Karandikar et al, 2015
Properties of reductases from P. aeruginosa PP4
ABAB-D-15-00530
Revised Manuscript
Supplementary material
Biochemical Characterization of Inducible ‘Reductase’ Component of
Benzoate Dioxygenase and Phthalate Isomer Dioxygenases from
Pseudomonas aeruginosa strain PP4
Rohini Karandikar, Abinaya Badri and Prashant S.Phale
Department of Biosciences and Bioengineering, Indian Institute of Technology-Bombay,
Powai, Mumbai 400 076, India
 Corresponding author
Department of Biosciences and Bioengineering,
Indian Institute of Technology-Bombay
Powai, Mumbai 400076, India.
E-mail: pphale@iitb.ac.in
Soil isolate Pseudomonas aeruginosa strain PP4 utilizes phthalate isomers and
benzoate as the sole carbon source. The degradation is initiated by a ring-hydroxylating
dioxygenase consisting of an ‘oxygenase’ and ‘reductase’ component. Reductase components
from strain PP4 for phthalate isomer or benzoate grown cells were partially purified using
ion-exchange chromatography followed by gel-filtration chromatography.
Elution profiles of partially purified reductases from phthalate and terephthalate
grown cells are depicted in Fig.S1 and S2, respectively. Fig S1a, b and c are elution profiles
of reductase from Q Sepharose, DEAE Sephacel and Sephacryl S-100 column, respectively,
for phthalate grown cells. In this case, the reductase eluted in a range of 70-150 mM
(NH4)2SO4. Fig.S2a-c are elution profiles for reductase from Q Sepharose, DEAE Sephacel
and Sephacryl S-100 column, respectively, for terephthalate grown cells, where the reductase
eluted in a range of 50-150 mM (NH4)2SO4. In the gel filtration chromatography, the
reductases from phthalate and terephthalate grown cells eluted with an elution volume (Ve) of
38 and 37 ml, respectively. Thus, the elution profiles for partially purified reductase from
phthalate and terephthalate grown cells are highly similar to those from isophthalate grown
cells (See main manuscript, Fig. 4a-c).
The activity staining and SDS-PAGE profiles for reductases partially purified from phthalate
and terephthalate grown cells are shown in Fig.S1d,e and Fig.S2d and e, respectively.
According to the activity staining profiles (Fig.S1d and S2d), the partially purified reductases
from phthalate as well as terephthalate grown cells show a prominent reductase band at R f
1
Karandikar et al, 2015
Properties of reductases from P. aeruginosa PP4
0.55 and 0.56, respectively. The activity staining pattern in both the cases is similar to that
shown by reductase partially purified from isophthalate grown cells (See main manuscript,
Fig.4d). Similarly, the SDS-PAGE profiles (Fig.S1e and S2e) for reductases partially purified
from phthalate and terephthalate grown cells appear similar to each other as well as to the
SDS-PAGE profile for reductase from isophthalate grown cells (See main manuscript,
Fig.4e).
b
2.0
1.5
40
1.0
20
0.5
0.0
0
0
5
10
15
20
30
0.25
2.0
25
0.20
0.15
0.10
0.05
20
1.5
15
1.0
10
0.5
0.00
5
0.0
25
0
0
5
Fractions
d
0.70
35
0.65
30
0.60
25
20
0.55
15
0.50
10
0.45
5
0.40
0
34
36
38
40
20
2
3
4
M
1
2
3
43
29
20
14
42
Fractions
Fig. S1 Purification of reductase component from phthalate grown cells of strain PP4
Panels (a), (b) and (c) represent elution profiles from Q Sepharose, DEAE Sephacel and
Sephacryl S-100 columns. Symbols denote (○), protein OD at 280 nm; (▲), reductase
activity; (●), concentration of (NH4)2SO4. Panels (d) and (e) depict in-gel activity staining
and SDS-PAGE profiles for the partially purified ‘reductase’ component from phthalate
grown cells. Lanes denote, 1,Cell-free extract; 2,Q Sepharose pool; 3,DEAE Sephacel pool;
4,Sephacryl S-100 pool.
2
0.15
0.10
0.05
0.00
97
66
Rf
0.55
0.20
25
(kDa)
0.35
32
10
15
Fractions
0.25
e
1
Reductase ( mol min-1ml-1)
Protein (A280)
c
0.30
4
(NH4)2SO4 (M)
60
2.5
Reductase ( mol min-1ml-1)
Protein (A280)
2.5
0.30
Protein (A280
80
(NH4)2SO4 (M)
3.0
Reductase ( mol min-1ml-1)
a
Karandikar et al, 2015
Properties of reductases from P. aeruginosa PP4
1.5
30
1.0
20
0.5
10
0.0
0
0
5
10
15
20
Fractions
25
30
0.3
2.0
25
0.2
0.1
0.0
15
1.0
5
0.0
0
5
10
d
0.4
4
0.3
0.2
Reductase ( mol min-1ml-1)
8
34
36
38
40
Fractions
42
44
30
1
2
0.0
2
3
M
4
3
97
66
Rf
43
0.56
29
0
32
25
(kDa)
12
0.5
15
20
Fractions
0.1
e
1
0.6
0.2
10
0
c
0.3
20
1.5
0.5
30
0.4
20
46
14
Fig. S2 Purification of reductase component from terephthalate grown cells of strain
PP4 Panels (a), (b) and (c) represent elution profiles from Q Sepharose, DEAE Sephacel and
Sephacryl S-100 columns. Symbols denote (○), protein OD at 280 nm; (▲), reductase
activity; (●), concentration of (NH4)2SO4. Panels (d) and (e) depict in-gel activity staining
and SDS-PAGE profiles for the partially purified ‘reductase’ component from terephthalate
grown cells. Lanes denote, 1,Cell-free extract; 2,Q Sepharose pool; 3,DEAE-Sephacel pool;
4,Sephacryl S-100 pool.
The properties of reductases from phthalate isomer-grown cells were highly similar to each
other as observed in Table 2, (see main manuscript). These properties were different for
reductase from benzoate grown cells. Table S1 and S2 summarize the purification of
3
4
(NH4)2SO4 (M)
40
2.5
Reductase ( mol min-1ml-1)
2.0
0.4
Protein (A280)
50
(NH4)2SO4 (M)
2.5
Reductase ( mol min-1ml-1)
b
Protein (A280)
Protein (A280)
a
Karandikar et al, 2015
Properties of reductases from P. aeruginosa PP4
reductase component from phthalate and terephthalate grown strain PP4. Reductase from
phthalate and terephthalate-grown cells was purified with a yield of 9 and 3 %, respectively.
Table S1 Purification table for partially purified reductase from strain PP4 grown on phthalate
Step
Volume
Total activity
Total protein
Specific activity
Fold
Yield
(ml)
(µmol min-1)
(mg)
(µmol min-1mg-1)
Cell-free extract
12
328
110
3.0
1
100
Q Sepharose
5
153
24
6.5
2
47
DEAE Sephacel
1
87
7
12
4
27
Sephacryl S-100
1
31
0.8
39
13
9
(%)
Table S2 Purification table for partially purified reductase from strain PP4 grown on terephthalate
Step
Volume
Total activity
Total protein
Specific activity
Fold
Yield
(ml)
(µmol min-1)
(mg)
(µmol min-1mg-1)
Cell-free extract
12
348
93.6
3.7
1
100
Q Sepharose
5
145
18
8.1
2.2
42
DEAE Sephacel
1
34
3
11.3
3.05
10
Sephacryl S-100
1
12
0.4
30
8.1
3
(%)
The partially purified reductase component from phthalate and terephthalate grown cells were
characterized for kinetic constants Km and Vmax by measuring initial velocities by varying the
concentration of substrate DCPIP from 5 to 150 µM. The kinetic constants were calculated
using Enzyme Kinetics 11.0 software (SigmaPlot 12.3) and determined using MichaelisMenten and Lineweaver-Burke plot using equation V=Vmax[S]/Km + [S]. Apparent Km values
were 39.3 and 40 µM, whereas Vmax values were 45 and 48.3 µmol min-1mg-1 for reductases
partially purified from phthalate and terephthalate grown cells, respectively. The plots for
kinetic constants for reductases from phthalate and terephthalate grown cells are depicted in
Fig. S3a and b, respectively.
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Karandikar et al, 2015
Properties of reductases from P. aeruginosa PP4
a
20
1/Rate (µmol/min/mg)
Rate (µmol/min/mg)
30
10
0.20
0.15
0.10
0.05
-0.04
0.00
0.04
0.08
0.12
0.16
1/DCPIP (µM)
0
0
40
80
120
DCPIP (µM)
160
b
30
0.30
1/Rate (µmol/min/mg)
Rate (µmol/min/mg)
40
20
10
0.25
0.20
0.15
0.10
0.05
-0.05 0.00
0.05
0.10
0.15
0.20
0.25
1/DCPIP (µM)
0
0
40
80
120
DCPIP (µM)
160
Fig. S3. Kinetic properties of partially purified reductase from Pseudomonas aeruginosa
strain PP4. Panels (a) and (b) depict the Michaelis-Menten curve for reductase from phthalate
and terephthalate grown cells, respectively. Double reciprocal plots are shown in inset.
5