H. Rimal et al. / Archives of Biochemistry and Biophysics 585 (2015) 64e74
67
ampicillin (Amp) as the respective selection markers, and were
then incubated in an orbital shaker (180 rpm) at 37 C until the cell
chromatography (HPLC, SPD-M20A; Shimadzu, Tokyo, Japan). The
HPLC was performed under the following condition: Column,
mightysil RP-18 GP (the binary mobile phases, 250 mm ꢁ 4.6 mm
I.D., Kanto Chemical, Tokyo, Japan), composed of solvent A [pH 2.3,
with TFA (v/v)] in water and solvent B (100% methanol), were used
ꢀ
density was about 0.6 at OD600. The protein expression was induced
by the addition of 1 mM IPTG, and the cells were incubated for 48 h
ꢀ
at 20 C. The harvestation and lysis of the cell pellets were exactly
ꢂ1
the same as those for the DoxA.
in a flow rate of 1.0 ml min . Detection was carried out with a UV
detector (Shimadzu, SPD-20AV, and UVeVIS Detector) at 254 nm.
The catalytic DoxA parameters were optimized for temperature,
pH, time, protein concentration, and enzymatic thermostability to
find a suitable condition for a more effective substrate conversion.
2
.9. Purification of proteins
The DoxA, FDX1, FDX3, FDR2, PDX, and PDR were all purified
using the same protocol. The soluble protein fraction was purified
þþ
by using immobilized metal (Co ) affinity chromatography (IMAC)
3. Results
®
with a Talon resin. The protein lysate was mixed with resin and
kept on a shaker for 30 min in ice for proper binding. The protein-
bound resin was pre-equilibrated with 3-column volumes of
equilibrium buffer. Any nonspecifically-bound proteins were
washed off the column with 4-column volumes of washing buffer
containing 10 mM imidazole, before the bound protein was eluted
with 2-column volumes of elution buffer with 100 mM imidazole.
The eluted proteins were concentrated by using centricon (Milli-
pore) of a respective size. Since stability is the one of the most
challenging factors of the in vitro study, we added 10% glycerol
along with 1 mM PMSF and 0.1 mM EDTA in 50 mM sodium
phosphate buffer (pH 7.5) during the concentration of the proteins.
3.1. Protein structure, proteineprotein docking by Z-Dock, and
proteineprotein refining by R-Dock
The target sequence and its template protein structure that were
obtained from the PDB have been summarized in Table 2, which
includes the PDB ID, sequence identity, resolution, and source or-
ganism. Models of DoxA, FDX1, and FDR2 were generated using
homology modeling. The first step in the proteineprotein docking
was performed using Z-Dock (Fig. 2A and B); a total of 60 clusters
and 2000 poses were generated (data not shown). The most-
consistent cluster no. 1, which has the highest number of good
scoring poses as scored by Z-Rank, was chosen for an R-Dock
refinement. Out of all of the poses in cluster no.1, only 5 top-scoring
poses were made into a single group and further refined by R-Dock.
The docked poses generated after Z-Dock were between DoxA-
FDX1 and FDX1-FDR2 (data not shown). R-Dock helped to re-rank
(refine) the Z-Dock hits based on the multi-staged CHARMm en-
ergy minimization method and is a very effective method for
determining the best complex in terms of energy. The E-RDock
score that was calculated in terms of the energy of the 5 poses
subjected to the R-Dock refinement are enlisted in Table 3. The E-
RDocks is given by the following molecular formula:
E_sol þ beta*E_elec2, whereby “E_sol” is the desolvation energy of
the protein complex and “E_elec2” are the electrostatic energies of
the protein complex after the second CHARMm minimization.
ꢀ
Aliquots of the purified protein were prepared and frozen at ꢂ20 C
until use. The purified proteins were also analyzed using 15% SDS-
PAGE.
2.10. Determination of protein concentration and CO-binding CYP
assay
To measure the CYP concentration in the cell culture, the protein
was diluted in 50 mM of sodium phosphate buffer containing 10%
v/v) glycerol. A few crystals of sodium dithionite were added and
(
the protein was divided into two cuvettes. The sample cuvette was
saturated with about 30 bubbles to 40 bubbles of CO at a rate of 1
bubble per second, and was scanned between 400 nm and 500 nm
at room temperature using a Shimadzu 1601PC Spectrophotometer.
The CYP content was measured from the difference in the spectrum
at 450 nm and 490 nm using an extinction molecular coefficient of
3.2. Calculation of interaction energy on the proteineprotein model
ꢂ1
ꢂ1
ε
450e490 ¼ 91 mM cm [22]. The PDR concentration was deter-
mined as the average of the concentrations calculated from each of
the three wavelengths 378 nm, 454 nm, and 480 nm using the
extinction coefficients (ε) 9.7 mM cm , 10.0 mM cm , and
The interaction energy between DoxA-FDXs and FDRs-FDXs was
calculated by using CHARM. The average interaction energy of the 5
refined poses between DoxA-FDXs was found to be optimal with
DoxA and FDX1 with an energy of ꢂ996.34 kcal/mol, and between
FDRs-FDXs, the average was found to be optimal among FDX1 and
FDR2 with an interaction energy of ꢂ943.29 kcal/mol (Table 3).
ꢂ1
ꢂ1
ꢂ1
ꢂ1
ꢂ1 ꢂ1
8
.5 mM cm [23]. The PDX concentration was determined as the
ꢂ1
ꢂ1
average concentration calculated with ε415 ¼ 11.1 mM cm and
ꢂ1
ꢂ1
ε
455 ¼ 10.4 mM cm [23]. The FDX1 and FDX3 concentrations
ꢂ1
ꢂ1
were estimated at 465 nm with ε ¼ 8.42 mM cm [24]; for the
3 4
Ligands like heme and Fe eS were excluded from the energy
FDR2, the concentration was determined at 456 nm with
calculation because calculating binding energy with ligands leads
to instability; moreover, protein-binding energy is much stronger
than the binding energy of the ligands inside a protein. According to
the previously mentioned in silico analysis, we finally concluded
that a more effective electron-transport could be achieved with the
following: NADH / FDR2 / FDX1/FDX3 / DoxA (Fig. 3).
ꢂ1
ꢂ1
ε ¼ 7.1 mM cm [25].
2.11. Enzymatic bioconversion of DNR to DXR by DoxA
The DoxA activity was determined using the DNR substrate. The
reaction mixture consisted of 1
FDX/PDX/FDX3), 1 FDR (FDR2/spinach FDR/PDR), 4 mM
glucose-6-phosphate, 5 U glucose-6-phosphate dehydrogenase,
00 ng catalase, 10 mM MgCl , and 100 M substrate, and the re-
action was initiated by adding 250
mM DoxA, 5 mM FDX (FDX1/spinach
mM
3.3. Measuring of distance between ligands involved in proteins
1
2
m
The distances between the ligands were measured based on the
R-dock results, whereby the ligands were inserted from the ho-
mology model that was made using DS 3.5 to allow the distances to
be measured. In the case of DoxA-FDX1 (Fig. 2C), the distance
measured between the Fe of DoxA and the FeeS cluster of FDX1 was
m
M NADH in 50 mM sodium
ꢀ
phosphate buffer. The reaction mixtures were incubated at 30 C for
2
h; afterward, the reaction mixture was stopped with 100 mM
Tris-buffer and extracted with a double volume of methanol and
chloroform at a ratio of 1:9. The extracted solution was subse-
quently dried under nitrogen gas and was again diluted with
methanol. The product was analyzed using high performance liquid
ꢀ
14.59 A. The active site of DoxA-FDX1 showed some residues,
whereby ARG57, ALA61, HIS104, HIS107, ALA111, PHE114, ASN115,
PRO116, ASP362, GLY363, PRO364, TYR366, and GLN371 emerged
Rimal, Hemraj
