Journal of Agricultural and Food Chemistry
Article
with S-adenosyl-L-homocysteine (SAH) and Mg2+ was performed in the
same method as apo NkCOMT, except for the addition of 5 mM SAH
and 10 mM Mg to the protein solution. The crystals of the best quality
MATERIALS AND METHODS
■
Enzyme Expression and Purification of NkCOMT. The
expression and purification of NkCOMT were performed by following
the published protocols with some changes.
the Niastella koreensis strain from Korean Collection for Type Cultures
KCTC, Republic of Korea). The NkCOMT gene was amplified by
2+
appeared in 18% PEG8000, 0.1 M MES/sodium hydroxide pH 6.0, and
35,36
In brief, we purchased
2+
0
.2 M calcium acetate supplemented with 5 mM SAH and 10 mM Mg .
Data Collection and Structure Determination of NkCOMT.
(
The NkCOMT crystals of the best quality were transferred to
cryoprotectant solution containing 25% (v/v) glycerol. The crystals
were harvested with a loop of 0.2 mm diameter and flash-frozen in a
nitrogen gas stream at 100 K. X-ray diffraction data were collected at the
7A beamline of the Pohang Accelerator Laboratory (PAL, Republic of
Korea), equipped with a Quantum 270 CCD detector (ADSC, USA).
All data were indexed, integrated, and scaled together using the
polymerase chain reaction (PCR) and subcloned into a pET30a
expression vector by NdeI and XhoI restriction sites. An Escherichia coli
R
BL21(DE3)-T1 strain transformed with the pET30a:NkCOMT
vector was grown to an OD600 of 0.7 in an LB medium containing 50
mg/L kanamycin at 312 K, and protein expression was induced by 0.5
mM 1-thio-β-D-galatopyranoside (IPTG) followed by further incuba-
tion for 20 h at 293 K. The enzyme purification was performed using a
Ni-NTA agarose column (Qiagen) and size-exclusion chromatography
using a Sephacryl S-300 column (320 mL, GE Healthcare). The
purified protein was concentrated to 37 mg/mL using a concentrator
37
HKL2000 software package. Crystals of NkCOMT belonged to the
space group P12 1 with unit cell parameters a = 38.27 Å, b = 97.08 Å, c =
1
1
06.52 Å, α = γ = 90.0°, and β = 93.4. With four molecules of NkCOMT
in the asymmetric unit, the crystal volume per unit of protein mass was
(
Amicon Ultra Centrifugal Filter, 10 kDa pore size). All purification
procedures were performed at 277 K.
3
−1
2
4
.05 Å Da , which indicates a solvent content of approximately
38
0.16%. Crystals in complex with SAM belonged to the space group
Thermal Stability Detection of NkCOMT. Thermal stability of
NkCOMT with various metal ions was measured by melting curves
with a protein thermal shift dye (Applied Biosystems) in StepOnePlus
Real-Time PCR (Thermo Fisher Scientific) according to manufac-
turer’s instructions. NkCOMT (1 mg/mL, 20 μM) was incubated with
C 2 with unit cell parameters a = 166.18 Å, b = 93.18 Å, c = 43.34 Å, α =
1
1
γ = 90.0°, and β = 104.1. Assuming three molecules of NkCOMT per
3
asymmetric unit, the crystal volume per unit of protein mass was 2.26 Å
−
1
Da , which corresponds to a solvent content of approximately
5.52%. Crystals in complex with SAH and Mg belonged to the
38
2+
4
2
+
2+
2+
2+
2+
2+
2+
each of 1 mM metal ions (Mg , Ca , Mn , Fe , Ni , Cu , Zn ,
space group P2 2 2 with unit cell parameters a = 42.69 Å, b = 98.23 Å, c
1
1 1
2+
2+
Co , and Cd ) for 1 h at 277 K. The reaction mixture contained 5 μg
of the metal ion-protein, 1 × protein thermal shift dye (Applied
Biosystems), and 2 M Tris-HCl pH 8.0 in 20 μL. NkCOMT protein
denaturation signals were detected by increasing temperature from 298
to 372 K. Melting temperatures were determined from the first
derivative curve.
=
101.77 Å, and α = β = γ = 90.0°. Assuming two molecules of
NkCOMT per asymmetric unit, the crystal volume per unit of protein
mass was 2.22 Å Da , which corresponds to a solvent content of
approximately 44.61%. All three structures of NkCOMT were
determined by molecular replacement with the CCP4 version of
3
−1
38
39
40
MOLREP, using the structure of COMT from Streptomyces regensis
HPLC Analysis of NkCOMT Reaction Products. The reaction
samples were analyzed using a CMB-20A HPLC (Shimadzu), equipped
with a UV/Vis detector (SPD-20A) and a C18 column (Shimadzu
Shim-pack GIS ODS-I C18, 5 μm, 4.6 × 150 mm). Mobile phase A
consisted of water containing 0.1% formic acid, and mobile phase B was
(PDB code 5N5D) as a search model. Further model building was
40
performed manually using the program WinCoot, and refinement was
40
performed with CCP4 refmac5. The refined models of NkCOMT,
2
+
those in complex with SAM, and those in complex with SAH and Mg
were deposited in the Protein Data Bank with PDB codes of 7CVU,
41
100% acetonitrile. The flow rate was 1.5 mL/min for 4 min and changed
7CVV, and 7CVW, respectively.
to 2 mL/min from 4 to 12 min and 50 s followed by 1 mL/min. Mobile
phases were as follows: 0−8 min and 50 s, 5% B; 8 min and 50 s−10 min
and 30 s, 6% B; 10 min and 30 s−17 min and 50 s, 7.5% B; 17 min and
Size-Exclusion Chromatographic (SEC) Analysis. Analytical
size-exclusion chromatography was performed using a Superdex 200
10/300 GL column (GE Healthcare Life Sciences) with purified
NkCOMT protein solution (2 mg/mL, 40 mM Tris-HCl pH 8.0, and
150 mM NaCl). The molecular mass of the eluted NkCOMT sample
was calculated by a calibration curve. All experiments were performed in
duplicate and at 277 K of column temperature.
5
0 s−21 min, 20% B; 21−23 min, 15% B; 23−27 min, 22% B; 27−29
min, 20% B; 29−40 min, 100% B. Vanillic acid and iso-vanillic acid were
detected at 280 nm. All experiments were performed in duplicate at a
column temperature of 323 K.
Site-Directed Mutagenesis. The forward primers for
E211R
E211K
NkCOMT
and NkCOMT
were 5′-ATGGTAGGTG-
RESULTS AND DISCUSSION
TAAAAAGATACGATGGAATGGCT-3′ (nucleotide 616−648, for-
ward) and 5′-ATGGTAGGTGTAAAAAAATACGATG-
GAATGGCT-3′ (nucleotide 616−648, forward). The reverse primers
■
Metal Preference of NkCOMT. To investigate the
biochemical properties of NkCOMT, we expressed and purified
the NkCOMT protein by Ni-NTA and size-exclusion
E211R
E211K
for NkCOMT
and NkCOMT
were 5′-AGCCATTCCATCG-
TATCTTTTTACACCTACCAT-3′ (nucleotide 616−648, reverse)
2+
chromatography. It has been reported that Mg is required
and 5′-AGCCATTCCATCGTATTTTTTTACACCTACCAT-3′
for enzyme catalysis for most of the known OMTs, and other
metal ions are required for a few enzymes. In order to investigate
the metal preference of NkCOMT, we added 1 mM of various
(
nucleotide 616−648, reverse), respectively. Site-specific mutations
were constructed using a QuikChange kit (Stratagene), and sequencing
was performed to confirm correct incorporation of the mutations.
Mutant proteins were purified with the same method with wild-type
NkCOMT.
2
+
2+
2+
2+
2+
2+
divalent metal ions, such as Mg , Ca , Mn , Fe , Ni , Cu ,
2
+
2+
2+
Zn , Co , and Cd , to the EDTA-treated reaction mixtures
containing protocatechuate (PCA) as a substrate (Figure 1A)
and measured the enzyme activity by monitoring the amount of
produced vanillic acid and iso-vanillic acid (Figure 1B). We
detected a residual activity even without the addition of metal
ions, indicating that a trace amount of metal ions remained in the
protein despite EDTA treatment (Figure 1B). We observed the
Crystallization of NkCOMT. For crystallization screening,
commercial crystal-screening kits were used, including Index, PEG
ion I and II (Hampton Research), and Wizard Classic I and II (Rigaku
Reagents), using the sitting-drop vapor diffusion method with an MRC
crystallization plate (Molecular Dimensions) at 295 K that was
35,36
published in previous paper.
The apo NkCOMT crystals of the best
quality appeared in 16% PEG3350 and 8% Tacsimate pH 6.0. The
crystallization screening of NkCOMT in complex with S-adenosyl-l-
methionine (SAM) was performed in the same method as apo
NkCOMT, except for the addition of 10 mM SAM to the protein
solution. The crystals of the best quality appeared in 20% PEG4000,
2+
highest activity with Mg among the divalent metal ions (Figure
2
+
2+
1
B). We also detected significant activities with Co , Zn ,
2
+
2+
2+
2+
Cu , and Cd , and among these metal ions, Zn and Cu
showed relatively higher activities (Figure 1B). We also
measured the ratio of para- and meta-forms of the products.
2
1
0% 2-propanol, and 0.1 M sodium citrate pH 5.6 supplemented with
0 mM SAM. The crystallization screening of NkCOMT in complex
2
+
When Mg was used, we observed the para- and meta-form
2
532
J. Agric. Food Chem. 2021, 69, 2531−2538