A R T I C L E S
Batabyal and Yeh
explored in Vitro, due to the technical difficulties associated
with its tendency to aggregate in free solution and to form
inclusion bodies when expressed in E. coli. By including L-Trp
throughout the preparation steps, we were able to produce
sufficient amounts of active recombinant hTDO enzyme for
spectroscopic studies (Batabyal et al., manuscript in preparation).
Here, we report the first thorough structural and functional
characterization of hTDO with resonance Raman and optical
absorption spectroscopies. In contrast to the monooxygenase
chemistry of P450s, the dioxygenase reactions of TDO and IDO
are relatively unexplored. Our results provide the first glimpse
of the mechanistic insights of the dioxygenase chemistry carried
out by the two enzymes. Recently, hIDO has been recognized
as an important drug target for cancer and neurological
disorders;8,25,26,35-37 the differences between hIDO and hTDO
revealed in this work hence offer invaluable structural informa-
tion for the design of new inhibitors selective for hIDO.
excitation from a Kr ion laser (Spectra Physics, Mountain View,
CA) was focused to a ∼30 µm spot on the spinning quartz cell
rotating at ∼1000 rpm. The scattered light, collected at right
angle to the incident laser beam, was focused on the 100-µm
wide entrance slit of a 1.25 m Spex spectrometer equipped with
a 1200 grooves/mm grating (Horiba Jobin Yvon, Edison, NJ),
where it was dispersed and then detected by a liquid-nitrogen
cooled CCD detector (Princeton Instruments, Trenton, NJ). A
holographic notch filter (Kaiser, Ann Arbor, MI) was used to
remove the laser scattering. The Raman shift was calibrated by
using indene (Sigma) and an acetone/ferrocyanide (Sigma)
mixture as the references for the 200-1700 and 1600-2200
cm-1 spectral windows, respectively. The laser power was kept
at ∼5 mW for all measurements, with the exception of that used
for the CO complexes, which was kept at ∼1-2 mW to avoid
photodissociation of the heme-bound CO. The acquisition time
was ∼20-30 min for all of the spectra obtained, with the
exception of that used for the measurements of the νC-O mode,
which was ∼1-2 h.
Materials and Methods
The ferrous derivative was prepared by reduction of the ferric
protein, pre-purged with Ar gas, with dithionite under anaerobic
conditions. The CO- or NO-bound ferrous complexes were
obtained by gentle purging CO or NO gas on the surface of the
solution containing the ferrous form of the enzyme under
anaerobic conditions. The concentration of the protein samples
used for the Raman measurements was ∼15-50 µM in pH 7.0
phosphate buffer (100 mM). The 12C16O and 14N16O were
obtained from Tech Air (White Plains, NY), and the 13C16O
and 15N16O were purchased from Icon Isotopes (Summit, NJ).
Expression and Purification of hTDO. The details of protein
expression and purification are described elsewhere (Batabyal
et al., manuscript in preparation). Briefly, the hTDO protein,
with truncated N- and C-terminal tails, plus a 6X-His tag
extension at the C-terminus, were overexpressed in E. coli BL21
Star (DE3) cells by using the pET14b vector (Stratagene, La
Jolla, CA). The recombinant protein was purified by affinity
chromatography with an Ni-NTA column (Novagen). The
protein was eluted with 250-300 mM imidazole (Sigma) in 50
mM phosphate buffer (pH 7.8), which was subsequently
removed by dialysis. To stabilize the protein, 10 mM L-Trp was
present throughout the purification procedure. The protein thus
collected was stored in 50 mM phosphate buffer (pH 7.8, 10
mM L-trp) with 10% glycerol at liquid nitrogen temperature
until use. To ensure that the protein was in its ferric L-Trp free
state, before each experiment, it was oxidized with potassium
ferricyanide, and subsequently passed through a sephadex G25
column to remove the ferricyanide, L-Trp and glycerol.
Activity Assay. For the activity measurements, the ferric
hTDO was rapidly mixed with a desired amount of D-Trp or
L-Trp in the presence of 100 µM sodium ascorbate in pH 7.0
phosphate buffer (100 mM) at room temperature. The final
concentration of hTDO was 0.5 µM. The reaction was followed
by monitoring the rate of product formation at 321 nm (ꢀ )
3750 M-1 cm-1 for N-formyl kynurenine).12 The Km and kcat
values were determined by Michaelis-Menten curve fit of the
data by using Origin 6.1 software (Microcal Software, Inc., MA).
All chemical reagents were obtained from Sigma-Aldrich and
were of the highest available purity.
Results and Discussion
Optical Absorption Spectra of hTDO. Figure 2A shows
the optical absorption spectra of the exogenous ligand-free ferric
and ferrous derivatives, as well as the CO-bound ferrous
derivative of hTDO. The ferric protein shows a Soret maximum
at 406 nm and a visible band at 633 nm (Table 1), typical for
a six-coordinate water-bound heme species. The ferrous protein
shows a Soret maximum at 432 nm and a single visible band at
556 nm, consistent with a five-coordinate high-spin ferrous heme
species. The CO-bound protein, on the other hand, exhibits a
Soret maximum at 420 nm and R and â bands at 568 and 538
nm, respectively, indicating a six-coordinate low-spin heme.
L-Trp-binding to hTDO induces small changes to the optical
absorption spectra, as shown in Figure 2B. The ferric derivative
shows a 1-nm shift of the Soret maximum to 407 nm, which is
associated with the disappearance of the visible band at 633
nm. The ferrous and CO-derivatives also exhibit 1-nm shift in
the Soret maximum to 433 and 421 nm, respectively, and the
visible bands shift to 555 and 567/537 nm, respectively.
Similar measurements were carried out for the NO-bound
ferrous complex, which shows Soret and R/â bands at 416 nm
and 573/542 nm, respectively, for the substrate-free enzyme.
L-Trp binding to the NO-bound hTDO induces 2 nm shift of
the Soret maximum to 418 nm, whereas the R/â bands stay
unaltered (see Figure S2 of the Supporting Information).
As listed in Table 1, the electronic transition bands of hTDO
revealed in this work are comparable to those of other heme-
containing proteins with histidine as the heme proximal ligand,
including hIDO. Nonetheless, the spectral perturbation intro-
duced by L-Trp binding in hTDO appear to be much less
Spectroscopic Measurements. The optical absorption spectra
were taken on a spectrophotometer (UV2100U) from Shimadzu
Scientific Instruments, Inc. (Columbia, MD) with a spectral slit
width of 1 nm. The resonance Raman spectra were taken on
the instrument described elsewhere.38 Briefly, the 413.1 nm
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Armstrong, R. S.; Lay, P. A. Biochemistry 2004, 43, 4892-4898.
(35) Gaspari, P.; Banerjee, T.; Malachowski, W. P.; Muller, A. J.; Prendergast,
G. C.; DuHadaway, J.; Bennett, S.; Donovan, A. M. J. Med. Chem. 2006,
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(36) Pereira, A.; Vottero, E.; Roberge, M.; Mauk, A. G.; Andersen, R. J. J.
Nat. Prod. 2006, 69, 1496-1499.
(37) Botting, N. P. Chem. Soc. ReV. 1995, 24, 401-412.
(38) Egawa, T.; Yeh, S. R. J. Inorg. Biochem. 2005, 99, 72-96.
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15692 J. AM. CHEM. SOC. VOL. 129, NO. 50, 2007