H. Dong et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e7
3
was determined at 313 K in BrittoneRobinson buffer (20 mM, pH
4.0e12.0). The optimum temperature was determined by using
20 mM Britton-Robinson buffer (pH 10.0) from 283 to 333 K. The
pH-dependent thermostability of PaAlr was determined by incu-
bating the sample proteins at 313 K in 20 mM BrittoneRobinson
buffer (pH 8.0, 9.0, 10.0, and 11.0) for 0e20 h. The residual activity
was then measured by standard assay (pH 10.0, 313 K). The ther-
mostability of PaAlr was evaluated by measuring the residual ac-
tivity under standard conditions after pre-incubation of the
enzyme in 20 mM Britton-Robinson buffer (pH 10.0) at 303, 313,
and 323 K for 0e20 h.
replicate trees in which the associated taxa clustered together in
the bootstrap test (500 replicates) are shown next to the branches
[35]. The evolutionary distances were computed using the p-dis-
tance method and are displayed in the units of the number of
amino acid differences per site. The analysis involved 13 amino acid
sequences. All positions containing gaps and missing data were
eliminated. A total of 333 positions were present in the final
dataset. Evolutionary analyses were conducted in MEGA X [36].
3. Results and discussion
The substrate specificity of PaAlr was determined by measuring
the activity of the enzyme toward different L-amino acids. All re-
3.1. Optimal reaction pH and temperature of PaAlr
actions were conducted in 20 mM Britton-Robinson buffer (pH
10.0) containing 50 mM substrate at 313 K for 10 min.
The Km and Vmax of PaAlr were reported previously [17]. The Km
value of PaAlr was 4.1 mM for
The Vmax of PaAlr was 210 U$mgꢀ1 for
-alanine [17]. We further characterized other parameters of the
enzymatic activities of PaAlr.
The PaAlr (0.086 g) showed a maximum activity for
L
-alanine and 5.6 mM for
D-alanine.
L
-alanine and 140 U$mgꢀ1 for
Kinetic parameters of PaAlr were determined by measuring the
total amount of D- or
matography (HPLC) with spectrofluorometer [26,28]. The reaction
mixture (40 L) was composed of 20 mM Britton-Robinson buffer
(pH 10.0), 10 M PLP, and D- or -alanine in various amounts. The
L-alanine by high performance liquid chro-
D
m
m
L-alanine
m
L
as the substrate at pH 10.0 and 313 K (Fig. 1A and B, respectively).
The relative activity of PaAlr demonstrated over 70% of its
maximum activity in the pH range of 8.0e12.0 and temperature
from 303 to 323K.
Fig. 1C and D shows the temperature and pH dependence of the
enzyme stability of recombinant PaAlr (0.086 mg) respectively. The
enzyme was more stable at pH 8.0 and 9.0 than pH 10.0 and 11.0.
Except for pH 11.0, the first 1 h of incubation at pH 8.0, 9.0, and 10.0
helped to activate the enzyme. The half-life of protein PaAlr at pH
10.0 was approximately 4 h. The enzyme was most thermostable at
303 K. The relative activities decreased rapidly at 313 and 323 K.
The half-life of protein PaAlr at 313 K was about 2 h.
enzymatic reaction was initiated by adding purified protein PaAlr or
Britton-Robinson buffer as the negative control. The enzymatic
reaction remained at 313 K for 10 min, and then was terminated by
adding 40
of 2 M sodium hydroxide (NaOH), D- and
mixture were derivatized in a 0.28 M borate solution (pH 9.0)
containing 0.2% N-tert-butyloxycarbonyl- -cysteine (Boc-L-Cys)
(Fluka, Switzerland) and 0.2% o-phthaldialdehyde (OPA) (Sigma,
Germany) at 298 K for 2 min. The resultant mixture (10 L) was
separated using a Nova-Pack C18 column (4
m, 3.9 ꢁ 300 mm;
Waters, USA) at 313 K, and the D- and -alanine derivatives were
mL of 2 M HCl. After neutralizing the reaction with 40
mL
L-alanine in the reaction
L
m
m
L
detected by a fluorescence detector (RF20A, Shimadzu, Japan). The
kinetic parameters, Km and Vmax were determined by nonlinear
regression using the GraphPad Prism 5.0 software (http://www.
3.2. Substrate specificity of PaAlr
The substrate specificity of PaAlr toward various L-amino acids
2.3. Protein purification, crystallization, diffraction data collection
Table 1
and structure determination
Data collection and structure refinement statistics.
The PaAlr was purified and crystallized as previously reported
[29]. The crystals were quick-soaked in reservoir solution supple-
mented with 15% (v/v) glycol as a cryoprotectant for 15e20 s and
flash-cooled in liquid nitrogen at 100 K for data collection. The X-
ray diffraction data set was collected at 100 K using an in-house X-
ray source (Rigaku MicroMax-007 HF desktop rotating-anode X-ray
generator with a Cu target operated at 40 kV and 30 mA) and a
Saturn 944 þ detector with a 70 mm crystal-to-detector distance at
a wavelength of 1.5418 Å. A total of 360 diffraction frames were
collected with 0.5ꢂ oscillation per image at 100 K. The diffraction
data was indexed, integrated using iMosflm and subsequently
scaled using Scala in CCP4 suite [30].
The crystal structure of PaAlr was solved by the molecular
replacement method [31] using PaDadX (Protein Data Bank (PDB)
ID 1RCQ) as template. Structure refinement was performed using
REFMAC5 [32]. Model building was facilitated using Coot [33].
The coordinate file of the crystal structure of PaAlr was depos-
ited to the PDB with access code 6A2F.
Data collection
Space group
Unit cell parameters (Å,
P212121
a ¼ 74.24, b ¼ 78.30, c ¼ 154.07;
ꢂ
)
a
¼
b
¼
g
¼ 90.0
Resolution (Å)
Rmergea (%)
Average I/s(I)
No. of observed reflections
No. of unique reflections
Completeness (%)
50e2.50(2.64e2.50)
7.8(17.4)
8.0(4.1)
177754
28844
90.4(58.8)
6.2(3.7)
2.78
Redundancy
Matthews coefficient (Å3Daꢀ1
Solvent content (%)
)
55.78
2
Molecules per asymmetric unit
Refinement
Resolution (Å)
50e2.50
0.188/0.238
97.2
2.8
0
Rwork/Rfree
Ramachandran favored (%)
Ramachandran allowed (%)
Ramachandran outliers (%)
No. of atoms
Protein
Ligand/ion
5420
62
344
2.4. Phylogenetic tree construction
Water
R.m.s. Deviations
Bond lengths (Å)
Bond angles (ꢂ)
The evolutionary history was inferred using the neighbor-
joining method [34]. The bootstrap consensus tree inferred from
500 replicates [35] was used to represent the evolutionary history.
In this method, branches corresponding to partitions reproduced in
less than 50% bootstrap replicates are collapsed. The percentage of
0.005
1.098
ꢀ
ꢀ
PP
PP
a
Rmerge
¼
jIiðhklÞehIðhklÞi =
IiðhklÞ, where IiðhklÞ is an individual in-
hkl
i
hkl
i
tensity measurement, and IðhklÞ is the average intensity for all i reflections. Values in
parentheses are for the highesteresolution shell.
Please cite this article in press as: H. Dong, et al., Enzymatic characterization and crystal structure of biosynthetic alanine racemase from