Catalytic mechanism of 5-chlorohydroxyhydroquinone dehydrochlorinase from the YCII superfamily of largely unknown function

Article abstract of DOI:10.1074/jbc.M113.499368

Background:TftG is a YCII superfamily dehydrochlorinase that catalyzes conversion of 5-chlorohydroxyhydroquinone to hydroxybenzoquinone. Results:The TftG crystal structure in complex with product analog 2,5-dihydroxybenzoquinone illustrated the catalytic residues and mechanism. Conclusion:A His-Asp dyad and other conserved signature residues are implicated for catalysis and substrate binding. Significance:This is the first elucidation of a YCII superfamily protein mechanism, which helps explain their obscure nature.

Full text of DOI:10.1074/jbc.M113.499368

Enzymology:
Catalytic Mechanism of
5-Chlorohydroxyhydroquinone
Dehydrochlorinase from the YCII
Superfamily of Largely Unknown Function
Robert P. Hayes, Kevin M. Lewis, Luying
Xun and ChulHee Kang
J. Biol. Chem. 2013, 288:28447-28456.
doi: 10.1074/jbc.M113.499368 originally published online August 16, 2013
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THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 288, NO. 40, pp. 28447–28456, October 4, 2013
© 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
Catalytic Mechanism of 5-Chlorohydroxyhydroquinone
Dehydrochlorinase from the YCII Superfamily of Largely
Unknown Function*
Received for publication, July 8, 2013, and in revised form, August 5, 2013 Published, JBC Papers in Press, August 16, 2013, DOI 10.1074/jbc.M113.499368
Robert P. Hayes‡, Kevin M. Lewis‡, Luying Xun§1, and ChulHee Kang‡2
From the ‡Department of Chemistry and §School of Molecular Biosciences, Washington State University,
Pullman, Washington 99164
Background: TftG is a YCII superfamily dehydrochlorinase that catalyzes conversion of 5-chlorohydroxyhydroquinone to
hydroxybenzoquinone.
Results: The TftG crystal structure in complex with product analog 2,5-dihydroxybenzoquinone illustrated the catalytic resi-
dues and mechanism.
Conclusion: A His-Asp dyad and other conserved signature residues are implicated for catalysis and substrate binding.
Significance: This is the first elucidation of a YCII superfamily protein mechanism, which helps explain their obscure nature.
TftG, 5-chloro-2-hydroxyhydroquinone (5-CHQ) dehydro-
chlorinase, is involved in the biodegradation of 2,4,5-trichloro-
phenoxyacetate by Burkholderia phenoliruptrix AC1100. It
belongs to the YCII superfamily, a group of proteins with largely
unknown function. In this work, we utilized structural and
functional studies, including the apo-form and 2,5-dihydroxy-
benzoquinone binary complex crystal structures, computa-
tional analysis, and site-directed mutagenesis, to determine the
dehydrochlorination mechanism. The His-Asp dyad, which ini-
tiates catalysis, is strongly conserved in YCII-like proteins. In
addition, other catalytically important residues such as Pro-76,
which orients the His-Asp catalytic dyad; Arg-17 and Ser-56,
which form an oxyanion hole; and Asp-9, which stabilizes the
oxyanion hole, are among the most highly conserved residues
across the YCII superfamily members. The comprehensive
characterization of TftG helps not only for identifying effective
mechanisms for chloroaromatic dechlorination but also for
understanding the functions of YCII superfamily members,
which we propose to be lyases.
phosphorylation, leading to subsequent convulsions, hyper-
thermia, and possible death (3). 2,4,5-TCP is a product of the
biodegradation of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T)
(4, 5).
Early investigation of Burkholderia phenoliruptrix illustrated
that it could utilize 2,4,5-T as a sole carbon source. A 2,4,5-T-
negative mutant, PT88, was also characterized and found to
accumulate 5-chloro-2-hydroxyhydroquinone (5-CHQ) when
grown in the presence of glucose and 2,4,5-T (4, 6). Comple-
mentation of PT88 for growth on 2,4,5-T as a sole source of
carbon identified a cluster of genes (tftEFGH) essential for the
metabolism of the 5-CHQ intermediate (4–6). The revealed
biodegradation pathway of 2,4,5-T B. phenoliruptrix begins
with the conversion of 2,4,5-T to 2,4,5-TCP by the oxygenase
enzyme encoded by the tftA and tftB genes. 2,4,5-TCP is subse-
quently converted to 5-CHQ (4, 6, 7) by TftC and TftD. We
previously reported the crystal structures and reaction mecha-
nisms of TftC and TftD, which are reductase and monoxyge-
nase enzymes, respectively (8). 5-CHQ is then dehydrochlori-
nated into hydroxybenzoquinone (HBQ) by the enzyme TftG.
The resulting HBQ is enzymatically reduced to hydroxyhydro-
quinone by a quinone reductase and then to ␤-ketoadipate by
TftH and TftE, respectively (4). ␤-Ketoadipate, a common
metabolite in the biodegradation of aromatic compounds, is
further channeled into the TCA cycle for complete mineraliza-
tion (Fig. 1).
2,4,5-Trichlorophenol (2,4,5-TCP)3 is a toxic recalcitrant
pollutant introduced into the environment through its exten-
sive use as a herbicide, insecticide, and wood preservative (1, 2).
Similar to other chlorophenols, the major toxicity of 2,4,5-TCP
results from its ability to uncouple mitochondrial oxidative
TftG, a relatively small (11,166 Da) protein, catalyzes aro-
matic dehydrochlorination, but its mechanism has been
unknown. Sequence analysis suggests that TftG belongs to the
YCII superfamily. All members of YCII share a conserved His-
Asp dyad, of which the putative role has been suggested. This
rapidly expanding family also contains strongly conserved Arg
and Ser residues. The structural genomics effort has deter-
mined several crystal structures from this family, which con-
* This work was supported by the National Science Foundation (MCB
1021148 and DBI 0959778) and the M. J. Murdock Charitable Trust.
The atomic coordinates and structure factors (codes 4LBH, 4LBI, and 4LBP) have
been deposited in the Protein Data Bank (http://wwpdb.org/).
1 To whom correspondence may be addressed: School of Molecular Biosci-
ences, Washington State University, Pullman, WA 99164. Tel.: 509-335-
2787; Fax: 509-335-1907; E-mail: luying_xun@vetmed.wsu.edu.
2 To whom correspondence may be addressed: Dept. of Chemistry, Washing-
ton State University, Pullman, WA 99164. Tel.: 509-335-1409; Fax: 509-335-
8865; E-mail: chkang@wsu.edu.
3 The abbreviations used are: 2,4,5-TCP, 2,4,5-trichlorophenol; 2,4,5-T, 2,4,5- firmed similar placement of these residues, but functions of
trichlorophenoxyacetic acid; 5-CHQ, 5-chloro-2-hydroxyhydroquinone;
those proteins still remain unclear. Thus, a structural and
HBQ, hydroxybenzoquinone; 2,5-DHBQ, 2,5-dihydroxybenzoquinone; ITC,
isothermal titration calorimetry; MM, molecular mechanics; QM, quantum
mechanics; PDB, Protein Data Bank.
mechanistic understanding of TftG, a critical enzyme in the
biodegradation of 2,4,5-T, is useful not only for future informed
OCTOBER 4, 2013•VOLUME 288•NUMBER 40
JOURNAL OF BIOLOGICAL CHEMISTRY 28447

Catalytic Mechanism of a YCII-like Dehydrochlorinase
concentrated, and buffer-exchanged into 20 mM Tris (pH 8.5)
with 1 mM DTT by ultrafiltration in an Amicon 8050 cell with a
5-kDa cut-off polyethersulfone membrane (Millipore), loaded
onto a Mono QTM GL10/100 anion-exchange column (GE
Healthcare), and eluted at 200 mM NaCl with a linear NaCl
gradient of 0 to 2 M NaCl using a preparative HPLC (Akta
Explorer, GE Healthcare). Fractions containing TftG were
pooled, concentrated, and exchanged into 20 mM Tris (pH 7.5)
with 1 mM DTT. Final homogeneity of purified TftG was esti-
mated at Ͼ99%.
Selenium auxotroph B834 cells were also transformed with
the same tftG pET30-LIC plasmid. Expression and purification
of selenium-substituted TftG was carried out by the same pro-
tocol as native TftG.
FIGURE 1. Conversion of 2,4,5-trichlorophenoxy acetic acid to 2-hydroxy-
benzoquinone. TftAB converts 2,4,5-trichlorophenoxy acetic acid to 2,4,5-
TCP. TftCD converts 2,4,5-TCP to 2,5-dichlorobenzoquinone (not shown),
which is reduced to 2,5-DCHQ. TftD further dechlorinates 2,5-DCHQ to
5-chloro-2-hydroxybenzoquinone (not shown), which is reduced to 5-chloro-
2-hydroxyhydroquinone, the substrate of TftG. TftG catalyzes the dehydro-
chlorination of 5-chloro-2-hydroxyhydroquinone to the product 2-hydroxy-
benzoquinone, which subsequently enters the TCA cycle after several
previously established enzymatic steps. This figure was produced using
ChemDraw (version 12.0.2).
Data Collection and Structure Determination—TftG at 50
mg/ml in 20 mM Tris, 1 mM DTT, pH 7.5, was crystallized at
4 °C using the hanging drop vapor-diffusion method. 1.5 ␮l of
TftG was mixed with 1.5 ␮l of a reservoir solution containing
30% (w/v) polyethylene glycol 1500 and equilibrated against the
same reservoir solution. This condition was used for both
native TftG and selenium-substituted TftG crystals. Diffrac-
tion-quality crystals appeared after 3 weeks. Selenomethionyl-
TftG diffraction data were collected on a Rigaku FR-Eϩ
Superbright rotating anode dual wavelength x-ray source.
Cryoprotectant consisted of 20% (v/v) glycerol in reservoir
buffer. Initial data collection for selenium-single wavelength
anomalous dispersion phasing was conducted using chromium
radiation (2.29 Å) at 100 K and processed using d*TREK (9).
Experimental single wavelength anomalous dispersion phasing
was conducted using PHENIX AutoSol followed by one cycle of
model building in PHENIX Autobuild (10), resulting in an elec-
tron density map in which 94 of 100 residues were correctly
bioremediation strategies, but also in better understanding the
largely uncharacterized YCII superfamily. Here, we report the
crystal structures for both apo-form and the binary complex of
TftG with the product analog 2,5-dihydroxybenzoquinone
(2,5-DHBQ). This information, together with accompanying
enzymatic assays for site-directed mutants, illustrates the bio-
logical function and potential reaction mechanism, which
sheds light on the functions of YCII superfamily enzymes.
EXPERIMENTAL PROCEDURES
Chemicals—Chemicals were obtained from Sigma Aldrich or modeled. Higher resolution (1.75 Å) apo-form data were col-
Fisher Scientific. Crystallization screens were obtained from lected at the Advanced Light Source beamline 8.2.1. A binary
Hampton Research.
complex crystal of TftG with 2,5-dihydroxybenzoquinone was
Cloning and Enzyme Purification—The tftG gene was cloned formed by soaking an apo-form TftG crystal with 2,5-dihy-
into the pET30-LIC vector and expressed as a C-terminal His6 droxybenzoquinone and followed by cryoprotection in 30%
fusion protein. 100 ml of Luria broth supplemented with 30 (w/v) polyethylene glycol 1500 supplemented with 20% glyc-
␮g/ml kanamycin was inoculated with a freezer stock of erol. Binary complex diffraction data were also collected at
pET30A TftG in BL21(DE3) cells and incubated overnight at the Advanced Light Source (BL8.2.1) and processed using
37 °C with constant shaking at 250 rpm. The 100-ml culture HKL2000 (11). The atomic model provided by the selenomethio-
was then used to inoculate 1.5 liter of Luria broth medium. TftG nyl-TftG chromium data set was used for refinement with the
expression was induced by addition of isopropyl ␤-D-thiogalac- Advanced Light Source data described above. The TftG-Apo
topyranoside to 0.3 mM final concentration at mid-log phase structure had 100% of residues in Ramachandran favored regions.
(A600 ϳ 0.6). Following induction, the cells were further incu- Selenomethionyl-TftGhad99.19%of residuesinpreferred regions
bated for 12 h at 22 °C with constant shaking at 250 rpm. The and 0.84% in allowed regions with no outliers. The TftG⅐2,5-
cells were then harvested by centrifugation (3,000 ϫ g), after DHBQbinarycomplexstructurehad97.94%ofresiduesinfavored
which the pellet was frozen to promote cell lysis. The pellet was regions, 1.03% in allowed, and 1.03% outliers. The coordinates for
thawed at room temperature and suspended in a minimal TftG-apo, selenomethionyl-TftG, and TftG⅐2,5-DHBQ binary
volume of lysis buffer (50 mM Tris, 300 mM NaCl, 20 mM complex have been deposited in the Protein Data Bank (PDB)
imidazole, and 1 mM dithiothreitol, pH 8.0). The cell suspen- under codes 4LBH, 4LBI, and 4LBP, respectively.
sion was sonicated five times for 10 s each using a model 450 Multi-angle Light Scattering and Isothermal Titration Calo-
Sonifier௡ (Branson Ultrasonics), and the resulting lysate rimetry (ITC)—The weight average molecular mass of TftG was
cleared by centrifugation (20,000 ϫ g for 30 min).
measured by combined size exclusion chromatography and
Lysate was applied to a nickel-nitrilotriacetate column and multi-angle laser light scattering as described previously (12).
washed with several column volumes of lysis buffer. Elution Briefly, 200 ␮g of TftG was loaded onto a BioSep-SEC-S 2000
buffer consisted of lysis buffer supplemented with 250 mM column (Phenomenex) and eluted isocratically with a flow rate
imidazole. Eluted fractions containing TftG were combined, of 0.5 ml minϪ1. The eluate was passed through a tandem UV
28448 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 288•NUMBER 40•OCTOBER 4, 2013

Catalytic Mechanism of a YCII-like Dehydrochlorinase
detector (Gilson), Optilab digital signal processing interfero- ONIOM (16) calculations were assisted by the Toolkit to Assist
metric refractometer (Wyatt Technology), and a Dawn EOS ONIOM (17) The residues allowed to move during optimiza-
laser light scattering detector (Wyatt Technology). Scattering
data were analyzed using the Zimm fitting method with soft-
ware (ASTRA) provided by the instrument manufacturer.
Isothermal titration calorimetric reactions were carried out
in a VP-ITC instrument (MicroCal). The protein was prepared
for ITC by extensive buffer exchanging into titration buffer (20
mM Tris, pH 8.5). The concentration of protein in the calori-
metric reaction cell was diluted to 100 ␮M. All titrations were
performed at 25 °C with a stirring speed of 300 rpm and 29
injections (10-␮l each). Ligands were diluted into the same
titration buffer and injected into the cell containing TftG solu-
tion, and the heats of binding were recorded. Ligands were also
titrated against buffer to account for heats of dilution. Ligand
concentrations were adjusted to obtain significant heats of
binding. All samples were degassed prior to titration.
tions were Ile-5, Arg-7, Asp-9, Arg-17, Ile-18, Tyr-21–Leu-25,
Lys-32, Ile-35, Gly-38–Pro-40, Met-53, Gly-55–Leu-58, Phe-
71, Val-72, Asp-75–Phe-77, Leu-82, Phe-83, Gly-94–Asp-98;
all but the carbon of an additional C-terminal N-methyl cap
were allowed to move. 5-Chloro-2-hydroxyhydroquinone was
generated in GaussView (version 3.09) (18) and optimized with
tight convergence criteria, followed by a frequency calculation,
at the B3LYP (19–21)/aVDZ level of theory. Ligand harmonic
stretch and bend AMBER molecular mechanics (MM) force
field (22) parameters were generated from these frequencies
using the parafreq utility (23); chlorine non-bonded parameters
were taken from the parmpol12 parameter set as found in
AmberTools 13 (22), and dihedrals/improper torsions involv-
ing chlorine were assigned by analogy with the hydroxyl OH
atom type. The geometry-optimized ligand was inserted into
the N-methyl-capped TftG product⅐analog complex crystal
structure with Lys-95 converted to Ala, and hydrogen atoms
were added using the PHENIX ReadySet! program (10), with
His-24 as N␦-His and His-96 doubly protonated; all other his-
tidines were kept singly protonated. This model was converted
into a dimer via symmetry operations, followed by optimization
of its key residues (listed above), the ligand, and all hydrogen
atoms using PM7 semi-empirical theory (25) with the
MOZYME linear-scaling method (26) as implemented in
MOPAC2012 (27). Restrained electrostatic potential charges
(28) were then fitted to the MOZYME-optimized Arg-7/His-
24/Asp-75 and Asp-9/Arg-17/Ser-56 trios via Merz-Kollman-
Singh (29) electrostatic potential fitting (overlay 6/33 ϭ 2) at
the CAM-B3LYP (30)/aVDZ level of theory, generating the
ONIOM input structure. Hydrogen atom geometries of the
input structure were first reoptimized using a two-layer
ONIOM scheme with PM6 semi-empirical theory (31) for the
model system (defined as the ligand and the side chains of
Arg-7, Arg-17, Asp-9, Asp-75, Asp-96, His-24, His-96, Ser-56,
and Tyr-21) and the AMBER MM force field for the real system.
Following this, the ligand and key residues were optimized
using a three-layer ONIOM scheme with CAM-B3LYP/aVDZ
for the model system, PM6 for the intermediate system, and the
AMBER force field for the real system. Residues included in the
model and intermediate systems, with their approximations
shown as (model/intermediate), were Arg-7 and Arg-17 (N-
methylguanidinium/N-propylguanidinium); Asp-9 and Asp-75
(acetate/propanoate); Tyr-21 (phenol/4-ethylphenol); His-24
(1H-imidazole/4-propyl-1H-imidazole); Ser-56 (ethanol/etha-
nol); the Pro-76/Phe-77 peptide bond (N-methylacetamide, inter-
mediate layer only); and the C-terminal tail (His-96 as imidazo-
lium and Asp-98 as acetate/His-93 as Ala plus Gly-94 through the
N-methyl cap in its entirety).
Atomic Absorption Spectroscopy—A standard curve of ZnCl2
(0–100 ␮M, 20 mM Tris, pH 7.5) was generated using a Shi-
madzu AA-6200 atomic absorption spectrophotometer at the
atomic absorption wavelength of zinc (213.9 nm). TftG samples
were measured at 20, 40, 60, and 80 ␮M protein concentration
to detect any bound zinc.
Enzyme Assays of Mutant TftG—TftG mutants (R17A,
H24A, S56A, D75N, and H96A) were generated using a
QuikChange Lightning site directed mutagenesis kit (Strat-
agene) and transformed into XL-10 Gold ultra-competent cells.
DNA sequencing was used to confirm mutation, and the result-
ing plasmids were transformed into BL21(DE3) expression
cells. Purification of mutant TftG was the same as that of wild
type. 5-CHQ was enzymatically produced by TftD as described
previously (13). Briefly, purified TftD (1 mg/ml) and Esche-
richia coli flavin reductase (5 ␮g/ml) was incubated with 100
␮M 2,4,5-TCP in the presence of 10 ␮M FAD, 2 mM NADH, and
2 mM ascorbate in 100 ␮l of 40 mM potassium phosphate buffer
at 25 °C for 10 min. Flavin reductase utilized NADH to reduce
FAD to FADH2, and TftD oxidized 2,4,5-TCP to 5-CHQ with
O2 and FADH2 as the co-substrates. The complete conversion
of 2,4,5-TCP to 5-CHQ was confirmed by HPLC analysis. Then,
the reaction mixture was aliquoted to 20 ␮l each, and 1 ␮l of
TftG was added and mixed. The reaction was incubated at 25 °C
for 1 min, and 20 ␮l of 10% acetic acid in acetonitrile was added
to stop the reaction. After centrifugation to remove protein
precipitates, the supernatant was analyzed by HPLC for 5-CHQ
consumption. Assays were run in triplicate, and specific activ-
ities were calculated for TftG and its mutants.
Circular Dichroism Spectroscopy—CD spectroscopy was uti-
lized to investigate the effect of mutations on secondary struc-
ture. CD spectra for each wild-type and mutant TftG protein
was measured from 200 to 300 nm wavelength using an AVIV
202SF spectropolarimeter (AVIV Biomedical) at 25 °C. The
TftG samples were prepared in 5 mM potassium phosphate (pH
7.0) at a protein concentration of 10 ␮M.
RESULTS
The apo-form TftG crystallized in a hexagonal space group
with one molecule in the asymmetric unit (Table 1). A crystal-
lographic symmetry operation of monomeric TftG generated a
Quantum Mechanics/Molecular Mechanics Calculations—
Unless otherwise stated, all calculations were performed in the
gas phase using Gaussian 09 (G09) (14) and utilized double-␨
correlation consistent basis sets (15) with augmented functions tetramer, which was consistent with the tetrameric nature of
on all atoms but hydrogen (hereafter referred to as aVDZ). TftG in solution (Fig. 2). To obtain a binary structure of TftG, a
OCTOBER 4, 2013•VOLUME 288•NUMBER 40
JOURNAL OF BIOLOGICAL CHEMISTRY 28449

Catalytic Mechanism of a YCII-like Dehydrochlorinase
TABLE 1
Data collection and refinement statistics
Values in parentheses are for the highest-resolution shell.
TftG-SeMET
TftG-Apo
TftG binary complex
P622
Data collection
Space group
Cell dimensions
a, b, c (Å)
C2221
P622
135.24, 158.01, 50.39
90.00, 90.00, and 90.00°
23.61–2.21
90.32, 90.32, 51.26
90.00, 90.00, and 120.00°
50.00–1.75
90.20, 90.20, 50.92
90.00, 90.00, and 120.00°
50.00–1.87
␣, ␤, ␥
Resolution (Å)
Rsym
0.040 (0.193)
28.7 (6.3)
0.077 (0.266)
0.089 (0.226)
I/␴I
53.96 (12.08)
66.22 (20.65)
Completeness (%)
Redundancy
77.3 (13.5)
99.8 (96.1)
99.8 (99.6)
6.23 (3.30)
20.6 (17.3)
19.7 (15.9)
Refinement
Resolution (Å)
No. reflections
23.61–2.21
21,232
45.17–1.75
12826
45.09–1.87
10,571
R
work/Rfree
0.1704/0.2070
0.1859/0.2165
0.1887/0.2194
No. of atoms
Protein
2992
0
748
7
783
10
Ligand/ion
Water
272
79
78
B-factors
Protein
29.60
N/A
27.30
44.30
35.50
27.20
28.40
33.30
Ligand/ion
Water
32.90
R.m.s. deviations
Bond lengths (Å)
Bond angles
0.007
1.05°
0.008
1.08°
0.016
1.20°
FIGURE 2. Oligomeric nature of TftG in solution. Elution profile for TftG protein (2 mg mlϪ1) was monitored with multi-angle laser light scattering and was
shown as absorbance (left-Y axis) and molecular weight (right-Y axis) versus elution volume (ml). The solid line represents changes in absorption at 280 nm. The
thick black cluster in the middle of the peaks indicated the calculated molecular mass, which was extended to the right y axis for ease of interpretation. The
average molecular weight for the TftG peak was indicated.
product analog, 2,5-DHBQ, was diffused into an apo-form crys-
tal by a soaking approach.
A structural alignment between the apo-form and binary
complex with 2,5-DHBQ illustrated no large structural devia-
Global Structure—Apo-form TftG resembled a ferredoxin- tions (root mean square deviation, 0.193 Å). One noticeable
like core fold. Each subunit consisted of four ␤-strands (␤1–␤4) structural difference was the improved electron density corre-
arranged in an antiparallel ␤-sheet that was flanked by three sponding to residues 95–98 in the binary complex structure due
opposing ␣-helices (␣1, ␣3, and ␣4) (Fig. 3a). The tetrameric to its hydrogen bonding with the substrate.
assembly of TftG establishes through the interaction of two
ITC—ITC was employed to find any suitable substrate analog
dimeric ␣-␤-barrels (Fig. 3, b and c). Hydrogen bonding for obtaining binary complex crystals. Tested ligands included
between the ␤4 strands (residues 84–88) from two different hydroquinone, 5-chlorohydroquinone, catechol, and 2,5-
dimeric ␣-␤-barrels forms a continuous eight-stranded anti- DHBQ. The only compound with significant affinity was 2,5-
parallel ␤-sheet. There are also several electrostatic interac- dihydroxybenzoquinone (Fig. 4) with Kd of 11.4 ␮M, which led
tions such as Lys-8-Glu-89 and Arg-90 with the carbonyl of us to pursue a binary complex. The association of 2,5-DHBQ
Gly-47, which stabilize the tetramer.
with TftG had a considerable enthalpic contribution, ⌬H ϭ
28450 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 288•NUMBER 40•OCTOBER 4, 2013

Catalytic Mechanism of a YCII-like Dehydrochlorinase
FIGURE 3. Ribbon diagram representing the crystal structure of TftG. a, TftG monomer. Secondary structural elements have been numbered sequentially
as ␣1–␣4 and ␤1–␤4. N and C refer to the N- and C-terminal regions, respectively. ␣-Helices were shown in red, and ␤-strands were shown in green. The product
analog 2,5-dihydroxybenzoquinone in the active site is illustrated in orange. b and c, TftG tetramer. Two orientations illustrating arrangement of the TftG
tetramer and binding sites with 2,5-dihydroxybenzoquinone represented as a molecular surface. This figure was generated by open source PyMOL (version
1.1r1).
clear electron density for 2,5-DHBQ in this pocket. The polar
residues comprising the pocket are Arg-17, His-24, Ser-56,
Asp-75, His-96, and Asp-98. The C-terminal residues of the
adjacent subunit, His-96 and Asp-98, close off the active site
trapping the ligand inside (Fig. 5b).
His-24 and Asp-75 were proximate to each other, establish-
␦1
ing a catalytic dyad, such that the Asp-75 O was hydrogen
␦1
bonded to the His-24 N . The His-24 N⑀2 atom also established
a hydrogen bond with the 2-hydroxyl group of 2,5-DHBQ. In
addition, the side chains of Arg-17 and Ser-56 were both within
hydrogen bonding distance to each other and to 2,5-DHBQ at
its 1-carbonyl oxygen. The His-96 N⑀2 atom was within hydro-
gen bonding distance of either the 4-carbonyl oxygen or the
5-hydroxyl group of 2,5-DHBQ. Asp-98 was within hydrogen
bonding distance of the 4-carbonyl oxygen of 2,5-DHBQ (Fig.
5b). A hydrogen bond network continued away from the His-
24-Asp-75 and Arg-17-Ser-56 dyads, including a salt bridge
between the side chains of Arg-7 and Asp-75, and another
between the side chains of Asp-9 and Arg-17.
Quantum Mechanics/Molecular Mechanics (QM:MM) Sub-
strate Modeling—Quantum mechanics/molecular mechanics
was employed to investigate placement of the native substrate
5-CHQ and surrounding catalytic residues. The r.m.s.d
between the crystal structure and three-layer ONIOM-opti-
mized structures was 0.3 Å. The orientations of the key catalytic
FIGURE 4. Measurement of substrate/substrate analog binding through
ITC experiments. The trend of heat released by serial injections of substrates
into TftG was monitored. 2,5-Dihydroxybenzoquinone (diamond) showed a
typical heat-releasing pattern. The solid line represents the least square fit of
the data. Hydroquinone (square), 5-chlorohydroquinone (circle), and catechol
(triangle) did not show any significant affinity to TftG.
residues and other residues forming the active site pocket
showed little change, with the major changes being due to
movement of the C-terminal tail. The position of 5-CHQ had
similar orientation to the crystallographic position of the prod-
Ϫ6.5 kcal molϪ1, likely due to the observed hydrogen bonds
between ligand and residues of TftG, which was later confirmed
in the binary crystal structure. In addition, the positive entropic
contribution, ⌬S ϭ 0.79 cal molϪ1 degreeϪ1 was observed, sug-
gesting the liberation of water molecules from the active site
upon ligand association.
uct analog 2,5-DHBQ with the chlorine atom facing out of the
binding pocket toward the C-terminal tail (Fig. 5c).
Activity Assays and CD Spectra of TftG Mutants—Activity
assays of R17A, H24A, S56A, and H96A site-directed mutants
were performed to test their potential catalytic roles (Table 2).
The mutant D75N could not be tested due to solubility issues.
The H24A mutant exhibited the least amount of activity, which
was Ͻ1% specific activity relative to wild type. R17A and H96A
also displayed quite low activity (ϳ1%), and S56A retained 5%
Active Site—As indicated in Figs. 3 and Fig. 5a, a distinct
solvent accessible pocket was noticed between ␤2-␤3 and ␣1 in
apo-form TftG. Coincidentally, the Fo Ϫ Fc maps calculated
from the 2,5-DHBQ-soaked crystal diffraction data showed activity.
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Catalytic Mechanism of a YCII-like Dehydrochlorinase
FIGURE 5. TftG active site and ligand binding. a, molecular surface of TftG-Apo tetramer. The apo-form structure illustrated a distinct solvent accessible
pocket (shown in the black squares), which functions as the active site in TftG. This figure was generated by open source PyMOL (version 1.1r1). b, active site of
TftG⅐2,5-dihydroxybenzoquinone binary complex. Shown is a 2Fo Ϫ Fc map covering substrate and hydrogen bonded residues at a contour level of 1.0␴. c,
computational results for position of 5-CHQ. Shown is an overlay of the crystallographic and three-layer ONIOM-optimized active site catalytic residues and
substrate. Carbons are shown in gray for the crystallographic structure with bound 2,5-DHBQ and light blue for the ONIOM-optimized protein and 5-CHQ sites.
Catalytic residue-substrate hydrogen bond distances are shown.
TABLE 2
Specific activity of TftG active site mutations
Specific
activity
% Specific activity
(relative to WT)
Enzyme
Ϫ1
␮mol minϪ1 mg
WT
66.877 Ϯ 0.993
1.102 Ϯ 0.057
0.420 Ϯ 0.046
3.422 Ϯ 0.330
0.938 Ϯ 0.068
100
R17A
H24A
S56A
H96A
1.6%
Ͻ1.0%
5.1%
1.4%
The overall secondary structure as evaluated by CD spectra
appeared unperturbed relative to wild-type for all mutants
except H24A mutant (Fig. 6). The crystal structure illustrated
the residue His-24 was located in ␣1 and Asp-75 was located at
the C-terminal end of ␣3. The side chains of those residues,
contributed from two different helices, were within hydrogen
bond distance and thus appeared to be important for structural
integrity of the active site pocket. Considering the location of
the functional imidazole ring of His-24 and its mutant CD data,
it is likely that His-24 could be important for both structural
and catalytic roles.
FIGURE 6. CD spectra for TftG wild-type and mutants. The CD spectra were
recorded from 200 to 300 nm for wild-type (diamonds), R17A (triangles), H24A
(filled circles), S56A (empty circles) and H96A (squares) using an AVIV 202SF
spectropolarimeter (AVIV Biomedical) at 25 °C at a concentration of 10 ␮M.
The second highest match was 5-chloromuconolactone deha-
DISCUSSION
logenase of Rhodococcus opacus 1Cp (PDB code 3ZNU) (34),
Structural Homologs of TftG—TftG is a recently discovered with a Z-score of 10 but displayed lower sequence identity
dehydrochlorinase enzyme that catalyzes a cofactor-indepen- (23%). Another protein of unknown function, TM1266 from
dent dehydrochlorination of 5-CHQ to produce 2-hydroxy- Thermotoga maritima (PDB code 2NZC), shared reasonable
1,4-benzoquinone. There has been a critical gap in structural structural similarity with a Z-score of 8.5 and a sequence identity
and mechanistic information regarding the dechlorination of of 11%. All of the following proteins in the DALI search list had
chlorocatechols such as 5-CHQ, hampering an overall under- substantially decreased Z-scores and sequence identity, which
standing of their catabolic mechanism.
included a bacterial actinorhodin biosynthesis monooxygenase
To firmly establish a classification of TftG and to find any (ActVa-Orf6) (35), bacterial muconalactone isomerase (36) and
structural homologs, a DALI (32) search was performed against the C-terminal domain of archaeal LprA (37).
deposited structures in the PDB. The search found several pro-
Detailed structural investigation of TftG with a manual
teins with similar structures but relatively low sequence iden- superposition of HI0828 protein (PDB code 1MWQ) illustrated
tity to TftG. All of the matches had promiscuous or unknown a similar active site pocket as shown in TftG with the exception
functional roles. The highest match was the HI0828 protein of of Arg at position 21 instead of Tyr. However, HI0828 has a zinc
unknown function from Haemophilus influenza (PDB code ion at the active site, and a phosphohistidine has been proposed
1MWQ) (33), with a Z-score of 14.3 and 27% sequence identity. to be involved in activity. Atomic absorption spectroscopy was
28452 JOURNAL OF BIOLOGICAL CHEMISTRY
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Catalytic Mechanism of a YCII-like Dehydrochlorinase
conducted to test for the presence of zinc ions in TftG; however, meric structure of 5-chloromuconolactone isomerase was also
no zinc ions were indicated at all tested concentrations of TftG. quite different, existing as a decamer (Fig. 7b).
Although it was not listed in our DALI search due to low level
of structural similarity with TftG, LinA from Sphingobium
japonicum catalyzes a dechlorination reaction for ␥-hexachlo-
rocyclohexane (38, 39). LinA belongs to the SnoaL-4 superfam-
ily (cl17707), which are all polyketide cyclases that share the
SnoaL fold. LinA exists as a homotrimer, and each subunit
forms a cone-shaped ␣ϩ␤-barrel fold. Although LinA and TftG
share little similarity (18% sequence similarity and 4.6 Å root
mean square deviation in their C␣ positions), manual C chain
alignment of LinA and TftG displayed an approximately␣similar
location of the active site. In addition, the exceedingly hydro-
phobic substrate-binding pocket of LinA contains the same
proposed catalytic dyad of His-73 and Asp-25. The carboxyl
group of Asp-25 of LinA has been proposed to abstract a proton
from imidazole side chain of His-73, thereby increasing the
basicity of the imidazole nitrogen (38). The imidazole nitrogen
then acts as a catalytic base, abstracting a proton from the car-
bon of ␥-hexachlorocyclohexane ring, ultimately leading to a
spontaneous loss of chlorine from the ring (38). The oligomeric
structure of LinA was a trimer, further setting it apart from
TftG and 5-chloromuconolactone isomerase (Fig. 7c).
The lack of any zinc ion in the crystal structure of TftG and
supporting atomic absorption evidence shows that zinc is not
involved in TftG catalysis. It is possible that the presence of zinc
ion in the HI0828 active site is an artifact from the crystalliza-
tion condition and HI0828 could be a dehalogenase. However,
the HI0828 quaternary structure is a dimer, whereas the TftG
quaternary structure is a tetramer (Fig. 7a). Thus, our specula-
tion about HI0828 from Haemophilus influenza needs to be
tested.
The next match in our DALI search, 5-chloromuconolactone
dehalogenase (PDB code 3ZNU) belongs to the muconolactone
␦-isomerase (pfam02426) superfamily. Members of this protein
family (EC 5.3.3.4) are involved in the metabolism of catechols.
5-Chloromuconolactone dehalogenase has a ferredoxin-like
fold similar to TftG, but it does not have a conserved His-Asp
dyad distancing itself from TftG and YCII superfamily. Instead,
it has a conserved Glu-27 acting as a catalytic base. The oligo-
LinA, TftG, and 5-chloromuconolactone dehalogenase
belong to different enzyme superfamilies and use different sub-
strates. TftG catalyzes proton abstraction from an oxygen atom
of an aromatic compound, whereas 5-chloromuconolactone
dehydrohalogenase and LinA catalyze proton abstraction from
a carbon atom of non-aromatic substrates.
Sequence Alignment with Other YCII Family Members—To
further understand the relation of TftG to other members of the
YCII superfamily, we conducted a BLAST (24) search of all
non-redundant protein sequences. The results revealed many
YCII-like proteins, some with high identity (40–45%), but all
were of unknown function. Inspection of a sequence alignment
of the top 100 proteins illustrated a conserved signature
sequence of residues that were both implicated in TftG catalysis
and conserved throughout the YCII superfamily. An alignment
of the top 20 highest sequence identity proteins illustrates this
FIGURE 7. Monomeric and oligomeric state of known dehydrochlori-
nases. a, TftG; b, 5-chloromuconolactone dehalogenase of R. opacus 1Cp
(PDB code 3ZNU); and c, LinA from Sphingobium japonicum (PDB code 3A76).
This figure was generated by open source PyMOL (version 1.1r1).
FIGURE 8. Sequence alignment of TftG BLAST search results. The top 20 sequences were aligned with TftG, illustrating several conserved residues (shown in
boldface type) throughout the YCII superfamily. Residues that were conserved and implicated in TftG catalysis were highlighted yellow. M. magneticum, Magnetospi-
rillum magneticum; M. magnetotacticum, Magnetosprillum magnetotacticum; M. gryphiswaldense, Magnetospirillum gryphiswaldense; R. rubrum, Rhodospirillum
rubrum;P. gallaeciensis, Phaeobacter gallaeciensis;R. bacterium, Rhodobacteraceae bacterium;R. sphaeroides, Rhodobacter sphaeroides;R. capsulatus, Rhodobacter cap-
sulatus; S. meliloti, Sinorhizobium meliloti; S. fredii, Sinorhizobium fredii; L. aggregata, Labrenzia aggregata; A. brasilense, Azospirillum brasilense.
OCTOBER 4, 2013•VOLUME 288•NUMBER 40
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Catalytic Mechanism of a YCII-like Dehydrochlorinase
FIGURE 9. Proposed mechanism for dechlorination of 5-CHQ. The resting state of the enzyme contains water in the active site of TftG. I, upon 5-CHQ
association, water is liberated from the active site, and the C-terminal arm closes. The catalytic dyad His-24-Asp-75 begins the reaction through general base
catalysis as the imidazole nitrogen of His-24 abstracts a proton from the 2-hydroxyl group of 5-CHQ, leading to intramolecular hydrogen bonding between the
2-oxygen and 1-hydroxyl group and formation of an oxyanion at the 1-position, which is stabilized by an oxyanion hole formed by Ser-56 and Arg-17. II, ring
protonation occurs at the 5-chloro position of 5-CHQ by the general acid His-96 followed by deprotonation of the 4-hydroxyl group of 5-CHQ. III, electrons from
the 1-position oxyanion move causing the spontaneous loss of chloride. IV, the chloride ion leaves following a proton transfer from Asp-98, the C-terminal arm
opens, and the product 2-hydroxy-1,4-benzoquinone is released with subsequent active site regeneration.
signature sequence (Fig. 8). As expected, the His-24-Asp-75 important for functions throughout the YCII superfamily and
dyad was conserved throughout the family. In addition, Pro-76, could serve as its signature.
which is responsible for stabilizing and orienting the catalytic
Catalytic Mechanism of TftG for 5-CHQ—The crystal struc-
dyad in TftG, was conserved. Another set of residues conserved ture of the 2,5-DHBQ binary complex of TftG in combination
across the YCII superfamily were Asp-9, Arg-17, and Ser-56. with the QM:MM results for 5-CHQ, and the site-directed
Both Arg-17 and Ser-56 were responsible for forming an oxy- mutagenesis illustrated a plausible catalytic mechanism for
anion hole critical for catalysis in TftG. Asp-9 in TftG electro- TftG. The resting state of TftG likely contains water in the
statically interacts with Arg-17, ultimately keeping Arg-17 in active site as supported by our ITC results and the presence of
the proper orientation for oxyanion charge stabilization on the PEG in its apo-form crystal structure. The QM:MM results
substrate. In summary, Asp-9, Arg-17, His-24, Ser-56, Asp-75, confirmed that 5-CHQ would bind in a near identical angular
and Pro-76 are conserved throughout the YCII superfamily. orientation to 2,5-DHBQ as expected from the relatively tight
Expansion of the BLAST search/sequence alignment to include fit of the TftG active site. The computational results also illus-
dozens of lower identity proteins from YCII superfamily still trated the 1- and 2-hydroxyl groups of 5-CHQ oriented toward
maintains those residues, and thus this conservation of Asp-9, His-24 and the Arg-17/Ser-56, respectively. Upon association
Arg-17, His-24, Ser-56, Asp-75, and Pro-76 is likely quite of 5-CHQ, all water molecules are liberated from the active site,
28454 JOURNAL OF BIOLOGICAL CHEMISTRY
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Catalytic Mechanism of a YCII-like Dehydrochlorinase
radation of the herbicide 2,4,5-T by Pseudomonas cepacia AC1100. Gene
71, 267–277
and there is a concomitant closure of the active site by the
C-terminal residues of the neighboring subunit. The His-24-
Asp-75 catalytic dyad is responsible for starting the reaction
through general base catalysis. The imidazole nitrogen of
His-24 abstracts a proton from the 2-hydroxyl group of 5-CHQ,
leading to intramolecular hydrogen bonding between the 2-ox-
ygen and 1-hydroxyl group, stabilizing the resulting oxyanion at
the 1-position, which is further stabilized by an existing oxyan-
ion hole formed by Arg-17 and Ser-56 (Fig. 9, I). The other two
mutants, R17A and S56A, displayed 1.6 and 5.1% specific activ-
ity, respectively. The higher specific activity of S56A relative to
R17A is probably due to the fact that Arg-17 plays a more dom-
inant role in stabilizing the oxyanion at the 1-position of
5-CHQ.
7. Xun, L., and Wagnon, K. B. (1995) Purification and properties of compo-
nent B of 2,4,5-trichlorophenoxyacetate oxygenase from Pseudomonas
cepacia AC1100. Appl. Environ. Microbiol. 61, 3499–3502
8. Webb, B. N., Ballinger, J. W., Kim, E., Belchik, S. M., Lam, K. S., Youn, B.,
Nissen, M. S., Xun, L., and Kang, C. (2010) Characterization of chlorophe-
nol 4-monooxygenase (TftD) and NADH:FAD oxidoreductase (TftC) of
Burkholderia cepacia AC1100. J. Biol. Chem. 285, 2014–2027
9. Pflugrath, J. (1999) The finer things in x-ray diffraction data collection.
Acta Crystallogr. D Biol. Crystallogr. 55, 1718–1725
10. Adams, P. D., Afonine, P. V., Bunkóczi, G., Chen, V. B., Davis, I. W., Echols,
N., Headd, J. J., Hung, L. W., Kapral, G. J., Grosse-Kunstleve, R. W., Mc-
Coy, A. J., Moriarty, N. W., Oeffner, R., Read, R. J., Richardson, D. C.,
Richardson, J. S., Terwilliger, T. C., and Zwart, P. H. (2010) PHENIX: a
comprehensive Python-based system for macromolecular structure solu-
tion. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221
Ring protonation occurs at the 5-chloro position of 5-CHQ
by the general acid role of His-96, being followed by deproto-
nation of the 4-hydroxyl group of 5-CHQ (Fig. 9, II). This was
consistent with the specific activity of H96A (1.4%). The
QM:MM result also illustrated the 5-chloro position of 5-CHQ
oriented toward His-96. The resulting movement of electrons
from the 1-oxyanion causes a spontaneous loss of chloride ion
(Fig. 9, III). Opening of the C-terminal arm then allows for
release of the product 2-hydroxy-1,4-benzoquinone. The chlo-
ride ion leaves as HCl following a proton transfer from Asp-98
and the active site of TftG is regenerated (Fig. 9, IV).
11. Otwinowski, Z., and Minor, W. (1997) Processing of X-ray Diffraction
Data Collected in Oscillation Mode. Methods in Enzymology 276,
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12. Youn, B., Moinuddin, S. G., Davin, L. B., Lewis, N. G., and Kang, C. (2005)
Crystal structures of apo-form and binary/ternary complexes of Podo-
phyllum secoisolariciresinol dehydrogenase, an enzyme involved in for-
mation of health-protecting and plant defense lignans. J. Biol. Chem. 280,
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13. Gisi, M. R., and Xun, L. (2003) Characterization of chlorophenol 4-
monooxygenase (TftD) and NADH:flavin adenine dinucleotide oxi-
doreductase (TftC) of Burkholderia cepacia AC1100. J Bacteriol. 185,
2786–2792
14. Frisch, M. J., Trucks, G. W., Schlegel, H. B., Scuseria, G. E., Robb, M. A.,
Cheeseman, J. R., Scalmani, G., Barone, V., Mennucci, B., Petersson, G. A.,
Nakatsuji, H., Caricato, M., Li, X., Hratchian, H. P., Izmaylov, A. F., Bloino,
J., Zheng, G., Sonnenberg, J. L., Hada, M., Ehara, M., Toyota, K., Fukuda,
R., Hasegawa, J., Ishida, M., Nakajima, T., Honda, Y., Kitao, O., Nakai, H.,
Vreven, T., Montgomery, J., J. A., Peralta, J. E., Ogliaro, F., Bearpark, M.,
Heyd, J. J., Brothers, E., Kudin, K. N., Staroverov, V. N., Kobayashi, R.,
Normand, J., Raghavachari, K., Rendell, A., Burant, J. C., Iyengar, S. S.,
Tomasi, J., Cossi, M., Rega, N., Millam, J. M., Klene, M., Knox, J. E., Cross,
J. B., Bakken, V., Adamo, C., Jaramillo, J., Gomperts, R., Stratmann, R. E.,
Yazyev, O., Austin, A. J., Cammi, R., Pomelli, C., Ochterski, J. W., Martin,
R. L., Morokuma, K., Zakrzewski, V. G., Voth, G. A., Salvador, P., Dannen-
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Summary—TftG is a novel dehydrochlorinase enzyme. The
conserved His-Asp dyad proves to be catalytic performing a
unique dehydrochlorination reaction, together with the neigh-
boring polar residues. The active site residues implicated in
TftG catalysis form a signature sequence conserved throughout
the YCII superfamily. Considering the unique topology and
conservation of active site residues in TftG, it is very likely that
the proteins in YCII superfamily conduct lyase reactions (e.g.
dehydrochlorination) for hydroquinones or related structural
analogs.
Acknowledgments—We thank B. Webb and M. Nissen for help with
initial crystallization trials and preparation of selenomethionyl-de-
rivatized TftG.
15. Peterson, K. A., and Dunning, T. H. (2002) Accurate correlation consistent
basis sets for molecular core-valence correlation effects: the second row
atoms Al-Ar, and the first row atoms B-Ne Revisited. J. Chem. Phys. 117,
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