Supplementary file 2 for Kaltenbach at al., Reverse evolution leads to genotypic
incompatibility despite functional and active-site convergence
Kinetic parameters of all variants
Information about Supplementary Files 2A-G. These files give an overview of the kinetic
parameters of all PTE variants. Lysate measurements: Cells were grown in at least
duplicate and lysates sufficiently diluted (~1-10,000-fold) to determine initial rates of
paraoxon and/or 2NH hydrolysis at a substrate concentration of 200 μM, normalized to
cell density, corrected for the dilution factor, and averaged. This experiment was
repeated twice and the combined average determined. Purified enzyme measurements:
Variants were purified in at least duplicate in a 96-well format and sufficiently diluted (~
0.1 nM - 10 μM) to determine initial rates of paraoxon and/or 2NH hydrolysis at a
substrate concentration of 200 μM, corrected for the dilution factor, and averaged.
Selected variants were purified on a larger scale to determine initial rates over a range
of substrate concentrations and calculate Michaelis Menten parameters. For back-towild-type mutations, wild-type amino acid is shown in lower case italics.
1
Supplementary File 2A. Enzymatic activities of selected variants from the forward and reverse
evolution in lysate and using purified enzyme.
Variant
wtPTE-1[c]
R1
R2a
R2b
R3
R4
R5a
R5b
R6
R7a
R7b
R7c
R8
R9
R10
R11a
R11b
R12
R13a
R13b
R14
R18-1 [d]
R18-2 [d]
R19
R20
R21
AE
revR1
revR2
revR3
revR4
revR5
revR6
revR7a
revR7b
revR8a
revR8b
revR9
neoPTE
wtPTE-2[c]
Round
0
1
2
2
3
4
5
5
6
7
7
7
8
9
10
11
11
12
13
13
14
18
18
19
20
21
22
rev1
rev2
rev3
rev4
rev5
rev6
rev7
rev7
rev8
rev8
rev9
rev12
0
lysate [a]
relative to wtPTE
1.0
0.08±0.003
0.071±0.007
0.049±0.023
0.082±0.014
0.014±0.004
0.024±0.003
0.022±0.003
0.03±0.004
0.015±0.006
(7.1±0.2)! 10 -3
(8.2±0.4)! 10 -3
(3.2±0.5)! 10 -3
(1.5±0.3)! 10 -3
(2.0±0.01)! 10-3
(1.2±0.3)! 10 -3
(2.0±0.3)! 10 -3
(2.0±0.1)! 10 -3
(1.6±0.1)! 10 -3
(1.0±0.2)! 10 -3
(1.6±0.1)! 10 -3
(9.9±1.3)! 10 -5
(2.0±0.4)! 10 -5
(2.0±0.1)! 10 -5
(9.1±0.1)! 10 -6
(1.0±0.01)! 10-5
(1.0±0.1)! 10 -4
(2.4±0.1)! 10 -3
(9.4±4.0)! 10 -3
0.016±0.003
0.033±0.018
0.04±0.01
0.075±0.024
0.06±0.005
0.089±0.001
0.082±0.01
0.014±0.003
0.022±0.05
Paraoxon
purified[b]
kcat [s -1 ]
KM [µM]
kcat/KM [M-1s-1 ]
(1.3±0.1)! 10 3
2.3! 107
57±5
62±3
7±1
8.9! 106
160±50
13±3
1.2! 107
2.4! 107
440±10
18±1
200±60
21±3
9.5! 106
300±10
250±20
1.2! 106
60±1
310±20
1.9! 105
63±1
67±4
9.4! 105
65±2
120±20
4.6! 105
20±4
120±2
1.7! 105
1.2! 105
53±3
430±60
17±1
180±10
9.4! 104
10±1
300±80
3.3! 104
11±1
320±10
3.5! 104
5.7±0.2
240±20
2.4! 104
4.2±0.1
250±20
1.7! 104
4.2±0.1
240±20
1.8! 104
6.4±0.1
240±10
2.7! 104
17±0.1
480±10
3.5! 104
2.2±0.1
360±40
6.1! 103
0.8±0.02
640±50
1.3! 103
0.73±0.1
(1.3±0.2)! 10 3
560
0.69±0.03
480±50
1.4! 103
0.15±0.01 (1.0±0.1)! 10 3
150
0.11±0.02
750±250
150
0.07±0.02 (1.1±0.1)! 10 3
68
0.12±0.01 (1.2±0.1)! 10 3
100
0.85±0.02
790±40
1.1! 103
13±1
350±10
3.7! 104
34±1
200±10
1.7! 105
34±1
90±4
3.7! 105
24±1
88±4
2.7! 105
28±1
74±2
3.8! 105
35±1
73±2
4.7! 105
340±10
91±2
3.7! 106
140±10
66±2
2.0! 106
48±1
140±10
3.5! 105
420±10
110±10
3.9! 106
270±10
130±10
2.1! 106
5.5! 106
360±10
65±5
2NH
lysate [a]
relative to wtPTE
1.0
6.6±1.5
430±80
(2.1±0.6)! 10 3
430±90
(5.6±3.8)! 10 3
(1.3±0.7)! 10 4
(7.1±3.8)! 10 3
(1.5±0.7)! 10 4
(1.7±0.9)! 10 4
(2.3±1.1)! 10 4
(8.3±3.7)! 10 3
(6.1±2.2)! 10 4
(7.1±3.1)! 10 4
(1.0±0.4)! 10 5
(1.0±0.5)! 10 5
(1.3±0.3)! 10 5
(1.4±0.4)! 10 5
(2.4±0.7)! 10 5
(1.2±0.6)! 10 5
(2.3±1.0)! 10 5
(3.3±0.9)! 10 5
(1.9±0.6)!
(2.3±0.8)!
(1.6±0.6)!
(1.7±0.6)!
(9.7±5.3)!
(6.1±1.5)!
(8.9±3.7)!
(8.3±3.3)!
(1.2±0.3)!
(1.9±0.6)!
(2.4±0.5)!
(1.2±0.5)!
(1.9±0.7)!
(1.6±0.4)!
(2.1±0.3)!
(3.4±1.2)!
10 5
10 5
10 5
10 5
10 4
10 4
10 3
10 3
10 4
10 4
10 4
10 4
10 4
10 4
10 4
10 4
kcat [s -1 ]
0.035±0.002
0.27±0.01
2.9±0.1
24±3
3.3±0.1
54±2
65±3
54±1
130±10
110±10
74±2
28±1
73±2
350±10
650±20
340±10
250±10
400±10
420±20
470±10
490±10
880±30
500±30
390±10
560±20
150±10
870±50
670±10
500±30
99±2
69±1
77±2
280±10
300±10
240±1
230±10
70±1
200±1
110±10
0.026±0.001
purified[b]
KM [µM] kcat/KM [M-1s-1 ]
83±19
420
250±30
1.1! 103
220±10
1.3! 104
1.6! 105
150±30
230±20
1.4! 104
120±20
4.5! 105
120±10
5.4! 105
110±10
4.9! 105
190±20
6.8! 105
160±10
6.9! 105
5.7! 105
130±10
120±10
2.3! 105
140±10
5.2! 105
110±10
3.2! 106
190±10
3.4! 106
160±10
2.1! 106
110±10
2.3! 106
140±10
2.9! 106
120±10
3.5! 106
100±10
4.7! 106
88±22
5.6! 106
63±9
1.4! 107
89±10
5.7! 106
200±10
1.9! 106
110±20
7.6! 105
130±10
1.1! 106
130±20
6.5! 106
140±10
4.7! 106
38±9
1.3! 107
150±10
6.6! 105
120±10
5.8! 105
200±20
4.0! 105
130±10
2.1! 106
140±10
2.1! 106
160±4
1.5! 106
140±4
1.7! 106
170±10
4.2! 105
170±10
1.2! 106
180±20
6.1! 105
210±20
120
[a] Note that the kinetic parameters from variant wtPTE to R18-1 were published in (Tokuriki et al., 2012).
[b] Cells were grown in at least duplicate and lysates sufficiently diluted to determine initial rates of
paraoxon and/or 2NH hydrolysis at a substrate concentration of 200 μM, normalized to cell density,
corrected for the dilution factor, and averaged. The experiment was repeated twice and the combined
average determined. [c] For better comparison, wtPTE was repuried and remeasured in parallel with the
reverse evolution variants (wtPTE-2). [d] For better comparison, PTE-R18 was repurified and remeasured
in parallel with the subsequent variants (R18-2).
2
Supplementary File 2B. Kinetic parameters of wtPTE, AE, and neoPTE variants (Figure 4-7) in
lysate and using purified enzyme.
3
[a] For comparison, the effect of each mutation is given in the same direction, e.g. A45t means the effect
of introducing Thr into each background (AE-A45t, neoPTE-A45t). When the amino acid in question (Thr in
this case) is already present in a certain background, the effect is calculated as its reintroduction after
removal (reversion of wtPTE-t45A to wtPTE).
[b] Cells were grown in at least duplicate and lysates sufficiently diluted (~1-10,000-fold) to determine
initial rates of paraoxon hydrolysis at a substrate concentration of 200 μM, normalized to cell density, and
corrected for the dilution factor. This experiment was repeated twice and the average change relative to
the respective parent variant (wtPTE, AE, and neoPTE) and the standard deviation were determined. A ttest was performed to obtain p-values. Only mutants with an average >1.3-fold difference from the
respective parent mutant AND a p-value <0.05 are considered significant. Non-significant values are
underlined. Note that the only >1.3 fold change with non-significant p-value is AE+T341i (1.4-fold, p-value
0.15).
[c] Variants were purified in at least duplicate in a 96-well format and sufficiently diluted to determine
initial rates of paraoxon hydrolysis at a substrate concentration of 200 μM, corrected for the dilution
factor, averaged and the standard deviation determined.
[d] Phosphotriesterase activity was too low to be determined.
[e] No standard deviation is given because only a single measurement is shown.
4
Supplementary File 2C. Kinetic parameters of additional variants made to determine mutational
effects over the forward evolution in lysate and using purified enzyme. The mutational effects
are given in Figure 5 and 6 and were calculated by comparing each variant to its parent
indicated in the name (e.g. R8+I138M to R8) and as shown in Supplementary File 2D. The effect
of all mutations not listed could be calculated directly by comparing variants from the forward
evolution as shown in Supplementary File 2D.
Variant
AE
R8+I138m
R8+I199t
R18+V49a
R18+E77k
R18+M140l
R18+F313i
R20+A45t
R20+T137s
R20+V144e
R20+H180q
R20+T314m
R20+T341i
Paraoxon
lysate [a]
purified[b]
relative to AE
[1/sec]
1
0.015±0.003
850±280
5.8±1.0
190±40
3.4±0.4
9.1±0.1
0.073±0.001
10±1
0.079±0.004
14±8
0.12±0.06
100±10
0.83±0.04
1.5±0.2
0.022±0.013
2.6±0.1
0.023±0.002
2.3±0.2
0.021±0.001
8.9±0.7
0.30±0.01
2.6±0.1
0.029±0.001
2.4±0.1
0.022±0.001
[a] Cells were grown in at least duplicate and lysates sufficiently diluted (~1-10,000-fold) to determine
initial rates of paraoxon hydrolysis at a substrate concentration of 200 μM, normalized to cell density, and
corrected for the dilution factor. This experiment was repeated twice and the average change relative to
the respective parent variant AE and the standard deviation were determined. P-values are shown in
Supplementary File 2D.
[b] Variants were purified in at least duplicate in a 96-well format and sufficiently diluted to determine
initial rates of paraoxon hydrolysis at a substrate concentration of 200 μM, corrected for the dilution
factor, averaged and the standard deviation determined.
5
Supplementary File 2D. Mutational effects over the forward evolution in lysate and using
purified enzyme.
Mutation
t45A
a49V
k77E
a80V
s102T
s111R
l130V
s137T
i138M
l140M
e144V
t172I
v176M
q180H
t199I
a204G
d233E
h254R
s269T
l271F
l272M
i274S
i313F
m314T
i341T
Paraoxon
lysate [a]
T-test[a]
relative to parent p-value
1.3±0.1
0.72
1.1±0.1
0.14
1.0±0.1
0.58
1.3±0.4
0.19
0.51±0.07
1.7×10-4
0.80±0.15
0.03
1.0±0.1
0.68
0.74±0.07
1.1×10-3
0.36±0.1
2.0×10-3
0.74±0.05
0.41
0.84±0.08
0.04
2.2±0.4
4.4×10-3
1.0±0.1
0.72
0.18±0.02
4.9×10-5
1.6±0.6
0.06
1.2±0.2
0.10
0.45±0.04
0.01
0.072±0.022
3.0×10-5
1.8±0.3
4.4×10-3
1.9±0.1
3.0×10-6
0.48±0.16
6.4×10-3
1.0±0.2
0.78
0.10±0.01
7.8×10-9
0.73±0.07
1.0×10-3
3.3×10-3
0.80±0.08
Calculation
R20 / R20+A45t
R18 / R18+V49a
R18 / R18+E77k
R10 / R9
R21 / R20
R12 / R11b
R14 / R13a
R20 / R20+T137s
R8 / R8+I138m
R18 / R18+M140l
R20 / R20+V144e
R6 / R5b
AE / R21
R20 / R20+H180q
R8 / R8+I199t
R12 / R11a
R2b / R1
R1 / wtPTE
R6 / R5a
R14 / R13b
R9 / R8
R3 / R2a
R18 / R18+F313i
R20 / R20+T314m
R20 / R20+T341i
[a] Cells were grown in at least duplicate and lysates sufficiently diluted (~1-10,000-fold) to determine
initial rates of paraoxon hydrolysis at a substrate concentration of 200 μM, normalized to cell density, and
corrected for the dilution factor. This experiment was repeated twice and the average change relative to
the respective parent variant (shown in the column “Calculation”) and the standard deviation were
determined. A t-test was performed to obtain p-values. Only mutants with an average >1.3-fold difference
from the respective parent mutant AND a p-value <0.05 are considered significant. Non-significant values
are underlined. Note that l140M and t199I have a >1.3 fold effect but non-significant p-values.
6
Supplementary File 2E. Kinetic parameters of additional variants made to determine mutational
effects over the reverse evolution in lysate and using purified enzyme. The mutational effects
are given in Figure 5 and 7 and were calculated by comparing each variant to its parent
indicated in the name (e.g. revR3+T172I to revR3) and as shown in Supplementary File 2F. The
effect of all mutations not listed could be calculated directly by comparing variants from the
reverse evolution as shown in Supplementary File 2F.
Paraoxon
Variant
lysate [a]
purified[b]
relative to AE
[1/sec]
AE
1.0
0.015±0.003
AE+s308C
6.6±0.9
0.059±0.001
revR3
900±260
7.6±1.7
revR3+t172L
220±20
3.0±0.1
revR3+M130V
170±40
1.1±0.1
revR3+p135S
400±170
9.9±5.0
3
revR3+T314m
(1.3±0.5)! 10
19±5
3
revR4
(1.4±0.5)! 10
23±5
revR6+K293m
(3.9±2.0)! 10 3
32±13
revR9
(1.8±1.1)! 10 4 100±10
revR9+D174g
(7.0±4.0)! 10 3
70±14
3
revR9+q180H
(6.3±2.0)! 10
55±5
revR9+N258s
(9.2±4.9)! 10 3
69±13
revR9+H156y
(1.4±0.4)! 10 4
70±21
4
neoPTE+a49V (1.5±0.6)! 10
140±20
3
neoPTE+M306I (1.8±0.1)! 10
92±29
4
neoPTE
(2.0±0.8)! 10
110±40
[a] Cells were grown in at least duplicate and lysates sufficiently diluted (~1-10,000-fold) to determine
initial rates of paraoxon hydrolysis at a substrate concentration of 200 μM, normalized to cell density, and
corrected for the dilution factor. This experiment was repeated twice and the average change relative to
the respective parent variant AE and the standard deviation were determined. P-values are shown in
Supplementary File 2F.
[b] Variants were purified in at least duplicate in a 96-well format and sufficiently diluted to determine
initial rates of paraoxon hydrolysis at a substrate concentration of 200 μM, corrected for the dilution
factor, averaged and the standard deviation determined.
7
Supplementary File 2F. Mutational effects over the forward evolution in lysate and using
purified enzyme.
Mutation
V49a
V130M
p135S
y156H
I172t
g174D
H180q
a203E
s258N
F271l
m293K
I306M
s308C
T314m
Paraoxon
lysate [a]
T-test[a]
relative to parent p-value
1.7±0.3
0.12
5.4±1.6
4.9! 10-5
1.1±0.4
0.83
1.3±0.8
0.42
4.1±1.2
3.5! 10-3
2.6±1.6
0.04
2.9±1.8
0.03
2.9±1.2
5.4! 10-3
2.0±1.2
0.09
2.9±0.8
4.9! 10-4
0.9±0.4
0.8
11±5
8.2! 10-3
6.6±0.9
7.4! 10-8
2.4! 10-4
3.4±1.2
Calculation
neoPTE+a49V / neoPTE
revR3 / revR3+M130V
revR3 / revR3+p135S
revR9 / revR9+H156y
revR3 / revR3+t172L
revR9 / revR9+D174g
revR9 / revR9+q180H
revR6+K293m / revR4
revR9 / revR9+N258s
revR3 / revR2
revR6 / revR6+K293m
neoPTE / neoPTE+M306I
AE+s308C / AE
revR4 / revR3+p135S
[a] Cells were grown in at least duplicate and lysates sufficiently diluted (~1-10,000-fold) to determine
initial rates of paraoxon hydrolysis at a substrate concentration of 200 μM, normalized to cell density, and
corrected for the dilution factor. This experiment was repeated twice and the average change relative to
the respective parent variant (shown in the column “Calculation”) and the standard deviation were
determined. A t-test was performed to obtain p-values. Only mutants with an average >1.3-fold difference
from the respective parent mutant AND a p-value <0.05 are considered significant. Non-significant values
are underlined. Note that V49a and s258N have a >1.3 fold effect but non-significant p-values.
8
Supplementary File 2G. Kinetic parameters of variants made for combinatorial mutational
analysis (Figure 7) in lysate and using purified enzyme.
[a] Cells were grown in at least duplicate and lysates sufficiently diluted (~1-10,000-fold) to determine
initial rates of paraoxon hydrolysis at a substrate concentration of 200 μM, normalized to cell density, and
corrected for the dilution factor. This experiment was repeated twice and the average change relative to
the parent variant AE and the standard deviation were determined. A t-test was performed to obtain pvalues. Only mutants with an average >1.3-fold difference from the respective parent mutant AND a pvalue <0.05 are considered significant. Non-significant values are underlined.
[b] Fold changes between the single and double mutants of each series shown in Figure 7B (e.g.,
AE+p135S differs by 4.3-fold from the double mutant AE+p135S+I138m, AE+I138m differs by 2.6-fold). A ttest was performed to obtain p-values. Note that the two mutants AE+F271l+s308C and AE+M272l+s308C
have non-significant p-values compared to the “double mutant” in this series, AE+F271l+M272l+s308C.
[c] Values for AE+F271l are compared to the double mutant AE+F271l+F313l. Values for AE+s308C are
compared to the double mutant AE+I306f+s308C.
[d] AE+F271l+s308C and AE+M272l+s308C are “single mutants” of the parent AE+s308C and values are
compared to the “double mutant” AE+F271l+M272l+s308C.
[e] Variants were purified to determine initial rates of paraoxon hydrolysis over a range of substrate
concentrations and calculate Michaelis Menten parameters.
9
Supplementary File 2H. Absorption wavelength λ, extinction coefficient ελ, and leaving group
pKa values for substrates used for kinetic analysis. Values were taken from (Khersonsky and Tawfik,
2005).
! [nm]
"! [M -1 ][a]
pKa
Phosphotriesters
Paraoxon
2,4-dinitrophenyl diethyl phosphate
2-fluoro 4-nitrophenyl diethyl phosphate
3-fluoro 4-nitrophenyl diethyl phosphate
2,6-difluorophenyl diethyl phosphate
4-cyanophenyl diethyl phosphate
diethyl phosphate acetophenone
3-fluorophenyl diethyl phosphate
405
360
395
390
270
275
320
270
10510
9160
11080
11676
950
7495
8080
656
7.14
4.08
5.45
5.94
7.3
7.95
8.05
9.28
Arylesters
2-naphthyl hexanoate (2NH)
4-nitrophenyl acetate
4-acetoxy benzaldehyde
4-cyanophenyl acetate
4-acetoxy acetophenone
3-nitrophenyl acetate
3-cyanophenyl acetate
4-chlorophenyl acetate
Phenyl acetate
3,4-dimethyl phenyl acetate
320
405
330
275
320
340
295
280
270
276
800
10510
12620
7495
8080
840
1610
838
700
850
[b]
Substrate
7.14
7.66
7.95
8.05
8.39
8.61
9.38
10
10.36
[a] Extinction coefficients are given for a pathlength of 0.58 cm, which results from a 200 μL reaction
volume in a 96-well plate.
[b] 2NH was not used for linear free energy relationship analysis.
References
Khersonsky O, Tawfik DS. 2005. Structure-reactivity studies of serum paraoxonase
PON1 suggest that its native activity is lactonase. Biochemistry 44:6371-6382.
Tokuriki N, Jackson CJ, Afriat-Jurnou L, Wyganowski KT, Tang R, Tawfik DS. 2012.
Diminishing returns and tradeoffs constrain the laboratory optimization of an enzyme.
Nature Communications 3:1257.
10