 as
the query sequence (13). Nuclear magnetic resonance (NMR)
spectra were recorded in 100% H2O on a Varian Unity INOVA-
500 spectrometer as reported previously (17).
as a 4-OT homologue in what appeared to be a catabolic pathway
for aromatic hydrocarbons, but the homologue lacked Pro-1.2
Moreover, each organism had a second protein of similar length
in the vicinity of the hypothetical tautomerase with the amino-
terminal proline, but these sequences were not annotated as
tautomerase-like. Because it was not immediately obvious what
role these two proteins might play, the corresponding genes were
cloned from Chloroflexus aurantiacus J-10-fl, and the two pro-
teins were produced and characterized. The proteins form an Rβ
dimer, and the active tautomerase is a heterohexamer, which
converts 1 to 2, and phenylenolpyruvate [3 (Scheme 1)] to phenyl-
pyruvate (4). Mutagenesis analysis implicates βPro-1, RArg-12,
and RArg-40 as critical residues for these activities, whereas
βArg-11 and βArg-39 are not required for activity. A crystal
structure confirms the heterohexamer arrangement and shows
that the active site contains βPro-1, RArg-12, and RArg-40. De-
spite the mechanistic parallels and active site similarities, hh4-OT
lacks a significant property of 4-OT; it does not have a low-level
CaaD activity. This is the first reported 4-OT heterohexamer, and
its properties have implications for the evolution of other super-
family members.
Cloning of the R- and β-Subunits of hh4-OT from
Genomic DNA. Genomic DNA was extracted from 50 mg of
cells following a protocol described elsewhere (20). The coding
regions for the two subunits were amplified from genomic DNA
in separate PCRs.3 Each PCR mixture (50 μL) contained
genomic DNA (0.5 μg), two 50-phosphorylated primers for blunt
cloning (0.4 μM), dNTPs (0.3 mM), bovine serum albumin (BSA,
0.5 mM), Vent polymerase (0.5 unit), and the accompanying 10ꢀ
buffer (diluted to 1ꢀ). For the R-subunit, the forward primer
(RG1) had the sequence 50-GGACGGTGATATGCTACTTC-30
and the reverse primer (RG2) had the sequence 50-GTTCTGAA-
CAAACGAGTTAC-30. For the β-subunit, the forward primer
( βG1) had the sequence 50-CTCCACTTACGGTTCGTGTG-30
and the reverse primer ( βG2) had the sequence 50-CGCTT-
TACCCATCACCTATC-30. The primers were designed to match
the areas just upstream and downstream of the coding region of
each subunit. The PCR amplification protocol consisted of an
initial 5 min 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 10 min
elongation cycle at 72 ꢀC, and ending with a hold at 4 ꢀC. The gel-
purified PCR products were each ligated into an EcoRV-digested
pBluescript II SK-. An aliquot of each ligation mixture was used
to transform E. coli DH5R cells by electroporation. A positive
colony resulting from each transformation was selected on the
basis of PCR screening. The plasmid from each colony was
isolated, sequenced, and designated the R- or β-genomic clone.
Construction of the Bluescript hh4-OT Dicistronic Clone.
The R-subunit was amplified by PCR from the correspond-
ing R-genomic clone using the oligonucleotide 50-TAGTAGT-
AGGAATTCAAGAAGGAGATATACATATGCTACTTC-30
EXPERIMENTAL PROCEDURES
Materials. Chemicals, biochemicals, buffers, and solvents
were purchased from Sigma-Aldrich Chemical Co. (St. Louis,
MO), Fisher Scientific Inc. (Pittsburgh, PA), Fluka Chemical
Corp. (Milwaukee, WI), or EM Science (Cincinnati, OH). The
Centricon and Ultrafree centrifugal filter devices were obtained
from Millipore Co. (Billerica, MA). The synthesis of 2-hydroxy-
muconate (1) is reported elsewhere (4). Recombinant 4-OT,
cloned from the TOL plasmid of P. putida mt-2 and expressed in
Escherichia coli strain BL21-Gold(DE3), was purified by modi-
fying previously reported procedures (13, 14), as reported in the
Supporting Information. The Phenyl Sepharose 6 Fast Flow and
the Sephacryl-S100 High Resolution resins were obtained from
GE Healthcare (Piscataway, NJ). The Econo-Column chroma-
tography columns and Freeze ‘N Squeeze units, used for extrac-
tion of DNA from agarose gels, were obtained from Bio-Rad
Laboratories, Inc. (Hercules, CA). Enzymes and reagents used for
molecular biology procedures were obtained from New England
Biolabs, Inc. (Ipswich, MA). The sources for the components of
Luria-Bertani (LB) medium have been reported elsewhere (15).
Bacterial Strains, Plasmids, and Growth Conditions. C.
aurantiacus J-10-fl was provided by M. T. Madigan (16). Cells of
the strain were grown at 50 ꢀC under a tungsten light as described
elsewhere (16). The cells were stored at -80 ꢀC until they were
ready for use. E. coli strain DH5R was obtained from Invitrogen
(Carlsbad, CA). E. coli strain BL21-Gold(DE3) and pBluescript
II SK- were obtained from Stratagene (La Jolla, CA). The pET
vectors were obtained from Novagen (Madison, WI).
2The initiating methionine is the first amino acid according to the gene
sequence. However, proteins with a proline in the second position
undergo post-translational modification to remove the initiating
methionine, so thatproline becomestheamino-terminal aminoacid (12).
3The sequence of the genomic DNA was obtained from the NCBI
website (accession number NC_010175). The protein accession numbers
are YP_001634971 (gi|163846927) for the R-subunit (which appears first
in the genome) and YP_001634975 (gi|163846931) for the β-subunit.

5018 Biochemistry, Vol. 49, No. 24, 2010
Burks et al.
as the forward primer and the RG2 primer (above) as the reverse
primer. The forward primer contains nine nonspecific bases (to
enhance restriction enzyme digestion efficiency), a ribosome
binding site, an NdeI restriction site (underlined), and seven
additional gene-specific bases. The PCR mixture (50 μL) con-
tained the R-genomic clone (12 ng of plasmid), primers (0.2 μM),
dNTPs (0.2 mM), BSA, (0.5 mM), Vent polymerase (0.5 unit),
and the accompanying 10ꢀ buffer (diluted to 1ꢀ). The PCR
amplification protocol consisted of an initial 2 min 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, and ended with a 10 min elongation
cycle at 72 ꢀC, followed by a hold at 4 ꢀC. The gel-purified product
was phosphorylated and blunt cloned into pBluescript II SK- at
the EcoRV site. The resulting clone lacked the first 16 bases of the
forward primer and had the R-subunit oriented such that an
EcoRI site of the vector was at the 30-end of the gene.
The β-subunit was amplified by the PCR from the β-genomic
clone using the oligonucleotides 50-TAGTAGTAGGAATT-
CAAGAAGGAGATATACATATGCCGATGC-30 and 50-GA-
TGATGATCTCGAGGGATCCTCATTATTACTGCTGGT-
CTGGC-30 as the forward and reverse primers, respectively. The
forward primer contains nine nonspecific bases, an EcoRI
restriction site (underlined), a ribosome binding site, an NdeI res-
triction site (italicized), and seven gene-specific bases. The reverse
primer contains nine nonspecific bases, an XhoI restriction site
(underlined), a BamHI restriction site (italicized), three stop
codons, and 10 gene-specific bases. The PCR mixture (100 μL)
contained the β-genomic clone (1 μL of gel extract), the primers
(0.2 μM), dNTPs (0.2 mM), Vent polymerase (1 unit), and the
accompanying 10ꢀ buffer (diluted to 1ꢀ). Two separate reac-
tions were conducted using a PCR amplification protocol that
consisted of an initial 2 min denaturation cycle at 94 ꢀC, followed
by 25 cycles of 94 ꢀC for 45 s, 55 ꢀC for 45 s, and 72 ꢀC for 2 min,
and ended with a 10 min elongation cycle at 72 ꢀC, followed by a
hold at 4 ꢀC.
94 ꢀC, followed by 29 cycles of 94 ꢀC for 1 min, 55 ꢀC for 2 min,
and 72 ꢀC for 3 min, and ended with a 10 min elongation cycle at
72 ꢀC, followed by a hold at 4 ꢀC. The gel-purified PCR product
and the pET24a vector were treated with XbaI and XhoI
restriction enzymes, purified, and ligated using T4 DNA ligase.
Aliquots of the ligation mixture were used to transform com-
petent E. coli DH5R cells. Transformants were selected at 37 ꢀC
on LB/Kn (30 μg/mL) plates. Two colonies were randomly
chosen for sequencing. Both clones showed the correct sequences
for the R- and β-dicistronic gene arrangement. Plasmid DNA was
isolated from one clone and used as the expression vector in
E. coli BL21-Gold(DE3) cells.
Expression and Purification of hh4-OT and Construc-
tion, Expression, and Purification of the hh4-OT Mutants.
The expression, overproduction, and purification protocols for
the hh4-OT are provided in the Supporting Information. The
experimental procedures used for the construction, expression,
overproduction, purification, and mass spectral analysis of the
hh4-OT mutants are also provided in the Supporting Information.
Enzyme Assays and Kinetic Studies. The tautomerization
activities of hh4-OT were measured by monitoring the ketoniza-
tion of 1 to 2 and 3 to 4 in 10 mM potassium phosphate buffer
(pH 7.3) at 24 ꢀC (4, 5). Stock solutions (20 mM) of 2-hydroxy-
muconate (1) and phenylenolpyruvate (3) were made up in
ethanol and diluted (with ethanol) to 10 mM. The ketonization
of 1 to 2 was assessed by following the increase in absorbance at
236 nm (ε = 6580 M-1 cm-1) using substrate concentrations
ranging from 10 to 200 μM (4). The ketonization of 3 to 4 was
assessed by following the decrease in absorbance at 283 nm (ε =
18000 M-1 cm-1) using substrate concentrations ranging from
10 to 140 μM (5). An aliquot of enzyme was diluted into the
potassium phosphate buffer, yielding a final dimer concentration
of 1-191 nM for reactions using 1 and 100-1000 nM for
reactions using 3. Reactions were initiated by the addition of
substrate.
The product was cloned into the pBluescript construct con-
taining the R-subunit between the EcoRI and BamHI restriction
sites as follows. The gel-purified PCR product and the pBlue-
script construct containing the R-subunit were treated with
EcoRI and BamHI restriction enzymes, purified, and ligated
using T4 DNA ligase. Aliquots of the ligation mixture were used
to transform competent E. coli DH5R cells. Transformants were
selected at 37 ꢀC on LB/Ap (100 μg/mL) plates. The plasmid was
isolated from one clone, sequenced, and used for the production
of hh4-OT.
Cloning of the Dicistronic Gene for hh4-OT into
pET24a. The dicistronic gene for hh4-OT was amplified from
the pBluescript construct (containing the dicistronic gene) by the
PCR using the forward primer 50-TAGTAGTAGTCTAGAGG-
TATCGATAAGCTTG-30 and the reverse primer 50-GATGAT-
GATCTCGAGACTAGTGGATCC-30. The forward primer
contains nine nonspecific bases (for restriction enzyme digestion
efficiency), an XbaI restriction site (underlined), and 16 bases
specific for the 50 area upstream of the dicistronic gene in the
pBluescript construct. The reverse primer contains nine nonspe-
cific bases (for restriction site digestion efficiency), an XhoI restric-
tion site (underlined), and 12 bases specific for the 30 area down-
stream of the dicistronic gene in the pBluescript construct. The PCR
mixture (200 μL) contained the pBluescript construct (160 ng), the
primers (0.5 μM), dNTPs (0.2 mM), Taq polymerase (6 units), and
the accompanying 10ꢀ buffer (diluted to 1ꢀ). The PCR ampli-
fication protocol consisted of an initial 2 min denaturation cycle at
The spectrophotometric assay used to monitor the dehalo-
genation of trans- or cis-3-chloroacrylic acid was modified from
previously reported protocols (17, 21). Accordingly, reaction
mixtures containing 10 mM potassium phosphate buffer (pH
7.3), substrate [trans- or cis-3-chloroacrylic acid (0.5 mM)], and
enzyme (wild type, βR11A mutant, and βR39A mutant) were
incubated at room temperature. An aliquot of enzyme was
diluted into the potassium phosphate buffer, yielding final dimer
concentrations of 4.5, 5.0, and 4.9 μM for the wild-type, βR11A
mutant, and βR39A mutant enzymes, respectively. After 2 weeks,
there was no change in the absorbance at 224 nm.
1H NMR Spectroscopic Monitoring of the Incubation
Mixture Containing hh4-OT and trans-3-Chloroacrylic
1
Acid (5). The H NMR experiments were conducted as pre-
viously reported (17, 22), with the following modifications.
Accordingly, an aliquot (0.6 mL) of 5 (Scheme 2) was transferred
to an NMR tube from a stock solution made up in 100 mM
Na2HPO4 buffer. The aliquot contained 4 mg (0.04 mmol) of the
substrate. DMSO-d6 (30 μL) was also added to the tube. The pH
of the reaction mixture was then adjusted to 9.3 using small
amounts of an aqueous 1 M NaOH solution. Subsequently, an
aliquot of hh4-OT (50 μL of a 5.6 mg/mL solution of hh4-OT)
from a solution made up in 20 mM Na2HPO4 buffer (pH 7.3) was
added to the reaction mixture. 1H NMR spectra were recorded 10
days and 7 weeks after the addition of enzyme to the NMR tube.
At both intervals, the enzyme was examined for hh4-OT activity
using 1 and found to be active.

Article
Biochemistry, Vol. 49, No. 24, 2010 5019
Scheme 2
Table 1: Data Collection and Refinement Statistics for hh4-OT
Data Collection
space group
P31
unit cell dimensions
˚
a, b, c (A)
106.1, 106.1, 110.0
90, 90, 120
91.85-2.41
297700
Enzymatic Activity as a Function of the Oligomer State.
R = β = γ (deg)
resolution (A)
˚
The construction of pET24a vectors containing individual R- and
β-subunits is described in the Supporting Information. The genes
were then expressed separately in E. coli BL21-Gold(DE3) cells,
and the separate R- and β-subunits were partially purified and
examined for activity (using 1) before and after treatment with
8 M urea. Accordingly, the cells (∼2-4 g each) were suspended in
20 mM HEPES buffer (∼10 mL, pH 7.6). The individual suspen-
sions were sonicated and protease inhibitors added (see the
Supporting Information). The lysed cell mixtures were centri-
fuged for 45 min (30000g), followed by recovery of the super-
natants, which were then centrifuged for 3 h (264000g). The
individual supernatants (containing the R- or β-subunit) were
treated with urea in separate tubes when the supernatant (0.09
mL) was mixed with urea [0.4 mL of 10 M urea in 20 mM HEPES
buffer (pH 7.3)]. A small amount of NaCl [0.01 mL of 5 M NaCl in
20 mM HEPES buffer (pH 7.3)] was added to each sample. Rapid
dilution was achieved by mixing an aliquot (10 μL) with 1 mL of
10 mM KH2PO4 buffer (pH 7.3). The sample was then assayed
for activity using 1 (10 μL of a 20 mM solution of 1). In addition to
these samples, a mixture of the urea-treated R- and β-subunits
(∼1:1) was rapidly diluted and assayed, as described above. Each
sample was examined by SDS-PAGE (15% T/2% C gels) to
verify the presence of two proteins with the correct molecular
masses.
no. of reflections observed
no. of unique reflections
49737
R
merge (%)
7.2 (43.9)a
27.6 (4.1)a
98.6 (88.9)a
I/σ(I )
% completeness
Refinement
˚
resolution range (A)
91.85-2.41
47067
2670
no. of reflections in working set
no. of reflections in test set
R
work (%)
20.0
Rfree (%)
25.0
2
˚
average B factor (A )
32.1
protein
ions
31.70
59.60
32.80
water
rmsd
˚
bond lengths (A)
0.026
bond angles (deg)
2.23
no. of protein/water atoms
5447/249
aData for the last resolution shell are given in parentheses.
entry 1MWW), and a β-subunit hh4-OT homology model de-
rived from MODELER using the Pseudomonas sp. CF600 4OT
isozyme (PDB entry 1OTF) (25-27). WINCOOT was used for
model building, and REFMAC from the CCP4 software suite was
used for restrained TLS refinement using 36 TLS groups (24, 28).
The refinement statistics are listed in Table 1.
Crystallization and Structural Determination of hh4-
OT. The initial crystallization conditions for hh4-OT were
determined from the high-throughput screening of Qiagen Nextel
screens, Classics, Classics II, polyethylene glycol (PEG)s, and
PEGs II suites, with a Tecan Freedom Evo 200 liquid handling
robot. The hh4-OT [20 mg/mL in 10 mM Na/KPO4 buffer (pH
7.3)] was mixed in a 1:1 ratio with precipitant to give a total
volume of 2 μL in a sitting drop formatted microplate with a 100 μL
reservoir solution. Final crystals for hh4-OT were obtained by the
hanging drop vapor diffusion method with a 500 μL reservoir and
4 μL hanging drops that contained protein and precipitant, 0.25 M
(NH4)2SO4 and 4% PEG 4000, in a 1:1 ratio. Crystals appeared in
the course of 3-5 days.
Differential Scanning Calorimetry. Samples of hh4-OT
from C. aurantiacus J-10-fl and 4OT from P. putida mt-2 were
filtered through a 0.22 μm syringe filter following overnight
dialysis in 10 mM sodium phosphate buffer (pH 7.3). The protein
samples were subsequently diluted using the same buffer to
concentrations of 0.14 mg/mL (299 μM in heterohexamer) for
hh4-OT and 0.21 mg/mL (524 μM in homohexamer) for P. putida
mt-2 4-OT. Heat capacity measurements were taken using a VP-
DSC differential scanning calorimeter (Microcal, Northampton,
MA) over a temperature range of 10-125 ꢀC under ∼28 psi (1.9
atm). Samples were heated at a rate of 90 ꢀC/h. An accurate
baseline was derived from five buffer-buffer scans, which were
subtracted from five scans performed on each enzyme. Heat
capacity was plotted versus temperature to determine the tem-
perature of maximal heat capacity (Tm) for both protein samples
after normalization for the protein concentrations. Origin version
5.0 was used for all differential scanning calorimetry (DSC) data
analysis.
The X-ray diffraction data were collected for the hh4-OT
˚
crystals to 2.41 A resolution at the Argonne National Laboratory
(Argonne, IL) on Southeast Regional Collaborative Access
Team (SER-CAT) beamline 22-BM. Crystals were mounted on
nylon loops and submerged in a 4 μL drop of a cryo solution
containing 0.25 M (NH4)2SO4 and 25% PEG 4000. The crystals
were then immediately flash-frozen by being submerged in liquid
nitrogen. The flash-frozen crystals were mounted on a goniostat
under a stream of dry N2 at 100 K. X-ray exposures of 1 s per
degree of rotation about 200ꢀ omega were collected on a MAR
225 CCD detector. The X-ray data were processed and scaled
using HKL2000 (23). The data collection statistics are listed in
Table 1.
The molecular replacement solutions were determined using
PHASER (24). The overall search model was based on the
heterohexamer CaaD [Protein Data Bank (PDB) entry 1S0Y]
comprised of an R-subunit homology model produced by 3D-
Jigsaw using a 4OT homologue, Haemophilus influenzae (PDB
RESULTS
Sequence Analysis for the hh4-OT Homologues. A se-
quence similarity search of the NCBI database was performed
with the BLASTP program using the 4-OT amino acid sequence
from the TOL plasmid of P. putida mt-2 as the query se-
quence (13). The search yielded a sequence from Roseiflexus
sp. RS-1 (GenBank entry YP_001276901])that was annotated as
a tautomerase-like protein but showed a leucine rather than a
proline following the initiating methionine. Examination of the

5020 Biochemistry, Vol. 49, No. 24, 2010
Burks et al.
F
IGURE 1: Sequence alignment of the R- and β-subunits of the thermophilic hh4-OTs along with the 4-OT isozymes from P. putida mt-2 and
Pseudomonas sp. CF600 and the R- and β-subunits of CaaD (from Pseudomonas pavonaceae). The secondary structure elements are shown above
each set and were generated by a dictionary of protein secondary structure using the C. aurantiacus J-10-fl hh4-OT structure (41). The shading
indicates similar (orange), conserved (red), and absolutely conserved (red with green text) residues. (a) The sequences are ordered from the highest
to lowest percentage of sequence similarity with the R-subunit of C. aurantiacus J-10-fl (gi|163846927) (from top to bottom). Accordingly, the
R-subunit of Chloroflexus aggregans DSM 9485 (gi|219848927) is 59% similar, the R-subunit of Roseiflexus sp. RS-1 (gi|148656696) 58% similar,
the R-subunit of Roseiflexus castenholzii DSM 13941 (gi|156742377) 57% similar, the R-subunit of the Thermus thermophilus HB8 plasmid
(gi|55978425) 40% similar, 4-OT from P. putida mt-2 (gi|150974) 41% similar, 4-OT from Pseudomonas sp. CF600 (gi|1421033) 38% similar, and
the R-subunit ofCaaD from P. pavonaceae(gi|10637969) 29% similar. (b) The sequences are shown inthe order of the highest to lowestpercentage
of sequence similarity with the β-subunit of C. aurantiacus J-10-fl (gi|163846931) (from top to bottom). Accordingly, the β-subunit ofC. aggregans
DSM 9485 (gi|219848931) is 70% similar, the β-subunit of Roseiflexus sp. RS-1 (gi|148656700) 51% similar, the β-subunit of R. castenholzii DSM
13941 (gi|156742373) 50% similar, the β-subunit of the T. thermophilus HB8 plasmid (gi|55978421, one asterisk) 46% similar, 4-OT fromP. putida
mt-2 13.9% similar (two asterisks), 4-OT from Pseudomonas sp. CF600 15.3% similar (two asterisks), and the β-subunit of P. pavonaceae CaaD
(gi|10637970) 21% similar. Alignment and similarity calculation were obtained by using the CLUSTALW, TEXSHADE, BL2SEQ, and ALIGN
programs, which can be found at http://workbench.sdsc.edu/. The BL2SEQ settings were as follows: Matrix = BLOSUM62. The CLUSTERW
settings were as follows: Gap Opening Penalty = 11, Gap Extension Penalty = 1, and Lambda Ratio = 0.85. The single asterisk indicates that the
reported sequence is missing the initiating methionine and proline. A review of the corresponding gene indicated that the missing amino acids are
present. The double asterisk indicates that the ALIGN program was used.
genomic context for this gene (gi|148656696) using the genome
project link at the NCBI website revealed the presence of the genes
encoding enzymes similar to the ones surrounding 4-OT in the
TOL plasmid, including 2-hydroxymuconate semialdehyde dehy-
drogenase (which precedes 4-OT in the meta-fission pathway) and
4-oxalocrotonate decarboxylase and vinylpyruvate hydratase
(which follow 4-OT in the meta-fission pathway) (1-3). Using
the Roseiflexus sp. RS-1 sequence as the query sequence in a
BLAST search yielded several highly similar sequences (40-59%
sequence similarity) that were also annotated as tautomerase-like
proteins, but none had a proline following the initiating methio-
nine (Figure 1a). Examination of the genomic context of each also
showed genes encoding proteins typically associated with the
meta-fission pathway as well as a hypothetical protein with app-
roximately the same number of amino acid residues (63-73 amino
acids) (Figure 1b). Examination of the sequences of each of these
hypothetical proteins showed that they have a proline following
the initiating methionine, but apparently, the sequences do not
trigger the tautomerase designation by the BLAST domain search.
This study indicated that the first set of sequences (Figure 1a) likely
corresponds to the R-subunit of a hh4-OT and the second set of
sequences (Figure 1b) likely corresponds to the β-subunit of a hh4-
OT. The R- and β-subunits of C. aurantiacus J-10-fl hh4-OT are
∼44% similar and ∼22% identical in sequence.
individually into pBluescript expression vectors. A dicistronic gene
for the hh4-OT was then constructed by cloning the β-subunit into
the pBluescript vector containing the R-subunit. The five hh4-OT
mutants were constructed using the dicistronic pBluescript con-
struct. Finally, a high-expression level pET system was con-
structed by amplifying the dicistronic region from pBluescript
(using the PCR) and inserting the construct into a pET24a vector
at the XbaI and XhoI restriction sites.
The recombinant hh4-OT and the five mutants (RR12A,
RR40A, βP1A, βR11A, and βR39A) were purified in a three-
step protocol (heat treatment, anion exchange, and gel-filtration
chromatography). Typically, this procedure yielded ∼3-4 mg of
homogeneous protein (as assessed by SDS-PAGE) per liter of
cell culture. The individual subunits (when expressed, purified
separately, and subjected to urea treatment and rapid folding) did
not have detectable activity (using 1), but a mixture of subunits
exhibited activity (data not shown). These observations indicate
that the fully functional enzyme requires both subunits. The
crystallography studies were conducted using the hh4-OT pro-
duced from the pET24a vector. All other experiments were
conducted using hh4-OT (or the hh4-OT mutants) produced from
the pBluescript construct.
The purified proteins were analyzed by electrospray ionization
mass spectrometry (ESI-MS). The samples generate two major
signals in the mass spectrometer. The R-subunit of wild type has a
mass of 7730 Da, which corresponds to an expected 72-amino
acid product with a calculated mass of 7732 Da. The β-subunit of
Expression, Purification, and Characterization of hh4-
OT and Its Mutants. The R- and β-subunits were initially
cloned from C. aurantiacus J-10-fl genomic DNA and inserted

Article
Biochemistry, Vol. 49, No. 24, 2010 5021
Table 2: Kinetic Parameters for 4-Oxalocrotonate Tautomerases Using
2-Hydroxymuconate (1)a
Table 3: Kinetic Parameters for 4-Oxalocrotonate Tautomerases Using
Phenylenolpyruvate (3)a
enzyme
kcat (s-1
)
Km ( μM)
kcat/Km (M-1 s-1
)
enzyme
kcat (s-1
)
Km ( μM)
kcat/Km (M-1 s-1
)
hh4-OTb
RR12A
RR40A
βP1A
βR11A
βR39A
4-OT
3000 ( 100
43 ( 19
65 ( 14
70 ( 8
1033 ( 510
345 ( 100
17 ( 2
69 ( 7
135 ( 27
62 ( 8
4.3 ꢀ 107
4.2 ꢀ 104
1.9 ꢀ 105
2.1 ꢀ 106
5.1 ꢀ 107
5.4 ꢀ 106
6.5 ꢀ 107
hh4-OTb
RR12A
RR40A
βP1A
βR11A
βR39A
4-OT
13 ( 1
0.6 ( 0.2
44 ( 8
0.2 ( 0.04
17 ( 3
11 ( 1
121 ( 20
143 ( 80
152 ( 40
63 ( 22
198 ( 49
159 ( 24
199 ( 23
1.1 ꢀ 105
4.2 ꢀ 103
2.9 ꢀ 105
3.2 ꢀ 103
8.6 ꢀ 104
7.0 ꢀ 104
3.7 ꢀ 105
36 ( 1
3500 ( 100
733 ( 67
4000 ( 182
73 ( 6
aThe steady-state kinetic parameters were determined in 10 mM potas-
aThe steady-state kinetic parameters were determined in 10 mM potas-
sium phosphate buffer (pH 7.3) at 24 ꢀC. bErrors are standard deviations.
sium phosphate buffer (pH 7.3) at 24 ꢀC. bErrors are standard deviations.
the wild type has a mass of 7963 Da, which is 133 Da less than the
calculated mass of 8096 Da (for a 73-amino acid product). The
combined observations indicate that the β-subunit has undergone
a post-translational modification for the removal of the initiating
methionine whereas the R-subunit has not. The R- and β-subunits
of the mutants show the same pattern (data provided in the
Supporting Information). The initiating methionine is removed
by a methionyl aminopeptidase, and the removal is correlated
with the amino acid in the second position (12). Accordingly, in
E. coli, a proline or alanine in position 2 (as seen in the β-subunit)
results in a high likelihood of removal. However, if the second
position is occupied by a leucine (as seen in the R-subunit), the
likelihood of removal is very low (12).
Kinetic Characterization of Heterohexamer 4-OT. The
kinetic parameters for hh4-OT were determined using 2-hydroxy-
muconate (1) and phenylenolpyruvate (3) and compared to those
measured for the homohexamer 4-OT from P. putida (Tables 2
and 3). For both substrates, the Km values are comparable whereas
the kcat values for the homohexamer 4-OT are slightly higher (1.3-
and 6-fold higher using 1 and 3, respectively) than those measured
for hh4-OT. The higher kcat values are reflected in higher kcat/Km
values observed for the homohexamer 4-OT. The higher kcat
values may be due to the fact that hh4-OT is a thermophilic
enzyme and is not operating at its optimum temperature.
The hh4-OT was also incubated separately with trans- and cis-
3-chloroacrylic acid for 2 weeks. There was no change in the
absorbance at 224 nm for either compound. Using this spectro-
photometric assay, the hh4-OT does not appear to have low-level
dehalogenase activity. The absence of activity (and the low extinc-
tion coefficient at 224 nm) prompted us to monitor an incubation
mixture containing hh4-OT and 5 (Scheme 2) over a much longer
time span by 1H NMR spectroscopy. After 7 weeks, there was no
spectral evidence of malonate semialdehyde (6), its hydrate, acet-
aldehyde, 7, (resulting from nonenzymatic decarboxylation of 6), or
the hydrate of acetaldehyde.4 (The enzyme still retains tautomerase
activity using 1.) In contrast, ∼74% of compound 5 is converted
to acetaldehyde and its hydrate in the presence of 4-OT (using
approximately twice the amount of enzyme, 0.6 mg) in less than
6 days (22). Hence, there is no evidence of low-level CaaD activity
comparable to that of 4-OT in the hh4-OT.
and βArg-39) to the hh4-OT activity was investigated by con-
struction of the alanine mutants at each position and measurement
of the kinetic parameters using 1 and 3 (Tables 2 and 3). The data
show that replacing RArg-12, RArg-40, or βPro-1 with an alanine
has significant effects on kcat (using 1), with 70-fold (RR12A), 46-
fold (RR40A), and 83-fold ( βP1A) decreases, respectively, being
observed. There are increases in Km for the RR12A and RR40A
mutants (15- and 5-fold, respectively) and a small decrease in Km
for the βP1A mutant (4-fold). As a result, there are 1023-, 226-,
and 20-fold decreases in kcat/Km for the RR12A, RR40A, and
βP1A mutants, respectively. In contrast, replacing βArg-39 with
an alanine has a small effect (4-fold decrease in kcat, 2-fold increase
in Km, and 8-fold decrease in kcat/Km), and changing βArg-11 to an
alanine has a negligible effect on the kinetic parameters. These
results show that like 4-OT, two arginine residues (RArg-12 and
RArg-40) and the amino-terminal proline ( βPro-1) are required
for the 1,5-enol-keto tautomerase activity using 1.
With 3, significant effects on the kinetic parameters are
observed only for the RR12A and βP1A mutants. Replacing
RArg-12 or βPro-1 with an alanine reduces kcat 22- or 65-fold,
respectively. The RR12A mutant shows little change in Km, and
the βP1A mutant shows a 2-fold decrease. The values of kcat/Km
decrease 26-fold (RR12A) and 34-fold ( βP1A). There is a small
increase in kcat for the RR40A mutant (3.4-fold) and little change
in Km. As a result, the kcat/Km increases 2.6-fold. There are
smaller changes in the kinetic parameters for the βR11A and
βR39A mutants. These results show that only RArg-12 and βPro-
1 are required for the 1,3-enol-keto tautomerase activity using 3.
Crystal Structure of hh4-OT. The crystal structure of
hh4-OT was determined to confirm the oligomeric state and to
explore the structural implications of the heterohexamer. Crys-
tals of hh4-OT were grown using a precipitant solution of 0.25 M
˚
(NH4)2SO4 and 4% PEG 4000 and diffracted to 2.41 A. The
structure was determined in a P31 space group using molecular
replacement with a search model derived from a homology model
of a theoretical hh4-OT based on a heterohexamer CaaD (PDB
entry 1S0Y) scaffold (25), where the H. influenzae homologue
(PDB entry 1MWW) and Pseudomonas sp. CF600 4-OT isozyme
(PDB entry 1OTF) serve as templates for the R- and β-subunits,
respectively (Table 1). The resulting hh4-OT structure consists of
two heterohexamers per asymmetric unit.
Each heterohexamer contains three heterodimer units com-
posed of one R-subunit and one β-subunit in an alternating
fashion (Figure 2a). Both subunits have the signature 4-OT
superfamily β-R-β scaffold, but the R-subunit displays one
more β-sheet (encoded by residues Trp-51, Thr-52, and Val-53)
than the β-subunit, which facilitates an R-subunit-R-subunit
interaction (Figure 2b). Structural alignment of residues 2-50
Kinetic Characterization of the hh4-OT Mutants. The
importance of five residues (RArg-125, RArg-40, βPro-1, βArg-11,
4The nonenzymatic decarboxylation of malonate semialdehyde (6)
yields acetaldehyde. Malonate semialdehyde is not sufficiently stable to
accumulate in quantities that can be detected by 1H NMR spectroscopy
in the course of the lengthy incubation periods (22).
5The presence of the initiating methionine in the R-subunit increases
the sequence number of each residue by one.

5022 Biochemistry, Vol. 49, No. 24, 2010
Burks et al.
from each subunit of hh4-OT with the Pseudomonas sp. CF600
isozyme (PDB entry 1OTF) monomer resulted in a relatively
˚
low average rmsd of 1.5 A. However, alignment of the R- and
β-subunits of hh4-OT (using residues 2-50) has an average rmsd
˚
of 5.5 A. The difference in rmsd can be attributed to the preceding
and succeeding RA0 loops and the last 10 C-terminal residues of
the selected alignment region (Figure 2c).
Active Site of hh4-OT. Two regions (designated “type I” and
“type II”) located at the interfaces of the three heterodimeric
units could serve as potential active sites (Figure 2a). The three
type I sites form around RMet-1, where the RAβ2 loop in the
R-subunit, the β10RA0 loop in the β-subunit, and a 310 helix
constitute the three sides of this region (Figure 3a). The type I site
pocket is shallow, as it has been partially filled by RMet-1, and
contains no arginine side chains. In addition, the RMet-1 side
chain is inserted into a hydrophobic pocket where the hydroxyl
group of βThr-52 forms a polar interaction with the sulfur atom
of RMet-1. The βThr-52 position is typically occupied by a
residue with a bulky hydrophobic side chain such as Phe-50, as
seen in the Pseudomonas sp. CF600 and P. putida mt-2 4-OTs (27).
However, the presence of such a residue in the β-subunit (and, in
turn, at the active site) would clash with the side chain of RMet-1.
The absence of Phe-50 is also one of the factors that facilitates the
formation of a heterohexamer. The mutagenesis results (above)
coupled with these structural observations eliminate the type I
regions as active sites.
F
IGURE 2: Structural composition of hh4-OT. (a) Biologically signi-
ficant unit of hh4-OT viewed from the side and above. The R- and
β-subunits of a heterodimer are colored red and orange, respectively,
whereas the other two heterodimers are colored gray. The catalytic
βPro-1 is colored green and RMet-1 purple. Additionally, type I
(lavender) and type II (green) active sites are denoted as colored
boxed regions superimposed onto hh4-OT. (b) Ribbon diagram of a
single heterodimer using the colors defined above. (c) LSQKAB
alignment of a single heterodimer of hh4-OT in the colors (defined
above) superimposed on the Pseudomonas sp. CF600 4OT (PDB
entry 1OTF), colored gray with the N-terminus colored blue.
The three type II sites are formed at the other end of the
heterodimeric unit interface around βPro-1 (Figure 3b). As with
the type I sites, two of the type II active site sides are composed of
loops, but these loops are contributed from different subunits,
that is, the RA0β20 loop in the β-subunit and the β1RA loop in the
R-subunit. The third side of the type II active site is composed of
the intramonomeric R-subunit β2-β3 loop instead of a 310 helix
(Figure 3b). The type II sites closely resemble the active sites in
the Pseudomonas sp. CF600 and P. putida mt-2 4-OTs, as seen
when the structures are overlaid (Figure 3a,c) (27). Within the
type II sites, a positively charged hydrophilic pocket is created by
three arginines, RArg-12, RArg-40, and βArg-39, as well as βThr-
35. The general hydrophilic nature of this site allows it to be
occupied by either one or two sulfates in the crystal structure. The
mutagenesis results (above) and the sum of these structural
observations identify the three type II regions as the active sites
in the hh4-OT.
In addition to the charged and polar residues in the type II site,
two hydrophobic pockets are created. One of these sites is created
by the side chains of βPhe-29, βIle-33, and the side chain methyl
group of βThr-35. The other pocket is formed by the side chains
of RTrp-51 and RLeu-9. This second pocket is positioned in front
of the prolyl nitrogen of βPro-1 suggesting that this pocket could
reduce the pKa of βPro-1 to that observed in the P. putida mt-2
4-OT (∼6.4) (7).
the hh4-OT is likely a characteristic required for its stability in the
thermophilic environment, and there is structural evidence that
may explain the higher thermostability of the hh4-OT. The
interactions between the R- and β-subunits of the hh4-OT are
markedly more stable than those found in the homohexamer
4-OT. Specifically, these interactions are found in the core of the
protein, where hh4-OT exhibits a unique set of three interdimer
salt bridges between the R- and β-subunits, involving residues
βGlu-4 and RArg-5 of each dimer. These salt bridges form a core
for a network of hydrogen bonds involving the side chains of
RGln-45, RThr-7, and RGln-44 (Figure 4b). Salt bridges have
been shown previously to contribute significantly to the thermo-
stability of an enzyme, both on the surface and in the protein
core (29, 30). These salt bridges also prohibit the formation of
homohexamers composed of all R- or β-subunits. Such an
arrangement would result in unfavorable electrostatic interac-
tions between similarly negatively or positively charged amino
acids, depending on the subunit. In contrast to hh4-OT’s salt
bridge core, the homohexamer 4-OT from P. putida mt-2 relies
simply on water-mediated polar interactions among residues
Thr-43, His-6, and Gln-4 (Figure 4c). The lack of intersubunit
salt bridges in homohexamer 4-OT may be one factor that could
impair its stability in a thermophilic environment.
Thermostability of hh4-OT and 4-OT. Dynamic scanning
calorimetry (DSC) was used to determine the melting tempera-
tures (Tm) of hh4-OT and the homohexamer P. putida mt-2 4-OT
(Figure 4a). The heat capacity for both samples was measured
from 10 to 125 ꢀC at 28 psi. Corrected for buffer effects, the Tm of
the homohexamer P. putida mt-2 4-OT was found to be 78.8 ꢀC,
whereas the Tm of the heterohexamer C. aurantiacus 4-OT was
108.8 ꢀC (Figure 4a). The observance of only one peak for hh4-
OT suggests that the hexameric, dimeric, and secondary structure
denatured at approximately the same temperature with no
distinction between the R- and β-subunits. The higher Tm for
DISCUSSION
The homohexamer 4-OT from P. putida mt-2 and Pseudomonas
sp. CF600 has been extensively studied (4, 5, 7, 13, 14, 27, 31-37).
The results of these studies identified key mechanistic and struc-
tural elements, delineated their roles, and produced a working
hypothesis for the 4-OT mechanism (Scheme 3). In this mecha-
nism, Pro-1 is a general base that abstracts the 2-hydroxyl proton
(of 1) for delivery to the C-5 position with a high degree of
stereoselectivity (31-33). [In D2O, the 5S isomer of [5-D]2 is
produced (31).] Pro-1 can function as a general base because the

Article
Biochemistry, Vol. 49, No. 24, 2010 5023
F
IGURE 3: Active site of hh4-OT. (a) Divergent stereoview of the hh4-OT noncatalytic type I site formed by R-subunit E (red) and β-subunit D
(orange), with RMet-1 colored purple. The active site of the Pseudomonas sp. CF600 4OT (PDB entry 1OTF) is superimposed in gray. (b)
Divergent stereoview of the hh4-OT type II active site formed by R-subunit A (red) and β-subunit D (orange), with βPro-1 colored green. Water
molecules are depictedascyan spheres, and sulfate molecules are colored goldwith pinkoxygenmolecules. (c) Divergent stereoview ofthe hh4-OT
type II active site with Pseudomonas sp. CF600 4OT (PDB entry 1OTF) superimposed in gray.
prolyl nitrogen has a pKa of ∼6.4 so that it exists largely as the
uncharged species at pH 7.3 (33). The side chains of three residues
(Leu-80, Met-450, and Phe-500)6 constitute a hydrophobic pocket
in front of the prolyl nitrogen and as such are likely the major
groups responsible for the lowered pKa of Pro-1 (37). In accord
with this observation, changing Phe-50 to an alanine increases
the pKa of the prolyl nitrogen to 7.3, which is attributed to the
increased solvent accessibility around Pro-1 (37). The remaining
key catalytic residues are Arg-11 and Arg-39 (14, 35, 36). Arg-39 is
proposed to interact with the 2-hydroxyl group (of 1) and a C-1
carboxylate oxygen and has primarily a catalytic role. Arg-11 is
proposed to interact with the C-6 carboxylate group in a bidentate
fashion, which binds substrate and draws electron density away
from C-5, thereby creating a partial positive charge at this position
to facilitate protonation.
RArg-40 may also interact with the 2-hydroxyl group and stabilize
the developing carbanion charge after its deprotonation. The pKa
of the βPro-1 is likely comparable to that of Pro-1 in 4-OT because
of the hydrophobic pocket formed by RLeu-9 and RTrp-51
(comparable to Leu-8 and Phe-50, respectively, in P. putida mt-2
4-OT).
The results of the mutagenesis experiments with βPro-1, RArg-
12, and RArg-40 are in accord with the proposed mechanism and
parallel the results obtained for 4-OT. Thus, changing the rigid
secondary amine (i.e., βPro-1) to a more flexible primary amine
(i.e., βAla-1) primarily affects kcat and kcat/Km. This observation
suggests that the mutation affects the reaction chemistry, release
of product, or both (34). The decreased activity could be due to a
decrease in basicity coupled with a suboptimal positioning of the
catalytic base due to the increased flexibility of the primary
amine. The small decrease in Km for the βP1A mutant may be due
to the removal of bulk (e.g., the five-membered ring system),
making binding more favorable (if Km is a reflection of binding).
It is not unexpected to see activity for the βP1A mutant because
βAla-1 still has an amino-terminal group that can function as a
base. Both the P1G and P1A mutants of 4-OT retain a significant
amount of activity (34). Changing RArg-12 to an alanine affects
both Km and kcat, consistent with its role in binding (C-6) and
The structural homology, the positional conservation of key
groups, and mutagenic results suggest that the hh4-OT mecha-
nism parallels that of 4-OT. Accordingly, βPro-1 functions as the
general base, and the two arginine residues, RArg-125 and RArg-40,
interact with the C-6 and C-1 carboxylate groups, respectively.
6The primed residues refer to different subunits within the 4-OT
homohexamer.

5024 Biochemistry, Vol. 49, No. 24, 2010
Burks et al.
Scheme 3
RArg-12 to an alanine affects kcat, but not Km. This observation
argues against a binding role for RArg-12 and instead suggests a
catalytic role. The absence of the positively charged arginine at
this position could destabilize the developing carbanionic char-
acter of the intermediate after deprotonation of the 2-hydroxyl
group. The mutation may also affect the pKa of βPro-1 or the
positioning of a catalytic group or groups. The other arginine
residues (RArg-40, βArg-11, and βArg-39) do not appear to be
involved in binding or catalysis.
The results of modeling studies are consistent with the pro-
posed orientations in the active site (Figure 5). Modeling 1 into
the active site shows the respective interactions of the C-6
carboxylate and C-1 carboxylate groups with RArg-12 and
RArg-40 (Figure 5a). βPro-1 is proximal to the 2-hydroxyl
proton. Modeling 3 into the active site shows the proximity of
the C-1 carboxylate group to RArg-12 (Figure 5b). Again, βPro-1
is near the 2-hydroxyl proton (of 3). Candidates that might bind
the C-1 carboxylate group were not identified, but the interaction
between the phenyl ring of 3 and RTrp-51 is one possible binding
determinant.
Stereochemical experiments could shed further light on the
binding of 3. For example, if the monoacid 3 binds in a single
orientation (with the C-1 carboxylate end pointing toward RArg-
12), then a high degree of stereoselectivity is expected (at C-3 of 4)
when the reaction is conducted in D2O. If compound 3 binds in
two orientations (the C-1 carboxylate end pointing toward RArg-
12 or the C-1 carboxylate end pointing toward RArg-40), then a
mixture of stereoisomers is expected.
The characterization of the two tautomerase sequences of
C. aurantiacus J-10-fl and the conservation of key mechanistic
and structural residues suggest that the gene products in the four
other organisms (in Figure 1) will also form functional hetero-
hexamers with 1,5- and 1,3-keto-enol tautomerase activities using
1 and 3, respectively. These hh4-OTs are likely composed of three
heterodimers, where each dimer consists of an R- and β-subunit.
The heterohexamer structure presumably confers thermostabi-
lity. Finally, βPro-1, RArg-12, and βArg-40 are present in these
sequences and have roles analogous to those of C. aurantiacus
hh4-OT.
F
IGURE 4: Thermostability of hh4-OT and 4-OT. (a) Thermal dena-
turation of P. putida mt-2 4-OT and hh4-OT at pH 7.3 by differential
scanning calorimetry. Curves are derived from the average of five
DSC scans per protein with the baseline subtracted. (b) Internal
cavity of hh4-OT. The R- and β-subunits are colored red and orange,
respectively. Water molecules are depicted as cyan spheres. (c)
Internal cavity of 4-OT from P. putida mt-2 (PDB entry 4OTB)
aligned as described for panel b. Waters are depicted as red spheres.
catalysis (drawing electron density away from C-5) (7, 35, 36).
Changing RArg-40 to an alanine affects both Km and kcat, but the
effect on Km is not as severe as that seen for the RR12A
mutant (35, 36). Hence, the function of RArg-40 is predominantly
catalytic.7. Both results are consistent with those observed for
4-OT mutants.
The behavior of hh4-OT and its mutants with the nonphysio-
logical substrate 3, a monoacid, implicates βPro-1 and RArg-12
in catalysis. The reduced activity of the βP1A mutant can again
be ascribed to a decrease in basicity along with a suboptimal
positioning of the catalytic base, as discussed above. Changing
Thus far, the only other characterized heterohexamer in the
tautomerase superfamily is CaaD, which catalyzes the hydrolytic
dehalogenation of trans-3-chloroacrylate [5 (Scheme 2)] (17, 26).
Like the hh4-OT, CaaD is a trimer of dimers in which the three
active sites are composed of residues from R- and β-subunits (26).
Both subunits of CaaD have an amino-terminal proline, but only
βPro-1 is catalytic. Accordingly, the active site in one heterodimer
is composed of βPro-1, RArg-8, RArg-11, and RGlu-52 (Scheme 4).
7The pKa of Pro-1 in the R39Q mutant of 4-OT is 7.1 (36). A similar
pKa for Pro-1 in the RR40A mutant of hh4-OT could partially
contribute to its decrease in activity.

Article
Biochemistry, Vol. 49, No. 24, 2010 5025
F
IGURE 5: Comparison of hh4-OT substrate-bound models. (a) Divergent stereoview of the hh4-OT active site (PDB entry 3MB2) with
2-hydroxymuconate (pink) modeled in the site. β-Pro-1 is colored green, R-subunit A red, and β-subunit D orange. (b) Divergent stereoview of the
hh4-OT active site with phenylenolpyruvate (purple) modeled in the site. All other atoms are shown as described in panel a.
Scheme 4
Scheme 5
In the proposed mechanism, the arginine pair interacts with the
carboxylate group of the substrate to bind and polarize the
substrate, and RGlu-52 activates water for addition at C-3. These
actions produce the enediolate species 8, which can undergo two
fates (route A or B) (26, 38). In route A, βPro-1 provides a proton
at C-2 and the resulting species 9 collapses to 6 by the direct
expulsion of the chloride (26, 38). In route B, rearrangement of 8
with the elimination of chloride (i.e., an R,β-elimination) yields
enol 10, which can tautomerize to 6 (26, 38). Again, βPro-1 may
provide a proton at C-2.
additional arginine is proposed to assist in polarization of the
substrate and perhaps result in a preferred orientation of the
substrate where the interaction with the two arginines is now
favored over the interaction with a single arginine, Arg-39.
In contrast, hh4-OT does not have a low-level CaaD activity.
This observation is particularly curious because of the striking
similarities in the active sites, including the presence of the
key residues associated with the low-level CaaD activity of
4-OT (i.e., βPro-1 and RArg-12). One possible explanation for
the absent activity may reside with the presence of RTrp-51
instead of a phenylalanine, found in both CaaD (RPhe-50)
and 4-OT (Phe-50). A comparison of the active sites suggests that
the extra bulk of this residue in hh4-OT fills the cavity and
may exclude an appropriately placed water molecule or interfere
with the action of Pro-1. This observation implies that the other
hh4-OTs identified in this study (i.e., Figure 1) will not have a low-
level CaaD activity because tryptophan is conserved at this
position.
P. putida mt-2 4-OT has a low-level CaaD activity (as well as a
low-level cis-CaaD activity) (22).8 Mutagenic analysis implicated
Pro-1 as a critical residue for the CaaD activity, but not Arg-11.9
Hence, it was proposed that Arg-11 or Arg-39 interacted with the
C-1 carboxylate group of the substrate, thereby polarizing the
R,β-unsaturated acid and creating a partial positive charge at C-3
(Scheme 5). Water could then attack at C-3 with (or without) the
assistance of Pro-1 (22). Further support for this mechanism
came from the observation that changing Leu-8 in 4-OT to
an arginine enhanced the low-level CaaD activity (14). The
8The Pseudomonas sp. CF600 4-OT has not been examined for low-
level dehalogenase activity.
9Changing Arg-11 to an alanine increases the CaaD activity of
4-OT (22). Arg-39 presumably interacts with the carboxylate group of
the substrate in the R11A 4-OT.

5026 Biochemistry, Vol. 49, No. 24, 2010
Burks et al.
Although Phe-50 is critical for the tautomerase activity of
4-OT, this is the first indication that it may also be critical for the
low-level CaaD activity (of 4-OT). Changing the phenylalanine
to a tryptophan in 4-OT might diminish or eliminate the activity,
whereas changing the tryptophan to a phenylalanine might
introduce the activity in hh4-OT. Moreover, the newly intro-
duced activity could be enhanced by the RL9R mutation (com-
parable to the L8R mutation of 4-OT). The effects of these
mutations on the parent tautomerase activity are not known.
If the RW51F mutant of hh4-OT has measurable CaaD
activity that is enhanced by additional mutagenesis, it would
further support a scenario for the evolution of new enzymatic
activities demonstrated by Gerlt and colleagues (39, 40). It was
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activity in the tautomerase superfamily: Mechanistic and structural
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found that a single mutation in L-Ala-D,L-Glu epimerase (AEE),
an enolase superfamily member, introduced a low-level activity
of another superfamily member, o-succinylbenzoate synthase
(OSBS). Three additional mutations to the now functionally
promiscuous construct produced an enzyme with a rate accele-
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mimics (on some level) nature’s process for generating new
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ACKNOWLEDGMENT
We thank Dr. Michael T. Madigan for the generous gift of
C. aurantiacus J-10-fl. The cells were grown in the laboratory of
Dr. David Graham (Department of Chemistry and Biochemistry,
The University of Texas). Supporting institutions of the SER-
CAT 22-BM beamline at the Advanced Photon Source, Argonne
National Laboratory, can be found at www.ser-cat.org/members.
html.
€
€
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SUPPORTING INFORMATION AVAILABLE
Expression, overproduction, and purification protocols for
hh4-OT; experimental procedures used for the construction,
expression, overproduction, purification, and mass spectral ana-
lysis of the hh4-OT mutants and the construction and expression
of the separate subunits of the hh4-OT; and molecular modeling
studies. This material is available free of charge via the Internet at
http://pubs.acs.org.
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Murzin, A. G., and Whitman, C. P. (2004) Cloning, expression, and
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4-oxalocrotonate tautomerase- and YwhB-catalyzed hydration of 3E-
haloacrylates: Implications for evolution of new enzymatic activities.
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