BglB Variant Characterization Data

Hover over any column for a desciption of that parameter.
Click on any column header to sort the data by that value.

► Expand all averages to see data from individual experiments. ◄
☑ Rosetta/Foldit sequence numbering.
☐ Include noncurated data.


556 records found


View complete BglB sequence.
.pbb structural image
Color Key
relative scale −5      −4      −3      −2      −1      0        1
For kinetic constants,
the table is color-coded by relative log values of
1/KM, kcat, and kcat/KM compared to WT.
log 1/KM is used so that larger values are "better".

For T50 and TM values and Rosetta scores, a linear scale is used.

Blue shading indicates a variant that is "worse" than WT.
Orange shading indicates a variant that is "better" than WT.

Variants shaded black expressed (as confirmed by gel electrophoresis and/or yield > 0.1 mg/mL).
Variants marked with an asterisk (*) expressed, but no yield was recorded.

How were these data calculated?

Variant ▲ Yield, cE
(mg/mL)
KM (mm) kcat
(min−1)
kcat/KM
(mm−1min−1)
T50 (°C) Tm (°C) Rosetta
score
change
WT 2.24 ± 1.77 5.79 ± 1.33 529.1 ± 36.3 128.77 ± 40.13 39.4 ± 2.1 45.4 ± 0.9
W11F 0.71 5.33 ± 0.40 836.9 ± 16.4 156.97 ± 12.12
G12N
T13K * 36.0
T13S 0.71 ± 0.51 11.02 ± 1.10 809.2 ± 36.0 153.00 ± 19.79 38.2 ± 0.5 46.3 ± 0.6 2.8
S14A * 8.25 ± 1.02 261.9 ± 8.7 31.82 ± 4.11 40.2 ± 0.3 44.1 ± 0.0
T15A * 5.30 ± 0.48 504.4 ± 13.0 95.25 ± 8.88 38.2 ± 0.4 42.0 ± 0.0 9.3
S16A * 14.01 ± 0.40 125.6 ± 0.0 8.88 ± 0.22 39.4 ± 0.3 43.9 ± 0.0
S16N
S17A * 18.45 ± 3.72 694.9 ± 62.8 37.67 ± 8.23 41.7 ± 0.1
S17E * 7.32 ± 0.38 526.1 ± 6.5 71.87 ± 3.90 39.5 ± 0.1
S17N 0.56 1.61 ± 0.42 35.6 ± 2.1 22.13 ± 5.94 32.0 ± 0.2
Y18A * 31.55 ± 3.61 162.4 ± 6.5 5.20 ± 0.65 38.3 ± 0.2 40.6 ± 0.0
Q19A 1.25 1.68 ± 1.14 0.1 ± 0.0 0.08 ± 0.05 35.2 ± 0.7 -1.3
Q19C *
Q19M 0.43 0.00 ± 0.03 2.9 ± 0.4 2,964.50 ± 95,063.40 30.0 ± 18.8 46.4 ± 1.5 -18.7
Q19N 3.9
Q19P
Q19S * 36.8 ± 0.2 43.1 ± 0.0
Q19T 2.19 1.74 ± 0.83 0.1 ± 0.0 0.06 ± 0.03 -0.4
I20N 0.31 19.41 ± 4.87 44.5 ± 4.0 2.29 ± 0.61
E21C 5.4
E21D 0.41 4.55 ± 0.53 32.0 ± 1.0 7.04 ± 0.86 37.5 ± 0.3 37.5 ± 0.3 4.4
E21F 32.3
E21G 8.0
Variant Yield, cE
(mg/mL)
KM (mm) kcat
(min−1)
kcat/KM
(mm−1min−1)
T50 (°C) Tm (°C) Rosetta
score
change
E21H 22.3
E21I 32.6
E21K 27.7
E21N 6.4
E21R 16.9
E21T 6.5
E21W 383.6
E21Y 4.45 1.52 ± 0.20 184.4 ± 5.4 120.95 ± 16.52 40.3 ± 0.3 40.3 ± 0.3 27.1
D25Q 2.73 ± 2.96 3.09 ± 0.29 292.9 ± 7.2 90.27 ± 8.49 35.1 ± 287,764,175.7
E26K 0.62 6.81 ± 0.48 638.8 ± 12.9 93.81 ± 6.93 38.4 ± 0.3 -0.9
S32L
W34A
D35E 2.00 2.72 ± 0.31 16.3 ± 0.4 5.97 ± 0.70 11.6
D35W 0.46 2.80 ± 0.55 13.0 ± 0.6 4.66 ± 0.94 14.7
F37N 0.26 4.88 ± 0.15 8.3 ± 0.1 1.70 ± 0.06
P41L *
G46C 0.04 40.81 ± 25.63 4.4 ± 1.2 0.11 ± 0.07
C49S 0.85 6.63 ± 3.19 570.0 ± 74.3 86.02 ± 42.89 40.0 ± 0.0
V52D 0.20 4.76 ± 1.88 259.6 ± 16.1 54.55 ± 21.78 -2.6
V52G * 8.25 ± 0.54 562.9 ± 10.8 68.19 ± 4.55 39.3 ± 0.1
F57Y 2.75 5.11 ± 0.60 37.3 ± 1.1 7.29 ± 0.88 45.2 ± 0.1 -0.1
H58R 1.22 5.64 ± 0.68 665.5 ± 21.2 118.04 ± 14.68
H59K 1.50 2.08 ± 0.82 6,326.0 ± 604.1 3,046.47 ± 1,233.53 41.7 ± 0.5 1.8
H59R 1.35 4.83 ± 0.35 576.2 ± 10.7 119.32 ± 8.87
K61A 1.55 3.79 ± 0.40 167.6 ± 4.4 44.28 ± 4.78 38.7 ± 0.4 1.3
Variant Yield, cE
(mg/mL)
KM (mm) kcat
(min−1)
kcat/KM
(mm−1min−1)
T50 (°C) Tm (°C) Rosetta
score
change
K61C 2.86 ± 0.01 1.75 ± 0.23 115.8 ± 3.3 67.22 ± 8.78 38.1 ± 0.3 2.4
K61D 1.11 3.96 ± 0.39 116.3 ± 2.8 29.36 ± 2.96 38.3 ± 0.3 5.6
K61E 1.36 3.79 ± 0.28 141.6 ± 2.7 37.39 ± 2.87 38.8 ± 0.2 0.8
K61F 2.58 ± 0.71 3.29 ± 0.25 70.9 ± 1.6 23.62 ± 2.25 37.4 ± 0.5 7.6
K61G 1.36 ± 0.45 3.02 ± 0.21 102.4 ± 2.0 26.48 ± 2.04 38.0 ± 0.5 4.6
K61H 6.16 1.95 ± 0.19 164.0 ± 3.6 84.15 ± 8.28 38.2 ± 0.1 3.3
K61I 1.80 2.43 ± 0.19 104.7 ± 2.0 43.13 ± 3.46 38.4 ± 0.2 1.1
K61L 0.83 2.37 ± 0.30 60.7 ± 1.9 25.68 ± 3.33 40.6 ± 0.8 5.5
K61M 4.60 4.59 ± 0.53 200.1 ± 6.0 43.57 ± 5.17 38.0 ± 0.1 2.6
K61N 3.50 ± 0.22 4.16 ± 0.41 156.9 ± 4.4 37.68 ± 4.19 35.6 ± 7.2 3.1
K61P 1.49 2.90 ± 0.26 81.6 ± 1.8 28.19 ± 2.62 38.9 ± 0.3 11.7
K61Q 1.42 4.28 ± 1.38 137.0 ± 12.5 31.98 ± 10.68 39.3 ± 0.2 0.8
K61R 2.60 2.82 ± 0.25 109.8 ± 2.3 38.93 ± 3.50 39.2 ± 0.3 1.4
K61S 1.83 ± 1.38 2.75 ± 0.60 145.0 ± 5.3 79.58 ± 6.91 38.5 ± 0.2 46.2 ± 0.3 1.0
K61W 1.94 3.95 ± 0.43 74.2 ± 2.2 18.78 ± 2.13 38.2 ± 0.7 7.8
K61Y 0.47 1.86 ± 0.17 93.3 ± 1.9 50.18 ± 4.73 38.0 ± 0.5 7.7
D63A 0.01 0.33 ± 0.09 0.0
D63C 0.98 0.69 ± 1.31 0.0 ± 0.0 0.07 ± 0.14
D63F 0.77 4.13 ± 9.33 0.1 ± 0.0 0.01 ± 0.03 30.0 ± 96.9
D63M 0.36 ± 0.26 0.13 ± 0.81 0.1 ± 0.1 1.96 ± 14.10
D63N 0.44 0.28 ± 0.43 0.2 ± 0.0 0.56 ± 0.87
D63P 0.28
D63R 0.13 0.02 ± 0.00
D63S 0.35 0.09 ± 0.16 0.1 ± 0.0 1.39 ± 2.61
K68H 2.79 3.83 ± 0.38 397.6 ± 10.1 103.74 ± 10.74 46.8 ± 1.1 2.4
Variant Yield, cE
(mg/mL)
KM (mm) kcat
(min−1)
kcat/KM
(mm−1min−1)
T50 (°C) Tm (°C) Rosetta
score
change
F72A * 5.47 ± 0.28 502.2 ± 6.5 91.79 ± 4.76 38.6 ± 0.4
Y75F 0.11 0.44 ± 0.49 1.5 ± 0.3 3.51 ± 3.96 40.1 ± 1.7 2.0
Y75N 0.15 ± 0.06 4.98 ± 0.47 364.4 ± 11.9 102.68 ± 19.80 38.0 ± 1.2 10.8
R76A
R76L 0.54 43.36 ± 1.82 7.4 ± 0.1 0.17 ± 0.01
R76Q 0.70 6.62 ± 2.33 0.8 ± 0.1 0.13 ± 0.05 -0.1
F77W 0.38 73.32 ± 94.87 20.0 ± 14.3 0.27 ± 0.40
S78A 3.12 20.08 ± 1.37 190.0 ± 4.9 9.46 ± 0.69 37.5 ± 0.1 1.9
V79I 3.56 ± 0.08 3.21 ± 0.31 508.4 ± 11.5 158.50 ± 16.22 39.2 ± 0.2
A80P 0.30 0.17 ± 0.11 0.5 ± 0.1 2.94 ± 1.92 30.6 ± 10.1 190.9
A80S 1.57 ± 0.69 7.92 ± 0.74 1,146.2 ± 36.6 141.60 ± 12.37 40.5 ± 0.5 41.1 ± 0.4 2.7
A88K 0.79 4.46 ± 0.60 307.4 ± 10.5 68.93 ± 9.53
I90V 1.25 6.67 ± 0.28 790.7 ± 9.2 118.48 ± 5.19
I91A 0.42 ± 0.31 7.44 ± 2.58 603.9 ± 21.6 109.73 ± 11.98 38.5 ± 304,961.7 -2.8
I91C 5.45 3.58 ± 0.15 19.6 ± 0.2 5.47 ± 0.24 38.1 ± 0.2
I91D 0.72 11.21 ± 0.69 89.6 ± 1.8 7.99 ± 0.51 37.4 ± 183,653.0
I91E * 6.71 ± 0.79 692.7 ± 28.1 103.26 ± 12.77 39.7 ± 0.3
I91G 0.45 9.30 ± 3.31 93.7 ± 10.2 10.08 ± 3.75
I91H 3.69 11.59 ± 0.81 181.2 ± 4.1 15.64 ± 1.15 37.6 ± 221,464.0
I91K 0.31 3.73 ± 0.92 4,015.9 ± 262.1 1,075.53 ± 275.42 39.7 ± 0.3
I91M 0.87 9.04 ± 0.55 148.5 ± 2.8 16.43 ± 1.05 37.5 ± 0.3
I91N 1.50 9.39 ± 0.98 183.9 ± 5.9 19.59 ± 2.14 36.0 ± 0.2
I91Q 3.47 13.34 ± 0.58 209.0 ± 3.0 15.66 ± 0.72 37.8 ± 2,701,870.0
I91R 0.90 8.45 ± 2.50 207.6 ± 18.4 24.56 ± 7.59 36.2 ± 0.2
I91S 1.40 10.13 ± 3.43 299.2 ± 29.0 29.54 ± 10.41 36.8 ± 0.2
Variant Yield, cE
(mg/mL)
KM (mm) kcat
(min−1)
kcat/KM
(mm−1min−1)
T50 (°C) Tm (°C) Rosetta
score
change
I91V 1.88 10.44 ± 1.37 22.5 ± 0.9 2.16 ± 0.30 37.9 ± 88,473.6
I91Y 2.71 10.90 ± 0.60 248.8 ± 4.3 22.81 ± 1.31 35.8 ± 0.2
H101R * 10.62 ± 0.53 868.1 ± 13.0 81.83 ± 4.33 40.1 ± 0.1 48.2 ± 0.0
I106A 1.03 5.15 ± 0.34 732.2 ± 13.0 142.12 ± 9.74
L108E 2.77 ± 0.93 6.39 ± 0.81 387.5 ± 11.1 68.79 ± 5.53 43.3 ± 0.0 -0.5
L108M 2.35 4.14 ± 0.35 391.2 ± 8.6 94.54 ± 8.36 47.3 ± 0.1 0.6
L108N 2.91 4.27 ± 0.67 330.4 ± 13.4 77.38 ± 12.49 44.8 ± 0.0 0.0
L108S 0.26 2.76 ± 0.50 95.4 ± 4.1 34.55 ± 6.39 38.1 ± 0.5 -1.1
M114S 1.18 13.79 ± 1.07 257.2 ± 6.8 18.65 ± 1.53 5.0
T116I 3.42 29.39 ± 2.92 261.6 ± 10.7 8.90 ± 0.95 42.6 ± 0.5 -7.3
T116S 1.61 2.21 ± 0.44 138.1 ± 6.3 62.58 ± 12.74 43.1 ± 0.1 4.0
Y118F 2.03 ± 1.69 0.87 ± 0.86 108.4 ± 20.7 124.90 ± 126.20 44.5 ± 0.6 3.2
H119A * 15.10 ± 3.36 116.9 ± 8.7 7.79 ± 1.73 40.2 ± 0.2
H119E
H119M 0.55 17.02 ± 4.07 9.8 ± 0.8 0.57 ± 0.15
H119N * 23.22 ± 2.20 2.2 ± 0.0 40.3 ± 0.6
H119Q 0.80 67.26 ± 12.70 153.1 ± 16.0 2.28 ± 0.49 45.7 ± 0.6
H119T 0.84 66.09 ± 31.10 20.7 ± 5.2 0.31 ± 0.17
H119V 0.43 44.36 ± 5.88 5.2 ± 0.3 0.12 ± 0.02
W120A
W120D 0.18 12.34 ± 0.44 47.0 ± 0.5 3.81 ± 0.14
W120E 0.11 2.02 ± 0.76 8.9 ± 0.6 4.43 ± 1.69 40.9 ± 0.1 6.5
W120F * 16.08 ± 2.07 387.5 ± 17.3 24.03 ± 3.25 41.7 ± 0.4 50.7 ± 0.0
W120G 0.38 0.12 ± 0.00
W120H * 89.18 ± 4.31 69.3 ± 2.2 0.87 ± 0.00 40.1 ± 0.1 46.2 ± 0.0
Variant Yield, cE
(mg/mL)
KM (mm) kcat
(min−1)
kcat/KM
(mm−1min−1)
T50 (°C) Tm (°C) Rosetta
score
change
W120K 0.25 56.50 ± 10.55 1.7 ± 0.2 0.03 ± 0.01
W120L 1.43 0.61 ± 0.02 37.6 ± 633,161.0
W120M 1.46 50.86 ± 3.20 100.2 ± 3.2 1.97 ± 0.14 43.9 ± 0.1 5.2
W120N 0.59 ± 0.30 11.72 ± 8.19 3.0 ± 0.4 2.56 ± 1.49 48.2 ± 0.1 4.6
W120P 1.58 8.47 ± 0.24 1,210.9 ± 9.9 143.04 ± 4.25
W120Q 0.63 0.29 ± 0.02
W120T 0.46 0.37 ± 0.25 0.4 ± 0.0 1.01 ± 0.69 47.0 ± 2.1 -0.2
W120Y 3.68 ± 1.31 20.53 ± 2.92 4.6 ± 0.3 0.26 ± 0.05 34.8 ± 0.2 50.2 ± 1.2 11.1
D121F
Q124D 0.75 4.60 ± 0.46 193.8 ± 4.9 42.15 ± 4.39 36.2 ± 0.4 10.1
Q124G 0.25 6.17 ± 0.27 71.3 ± 0.9 11.55 ± 0.52 36.6 ± 0.2
Q124I 1.74 3.43 ± 0.22 152.5 ± 2.3 44.49 ± 2.96 40.4 ± 0.7 5.4
Q124P 0.75 4.90 ± 0.41 313.4 ± 6.9 64.02 ± 5.48 38.6 ± 0.4 336.0
Q124S 0.61 5.66 ± 0.77 141.2 ± 5.3 24.94 ± 3.52
Q124T 1.30 5.65 ± 0.46 181.6 ± 4.0 32.13 ± 2.69 39.7 ± 0.8 7.5
W125R 0.18 3.71 ± 0.99 62.4 ± 4.1 16.81 ± 4.60 41.3 ± 1.5 10.1
E127D 0.22 2.42 ± 0.32 136.6 ± 4.3 56.50 ± 7.77 39.2 ± 0.3
E129Q 0.90 5.17 ± 0.46 348.9 ± 8.3 67.49 ± 6.21 43.4 ± 0.0 -1.1
Q139K 0.43 ± 0.06 6.07 ± 0.79 60.1 ± 4.0 10.75 ± 3.00 45.2 ± 0.1 -2.7
K142R 0.54 4.57 ± 1.26 60.2 ± 4.3 13.17 ± 3.74
Y144W 1.70 2.48 ± 0.47 280.5 ± 12.4 113.00 ± 22.09 43.8 ± 0.4 -8.8
A145C 2.42 4.02 ± 0.28 368.6 ± 6.5 91.74 ± 6.52 44.3 ± 0.1 3.0
A145E 2.62 4.95 ± 0.38 295.1 ± 6.0 59.61 ± 4.74 40.8 ± 0.1
D150V 0.38 3.02 ± 0.71 1.0 ± 0.1 0.32 ± 0.08 39.6 ± 0.3 19.0
E154A 0.27 30.92 ± 13.46 112.2 ± 20.0 3.63 ± 1.71 30.0 ± 39.3 1.6
Variant Yield, cE
(mg/mL)
KM (mm) kcat
(min−1)
kcat/KM
(mm−1min−1)
T50 (°C) Tm (°C) Rosetta
score
change
E154D * 3.46 ± 0.76 720.9 ± 39.0 208.26 ± 47.19 38.8 ± 0.3 49.5 ± 0.0
E154F 0.72 6.49 ± 0.56 2,334.7 ± 61.1 359.48 ± 32.42
N157D 0.72 6.32 ± 0.56 330.1 ± 8.2 52.25 ± 4.80 47.5 ± 0.0 -1.0
N157H 2.06 2.17 ± 0.46 344.9 ± 16.7 159.00 ± 34.33 44.1 ± 0.0 -0.4
N157T 0.26 2.55 ± 0.59 19.7 ± 1.1 7.73 ± 1.83
N157Y 1.15 7.39 ± 0.93 1,508.5 ± 51.8 204.18 ± 26.62 40.8 ± 0.2 -1.6
W158N 2.79 4.95 ± 0.18 850.8 ± 7.9 171.78 ± 6.35
N160A 3.59 14.26 ± 1.61 402.4 ± 15.5 28.22 ± 3.37 44.1 ± 0.3 2.4
N160C 4.07 4.95 ± 0.66 499.7 ± 17.8 101.02 ± 14.02 42.9 ± 0.1
N160H 0.99 14.81 ± 0.91 144.3 ± 3.0 9.74 ± 0.63 41.2 ± 0.0 20.6
N160S 3.61 6.71 ± 0.56 393.5 ± 9.3 58.64 ± 5.07 42.5 ± 0.1
N160V 1.77 ± 0.61 6.09 ± 1.22 560.4 ± 32.8 96.39 ± 27.65 38.9 ± 118,735.5 4.2
N163A * 11.95 ± 0.91 6.5 ± 0.0 0.65 ± 0.00 41.2 ± 0.3 44.4 ± 0.0
N163C * 5.42 ± 0.32 21.6 ± 0.0 3.90 ± 0.22 38.0 ± 0.3 40.5 ± 0.0
N163D * 15.19 ± 1.41 10.8 ± 0.0 0.65 ± 0.00 38.5 ± 0.3
N163E
N163G 2.38 9.68 ± 0.47 2.4 ± 0.0 0.25 ± 0.01 41.7 ± 0.0 6.0
N163H * 43.8 ± 0.1 21.9
N163K
N163Q 0.10 7.01 ± 4.09 1.1 ± 0.2 0.16 ± 0.10
N163S 0.30 2.67 ± 0.41 1,758.0 ± 62.2 658.99 ± 104.13 38.1 ± 0.5
N163T 0.09 ± 0.06 23.01 ± 2.67 4.8 ± 0.2 0.21 ± 0.03 4.5
E164A * 1.01 ± 0.17 46.0 ± 0.1 53.9 ± 0.0
E164G
E164R
Variant Yield, cE
(mg/mL)
KM (mm) kcat
(min−1)
kcat/KM
(mm−1min−1)
T50 (°C) Tm (°C) Rosetta
score
change
P165A 0.20 3.73 ± 0.33 554.7 ± 12.5 148.77 ± 13.64 39.5 ± 0.2
Y166F 0.32 ± 0.05 5.06 ± 1.13 7.3 ± 0.5 1.45 ± 0.39
Y166H 1.31 ± 0.03 18.37 ± 1.30 564.1 ± 14.2 31.86 ± 2.31 44.3 ± 0.1 3.4
Y166K 0.34 2.24 ± 0.55 1.8 ± 0.1 0.80 ± 0.20 48.4 ± 0.5 11.4
Y166P
Y166S 1.64 6.67 ± 1.04 43.3 ± 1.9 6.49 ± 1.05 8.2
Y166W 1.90 7.58 ± 0.52 9.7 ± 0.2 1.28 ± 0.09
C167A * 14.56 ± 1.27 391.8 ± 10.8 26.84 ± 2.38 39.8 ± 0.5 51.1 ± 0.0
C167D 1.60 57.52 ± 10.92 87.6 ± 8.8 1.52 ± 0.33 46.4 ± 0.1 7.6
C167E 0.69 5.14 ± 0.20 183.5 ± 1.9 35.68 ± 1.44
C167H 0.36 3.73 ± 0.63 168.2 ± 6.2 45.12 ± 7.79
C167N 2.33 37.36 ± 2.90 487.4 ± 16.6 13.05 ± 1.11
C167Q * 4.92 ± 0.19 413.5 ± 4.3 84.00 ± 3.46 38.9 ± 0.1 48.2 ± 0.0
C167S 3.71 ± 1.43 71.46 ± 9.54 17.0 ± 1.3 0.18 ± 0.03 48.0 ± 0.2 3.0
A168N 0.42 7.31 ± 0.40 157.0 ± 2.4 21.46 ± 1.23
S169A 1.35 4.33 ± 0.45 515.6 ± 14.8 119.07 ± 12.78 44.8 ± 0.5 -2.2
S169D 0.13 68.87 ± 91.89 1.6 ± 1.1 0.02 ± 0.03
S169N 0.28 10.12 ± 5.32 1.6 ± 0.2 0.16 ± 0.09
I170V 1.22 10.02 ± 1.24 33,748.0 ± 1,296.6 3,368.93 ± 435.92 50.0 ± 195,726,000.0 0.9
I170Y 0.86 4.82 ± 0.46 432.8 ± 11.0 89.75 ± 8.93 35.0 ± 0.2 6.7
L171A * 11.09 ± 0.42 662.4 ± 6.5 59.75 ± 2.38 38.8 ± 0.5 42.9 ± 0.0
L171F 5.30 ± 2.22 3.41 ± 0.46 229.3 ± 5.0 83.41 ± 8.37 50.0 ± 0.0 1.6
L171I 0.69 18.69 ± 3.62 427.8 ± 29.3 22.89 ± 4.70
L171Q 1.12 7.99 ± 0.66 77.1 ± 1.8 9.66 ± 0.83
L171R * 3.36 ± 0.23 331.2 ± 6.5 98.50 ± 6.93 38.8 ± 0.3 44.6 ± 0.0
Variant Yield, cE
(mg/mL)
KM (mm) kcat
(min−1)
kcat/KM
(mm−1min−1)
T50 (°C) Tm (°C) Rosetta
score
change
L171Y 0.91 1.10 ± 0.12 410.4 ± 9.4 373.98 ± 42.07 37.2 ± 0.1
Y173L 0.17 1.28 ± 0.18 193.7 ± 7.4 151.01 ± 22.19 45.1 ± 1.1 -3.6
T175A 0.38 10.60 ± 0.79 425.0 ± 9.7 40.10 ± 3.13
T175C 0.33 14.14 ± 1.21 2,190.6 ± 61.4 154.88 ± 13.95
T175H 0.07 ± 0.03 2.55 ± 0.67 199.6 ± 13.2 127.56 ± 178.06 43.8 ± 4.5 10.0
T175K 2.03 6.21 ± 0.54 708.5 ± 16.7 114.14 ± 10.27
T175R * 3.59 ± 0.15 655.9 ± 6.5 182.71 ± 7.79 38.1 ± 0.1
E177A 6.12 6.03 ± 0.44 566.2 ± 8.6 94.24 ± 5.24 37.5 ± 0.2 50.3 ± 0.4 3.1
E177C 1.52 6.13 ± 0.26 70.8 ± 0.8 11.55 ± 0.51
E177D 1.95 ± 0.02 5.04 ± 0.58 404.1 ± 12.5 80.17 ± 9.60 37.9 ± 78,411.8
E177I
E177K * 6.19 ± 0.30 454.6 ± 6.5 73.39 ± 3.68 36.7 ± 0.3
E177L * 7.48 ± 0.36 549.9 ± 8.7 73.60 ± 3.68 39.6 ± 0.4
E177Q 0.58 8.17 ± 0.46 799.4 ± 13.0 97.81 ± 5.78
E177T 2.73 8.71 ± 0.59 504.9 ± 9.9 57.94 ± 4.08
H178A * 7.67 ± 0.73 93.1 ± 2.2 12.12 ± 1.08 39.6 ± 0.4
H178C 17.97 ± 17.93 0.98 ± 0.24 36.8 ± 3.8 37.27 ± 17.39 50.0 ± 975,144,000.0 3.5
H178K 0.67 25.15 ± 1.69 22.5 ± 0.6 0.90 ± 0.06 8.9
H178N 1.16 ± 1.06 4.80 ± 0.68 340.4 ± 14.2 107.79 ± 42.16 44.7 ± 4.2 3.3
H178R 0.09 4.96 ± 0.48 48.9 ± 1.4 9.85 ± 1.00
H178S 0.09 14.86 ± 0.61 51.0 ± 0.7 3.43 ± 0.15
H178W 1.02 ± 0.30 7.00 ± 1.17 331.7 ± 18.5 89.44 ± 20.44 160.8
H182K 5.12 4.43 ± 0.35 377.2 ± 7.7 85.12 ± 6.87 50.0 ± 0.0 -0.8
H182Q 5.63 5.81 ± 0.38 412.3 ± 8.1 71.02 ± 4.90 49.5 ± 0.7 2.0
H182R 6.90 ± 6.22 8.28 ± 0.73 604.2 ± 15.8 73.34 ± 5.95
Variant Yield, cE
(mg/mL)
KM (mm) kcat
(min−1)
kcat/KM
(mm−1min−1)
T50 (°C) Tm (°C) Rosetta
score
change
H182S 1.18 4.26 ± 0.23 333.9 ± 8.3 78.41 ± 4.66 48.5 ± 0.7 2.7
H182T 4.02 3.72 ± 0.29 191.0 ± 9.4 51.36 ± 4.74 49.3 ± 1.2 8.1
H182V 6.6
H182Y 2.90 8.33 ± 2.15 528.0 ± 53.4 63.39 ± 17.61 47.2 ± 0.3 3.0
E183H 0.86 6.40 ± 0.55 355.9 ± 8.3 55.64 ± 4.93
E183T 4.10 4.50 ± 0.25 581.3 ± 8.6 129.17 ± 7.51
E187A 8.07 5.94 ± 0.37 384.4 ± 8.6 64.76 ± 4.25 51.2 ± 0.6 2.3
E187F 6.13 5.32 ± 0.22 40.9 ± 0.5 7.69 ± 0.34 50.8 ± 0.6 1.8
E187I 0.40 6.86 ± 0.46 930.0 ± 17.6 135.57 ± 9.38 61.5 ± 2.7 30.2
E187T 2.84 6.15 ± 1.07 404.9 ± 20.1 65.81 ± 11.85 49.2 ± 0.8 5.4
A192S * 5.09 ± 0.18 775.0 ± 8.7 152.19 ± 5.63 39.2 ± 0.3
H194G 0.20 1.59 ± 0.30 91.5 ± 4.4 57.56 ± 11.06 7.0
H194L 0.24 ± 0.05 2.77 ± 0.38 133.3 ± 5.3 58.22 ± 11.21 37.4 ± 0.2 28.9
H194Q 0.16 6.08 ± 0.86 78.8 ± 3.1 12.96 ± 1.90 37.2 ± 0.3
H194V 1.04 4.27 ± 0.53 194.4 ± 6.3 45.52 ± 5.88 39.9 ± 0.5 22.1
H194Y 0.75 ± 0.01 5.62 ± 0.80 186.8 ± 6.7 33.86 ± 4.11 38.9 ± 0.3
C198A 3.03 4.73 ± 0.47 82.9 ± 2.2 17.52 ± 1.79 39.3 ± 0.2 -0.9
C198D 6.8
C198F 287.9
C198G 0.93 1.59 ± 0.34 18.0 ± 0.8 11.33 ± 2.49 37.2 ± 0.4 4.1
C198K 1.76 4.96 ± 0.18 84.9 ± 0.8 17.13 ± 0.65 36.7 ± 1.3 55.6
C198L 0.68 33.2
C198M 1.24 4.93 ± 0.67 80.9 ± 2.9 16.42 ± 2.31 36.1 ± 0.4 42.9
C198N 12.0
C198Q 0.65 1.57 ± 0.32 2.8 ± 0.1 1.78 ± 0.38 40.9 ± 1.1 16.7
Variant Yield, cE
(mg/mL)
KM (mm) kcat
(min−1)
kcat/KM
(mm−1min−1)
T50 (°C) Tm (°C) Rosetta
score
change
C198S 1.98 2.75 ± 0.39 113.1 ± 3.8 41.19 ± 5.96 38.6 ± 0.3 2.0
C198T 5.49 2.27 ± 0.24 132.1 ± 3.2 58.08 ± 6.26 35.9 ± 0.3 3.2
H206R 19.65 3.12 ± 0.25 150.0 ± 3.8 48.07 ± 4.02
T212E 1.21 ± 0.07 4.93 ± 0.62 384.1 ± 13.2 77.91 ± 10.34 46.5 ± 0.2 -0.2
I217S 1.08 5.46 ± 0.66 1,280.4 ± 42.3 234.72 ± 29.55 42.4 ± 0.1 6.4
T218A * 6.51 ± 0.94 381.0 ± 15.2 58.45 ± 8.66 40.1 ± 0.1 47.5 ± 0.0
T218Q 1.04 0.06 ± 0.00
T218S 1.11 14.39 ± 2.25 378.3 ± 20.2 26.28 ± 4.34 41.0 ± 0.2
L219Q 1.53 7.15 ± 0.16 1,266.0 ± 7.8 177.11 ± 4.03
N220A 0.62 ± 0.04 16.89 ± 10.22 178.5 ± 10.4 15.51 ± 1.60 38.8 ± 0.2 43.6 ± 0.3 1.7
N220C 1.39 1.76 ± 0.46 380.3 ± 21.9 215.89 ± 57.83
N220D 1.16 1.48 ± 0.30 100.3 ± 4.5 67.69 ± 14.11 35.0 ± 0.2
N220F 1.94 ± 0.75 1.39 ± 0.22 452.0 ± 10.7 497.98 ± 46.74 37.9 ± 15,536.4 -2.0
N220G * 33.30 ± 2.36 171.0 ± 4.3 5.20 ± 0.43 41.9 ± 0.2 47.4 ± 0.0
N220H * 5.14 ± 0.21 101.7 ± 0.0 19.70 ± 0.87 39.6 ± 0.4 46.8 ± 0.0
N220K 3.56 2.21 ± 0.41 351.6 ± 15.0 159.19 ± 30.49
N220L 2.70 ± 1.82 13.83 ± 3.62 324.7 ± 24.6 39.66 ± 10.68 36.7 ± 1.2
N220M 0.53 1.62 ± 0.15 352.5 ± 7.1 217.55 ± 20.40 35.3 ± 0.3 1.0
N220Q 0.29 4.46 ± 1.16 123.6 ± 9.0 27.72 ± 7.52 45.7 ± 4.0 2.6
N220R * 12.50 ± 1.39 13.0 ± 0.0 1.08 ± 0.22 39.5 ± 0.3 46.9 ± 0.0
N220S 0.53 6.00 ± 0.19 39.7 ± 0.2
N220W 0.69 ± 0.11 0.84 ± 0.22 165.3 ± 11.3 169.64 ± 46.01
N220Y 3.23 3.15 ± 0.14 481.6 ± 4.2 681.98 ± 46.27 35.9 ± 17.1 49.2 ± 0.0
M221A * 6.25 ± 0.60 448.1 ± 13.0 71.66 ± 7.14 40.4 ± 0.3 47.2 ± 0.0
M221K 0.20 ± 0.03 9.48 ± 0.59 114.2 ± 2.2 12.30 ± 0.88 46.5 ± 0.1 5.5
Variant Yield, cE
(mg/mL)
KM (mm) kcat
(min−1)
kcat/KM
(mm−1min−1)
T50 (°C) Tm (°C) Rosetta
score
change
M221N 1.50 5.59 ± 0.33 246.3 ± 3.9 44.07 ± 2.67 36.6 ± 0.1
M221Q 2.84 8.03 ± 0.20 1,008.5 ± 7.3 125.56 ± 3.32
M221R 0.24 14.98 ± 0.66 25.1 ± 0.4 1.67 ± 0.08 3.2
E222A * 0.63 ± 0.15 73.6 ± 4.3 116.90 ± 28.58 36.9 ± 0.1 36.7 ± 0.0
E222H * 8.54 ± 0.53 132.1 ± 2.2 15.37 ± 1.08 34.9 ± 0.2 40.2 ± 0.0
E222K * 7.22 ± 0.75 88.8 ± 2.2 12.34 ± 1.30 39.0 ± 0.4
E222Q * 12.16 ± 0.65 547.7 ± 8.7 45.03 ± 2.60 39.8 ± 0.3
E222R * 2.48 ± 0.44 34.6 ± 2.2 14.07 ± 2.60 39.1 ± 0.2
E222Y * 18.43 ± 3.14 10.8 ± 0.0 0.65 ± 0.22 37.1 ± 0.5
H223A 1.03 ± 1.07 5.10 ± 1.29 427.9 ± 40.6 78.89 ± 19.79 42.8 ± 2.7 41.6 ± 0.3 -2.5
P231K 2.98 4.57 ± 0.42 349.8 ± 8.5 76.53 ± 7.33 44.8 ± 0.2 0.2
A236D 43.0
A236E
A236V 1.03 ± 0.52 0.45 ± 0.31 1.2 ± 0.2 2.51 ± 1.90 36.2 ± 1.3
I238K 1.76 9.56 ± 1.74 2,004.1 ± 112.1 209.71 ± 39.95 39.4 ± 0.2
R240A 2.57 ± 1.04 11.85 ± 2.51 419.3 ± 20.1 35.94 ± 5.10 38.5 ± 0.2 2.3
R240D * 10.82 ± 0.47 231.6 ± 4.3 21.43 ± 1.08 37.2 ± 0.0 45.7 ± 0.0
R240E
R240G 0.30 23.66 ± 2.61 456.7 ± 19.1 19.31 ± 2.28
R240K 2.20 12.92 ± 2.43 726.3 ± 37.5 64.63 ± 9.16 39.5 ± 0.2 44.0 ± 0.0 2.2
D241H 0.28 9.97 ± 0.78 100.2 ± 2.4 10.05 ± 0.82
D241R 0.13 2.42 ± 0.46 5.1 ± 0.2 2.10 ± 0.41 1.3
F243H 1.15 ± 0.43 21.06 ± 6.11 552.5 ± 64.9 36.16 ± 5.42 39.4 ± 0.3 1.4
I244A 0.20 8.50 ± 1.59 451.3 ± 16.6 53.12 ± 10.12 40.4 ± 0.2 5.3
I244E * 5.97 ± 1.04 407.0 ± 19.5 68.19 ± 12.34 37.8 ± 0.1 45.8 ± 0.0
Variant Yield, cE
(mg/mL)
KM (mm) kcat
(min−1)
kcat/KM
(mm−1min−1)
T50 (°C) Tm (°C) Rosetta
score
change
I244K 3.48 14.07 ± 0.87 134.4 ± 2.8 9.55 ± 0.63 4.3
I244M 2.97 2.72 ± 0.59 236.0 ± 15.5 170.89 ± 111.12 6.8
I244N * 2.15 ± 0.13 223.0 ± 4.3 103.69 ± 6.49 36.8 ± 0.6 45.0 ± 0.0
I244Q 2.18 14.73 ± 0.94 1,166.6 ± 24.6 79.20 ± 5.31
I244T 3.11 4.22 ± 0.17 681.8 ± 6.9 161.68 ± 6.69
I244V 1.54 2.04 ± 0.16 518.2 ± 10.0 254.43 ± 21.17
W247V 0.16 7.62 ± 2.81 4.2 ± 0.4 0.55 ± 0.21
F248N 0.30 8.99 ± 2.33 8.1 ± 0.6 0.91 ± 0.25 50.3 ± 0.7 6.2
A249E
F253H 0.28 64.81 ± 42.05 297.9 ± 110.9 4.60 ± 3.44 3.6
F253I 2.66 ± 1.40 5.68 ± 0.58 442.8 ± 12.3 78.01 ± 8.21 44.8 ± 0.1 5.9
F253M 2.17 ± 1.17 5.99 ± 0.39 448.5 ± 8.2 37.49 ± 3.61 44.7 ± 0.1 6.8
F253S 0.82 1.65 ± 0.23 11,626.5 ± 341.4 7,052.98 ± 1,010.15 39.5 ± 0.4
F253Y 0.23 6.14 ± 0.61 271.2 ± 7.5 44.21 ± 4.56 1.4
Y257F 1.59 1.86 ± 0.67 45.4 ± 3.5 24.42 ± 9.03 38.3 ± 0.9 58.2 ± 30.6 1.1
D260E 0.88 10.56 ± 0.84 1,349.4 ± 33.7 127.83 ± 10.64
M261D
W264H 0.27 10.37 ± 1.48 124.0 ± 6.0 11.96 ± 1.80 45.2 ± 0.0 0.0
W264Y 0.16 4.66 ± 2.54 24.4 ± 3.5 5.23 ± 2.96
Y265F 0.30 11.19 ± 3.84 5.5 ± 0.6 0.49 ± 0.18 34.5 ± 0.8 30.7 ± 18.7 -0.6
T267E 2.77 4.58 ± 0.47 422.5 ± 11.4 92.19 ± 9.79 47.6 ± 0.2 -1.3
L269I 0.21 3.77 ± 0.53 176.9 ± 6.2 46.90 ± 6.80 40.4 ± 0.6 0.4
G278V 3.54 5.16 ± 1.58 463.0 ± 36.2 89.76 ± 28.41
M280I 0.54 5.70 ± 0.43 451.7 ± 9.1 79.19 ± 6.22
D288E 2.29 5.38 ± 0.48 772.8 ± 18.0 143.61 ± 13.13
Variant Yield, cE
(mg/mL)
KM (mm) kcat
(min−1)
kcat/KM
(mm−1min−1)
T50 (°C) Tm (°C) Rosetta
score
change
N293A 0.79 11.73 ± 1.02 9.3 ± 0.2 0.83 ± 0.04 40.8 ± 0.2 46.5 ± 0.1 -0.4
N293C * 19.80 ± 2.08 8.7 ± 0.0 0.43 ± 0.00 38.9 ± 0.4 47.0 ± 0.0
N293D
N293E * 18.9
N293F 86.3
N293G 3.88 ± 2.24 24.01 ± 2.80 1.1 ± 0.0 0.04 ± 0.01
N293H * 42.1 ± 0.0 162.0
N293I 0.64 1.43 ± 0.28 1.3 ± 0.1 0.92 ± 0.19 39.6 ± 1.3 77.0
N293K
N293L 67.3
N293M 0.55 25.03 ± 2.77 50.0 ± 2.2 2.00 ± 0.24 41.2 ± 0.1 20.7
N293P
N293Q *
N293R 0.37 0.70 ± 0.15 7.9 ± 0.3 11.25 ± 2.44
N293S 4.54 ± 1.63 2.11 ± 0.46 2.8 ± 0.2 2.90 ± 1.39 41.3 ± 0.8 47.8 ± 0.5 0.6
N293T 2.11 13.62 ± 1.65 29.2 ± 1.2 2.15 ± 0.27 38.3 ± 0.4 38.3 ± 0.4 11.8
N293V 1.64 ± 0.40 6.80 ± 0.91 0.1 ± 0.0 0.01 ± 0.00 37.9 ± 67,349.9
N293W 119.6
N293Y 0.61 48.85 ± 36.83 0.6 ± 0.2 0.01 ± 0.01 49.0 ± 0.8 87.2
Y294A * 4.98 ± 0.17 136.4 ± 2.2 27.49 ± 1.08 40.4 ± 0.2 43.3 ± 0.0
Y294F * 5.99 ± 0.32 601.8 ± 8.7 100.45 ± 5.63 40.8 ± 0.1 44.0 ± 0.0
Y294L
Y295A *
Y295G *
Y295N 1.59 18.93 ± 3.85 140.3 ± 10.5 7.41 ± 1.61
Variant Yield, cE
(mg/mL)
KM (mm) kcat
(min−1)
kcat/KM
(mm−1min−1)
T50 (°C) Tm (°C) Rosetta
score
change
T296A 2.99 47.09 ± 4.63 0.6 ± 0.0 0.01 ± 0.00
T296C 0.60 ± 0.43 10.65 ± 1.48 248.3 ± 15.2 21.43 ± 4.20
T296E
T296H 0.46 8.23 ± 1.10 101.5 ± 3.9 12.33 ± 1.72
T296R 0.91 1.27 ± 0.15 780.7 ± 23.1 615.34 ± 75.45 42.4 ± 0.1 0.8
T296S 2.46 3.91 ± 0.83 297.1 ± 18.2 76.00 ± 16.89 43.8 ± 0.3 0.4
T296Y 4.35 5.95 ± 0.52 497.2 ± 12.0 83.60 ± 7.60 45.4 ± 0.0 -1.1
R297A 0.62 2.36 ± 0.49 52.2 ± 2.6 22.07 ± 4.74 41.8 ± 0.1 5.9
S298E * 5.28 ± 0.05 662.4 ± 2.2 125.56 ± 1.30 39.6 ± 0.2
S298R 0.76 2.56 ± 1.14 435.2 ± 45.8 170.03 ± 77.66 39.4 ± 0.2
S298T 0.85 3.34 ± 0.37 766.1 ± 21.2 229.47 ± 26.45 39.4 ± 0.3
I299R 4.56 3.48 ± 0.55 263.8 ± 10.5 75.84 ± 12.43 3.2
I300N * 4.48 ± 0.32 569.3 ± 10.8 127.07 ± 9.31 38.6 ± 0.5
S302A 7.15 ± 5.51 5.53 ± 0.32 2,213.5 ± 43.4 400.47 ± 30.68
S302H 3.21 ± 2.02 4.81 ± 0.37 568.3 ± 14.0 133.50 ± 16.70
S302N 3.27 5.43 ± 0.16 718.7 ± 5.4 132.24 ± 3.92
A306K 1.55 4.71 ± 0.54 698.6 ± 21.3 148.21 ± 17.68 40.7 ± 0.2 -1.5
A306Q 2.08 3.75 ± 0.63 587.8 ± 25.0 156.87 ± 27.23 38.3 ± 0.8
A306S 1.26 ± 0.63 9.48 ± 1.00 460.1 ± 13.8 51.42 ± 5.25 40.4 ± 0.1 43.7 ± 0.1 -1.5
V311D 3.05 6.84 ± 0.65 295.0 ± 8.0 43.14 ± 4.25 41.8 ± 0.1 2.6
Q313A 1.25 4.45 ± 0.57 603.3 ± 19.7 135.49 ± 17.81 39.6 ± 0.1
Q313C 3.14 ± 1.54 5.54 ± 0.46 285.0 ± 5.3 49.25 ± 3.80 0.9
Q313E 4.20 6.76 ± 0.86 413.8 ± 14.9 61.19 ± 8.05 39.7 ± 0.1
Q313L 3.90 3.58 ± 0.23 363.5 ± 5.8 101.67 ± 6.76 40.1 ± 0.1
Q313N 0.37 6.14 ± 0.89 291.1 ± 11.4 47.42 ± 7.10
Variant Yield, cE
(mg/mL)
KM (mm) kcat
(min−1)
kcat/KM
(mm−1min−1)
T50 (°C) Tm (°C) Rosetta
score
change
Q313P 2.63 3.48 ± 0.55 126.7 ± 4.7 36.42 ± 5.92 4.3
Q313R * 3.58 ± 0.51 565.0 ± 19.5 157.82 ± 23.16 42.1 ± 0.3
Q313V 2.53 8.77 ± 0.68 900.2 ± 20.5 102.63 ± 8.35
H315D 0.32 0.68 ± 0.24 897.0 ± 54.3 1,312.84 ± 460.56 40.1 ± 0.1
H315E *
H315I 3.82 6.73 ± 0.32 588.1 ± 7.6 87.37 ± 4.25
H315K 0.38 6.14 ± 0.56 36.6 ± 0.9 5.96 ± 0.57
H315N 0.79 4.27 ± 2.31 63.5 ± 9.4 14.89 ± 8.35 50.0 ± 0.0 -1.5
H315P 0.84 5.39 ± 0.27 647.6 ± 9.8 120.14 ± 6.24 38.8 ± 0.2 -1.8
H315T 4.76 4.67 ± 0.11 658.5 ± 4.0 141.04 ± 3.48
M316K 0.35 3.77 ± 0.90 439.2 ± 25.9 116.43 ± 28.63
E318R 3.49 8.52 ± 0.97 5.3 ± 0.2 0.62 ± 0.07
T321S 1.84 9.44 ± 2.87 422.2 ± 42.1 44.72 ± 14.29 43.3 ± 0.4 2.4
M323A * 9.34 ± 0.80 342.0 ± 8.7 36.59 ± 3.25 37.1 ± 0.1 42.5 ± 0.0
M323C 0.32 16.52 ± 1.78 105.5 ± 4.0 6.39 ± 0.73 39.9 ± 0.3 4.5
M323D 1.11 4.44 ± 0.89 5.4 ± 0.3 1.21 ± 0.25 40.3 ± 0.1 5.4
M323I 5.27 8.15 ± 0.35 344.0 ± 4.4 42.22 ± 1.90 44.9 ± 0.1 6.4
M323K
W325A * 1.61 ± 0.23 23.8 ± 0.0 14.72 ± 2.16 40.1 ± 0.4 41.8 ± 0.0
W325C * 4.18 ± 0.53 8.7 ± 0.0 2.16 ± 0.22 39.3 ± 0.2 41.1 ± 0.0
W325D 2.22 27.09 ± 3.65 1,039.5 ± 55.4 38.38 ± 5.57
W325E 3.58 2.26 ± 0.28 1.6 ± 0.1 0.71 ± 0.09 38.0 ± 0.3 5.2
W325F 1.87 12.63 ± 3.27 116.0 ± 9.7 9.19 ± 2.50
W325G * 39.0 ± 0.6 10.0
W325H * 3.08 ± 0.43 28.1 ± 0.0 9.09 ± 1.30 37.7 ± 0.3 40.7 ± 0.0
Variant Yield, cE
(mg/mL)
KM (mm) kcat
(min−1)
kcat/KM
(mm−1min−1)
T50 (°C) Tm (°C) Rosetta
score
change
W325I 3.45 1.29 ± 0.16 6.2 ± 0.2 4.84 ± 0.61 39.6 ± 0.5 29.7
W325K 0.54 ± 0.22 3.58 ± 0.97 4.5 ± 0.4 0.92 ± 0.37 50.0 ± 11.9 3.6
W325L * 5.74 ± 0.35 88.8 ± 2.2 15.37 ± 1.08 40.4 ± 0.5
W325M 4.58 ± 2.45 5.36 ± 0.51 41.7 ± 1.1 7.54 ± 0.87 38.8 ± 0.1 42.8 ± 0.4 3.5
W325N 3.86 1.93 ± 0.15 4.4 ± 0.1 2.27 ± 0.18 40.4 ± 0.4 6.2
W325P 112.0
W325Q 2.02 ± 0.59 0.99 ± 0.21 9.0 ± 0.4 9.31 ± 2.23 39.1 ± 0.3 42.2 ± 0.0 3.9
W325R 2.05 14.18 ± 3.12 213.4 ± 15.7 15.05 ± 3.49 40.0 ± 0.2 42.7 ± 0.0 3.0
W325S 1.89 1.05 ± 0.21 6.1 ± 0.2 5.81 ± 1.17 42.0 ± 1.5 7.2
W325T 3.96 2.03 ± 0.26 7.0 ± 0.2 3.45 ± 0.46 38.2 ± 0.5 8.9
W325V 3.50 1.42 ± 0.16 5.6 ± 0.1 3.92 ± 0.45 39.1 ± 0.7
W325Y 3.02 2.41 ± 0.20 32.6 ± 0.6 13.52 ± 1.16 40.0 ± 0.2 5.1
H328K 1.65 ± 0.05 7.30 ± 2.91 338.8 ± 42.9 91.26 ± 33.42 38.8 ± 0.3 1.1
H328N 0.64 0.79 ± 0.36 3,454.4 ± 308.9 4,359.60 ± 2,002.14 37.2 ± 0.8 0.6
H328P 0.36 2.71 ± 0.67 4.3 ± 0.2 1.58 ± 0.40 51.0
P329N
S331A * 4.34 ± 0.11 668.9 ± 4.3 154.13 ± 4.11 39.3 ± 0.3 41.0 ± 0.0
S331G 2.43 5.82 ± 0.16 1,425.9 ± 14.4 244.86 ± 7.02
S331T 0.89 4.50 ± 1.38 27.4 ± 1.9 6.09 ± 1.91
Y333F 2.28 ± 0.52 1.93 ± 0.20 352.2 ± 11.0 192.18 ± 28.53 37.8 ± 59,885.7
K334D 1.20 6.11 ± 0.90 279.9 ± 11.1 45.78 ± 6.95
L336S 0.28 8.02 ± 0.47 189.2 ± 3.1 23.58 ± 1.43
E340A 1.85 ± 0.85 1.59 ± 0.22 408.6 ± 14.9 311.92 ± 110.17 38.3 ± 0.2 -6.8
E340S 0.81 3.54 ± 0.46 308.7 ± 10.1 87.21 ± 11.72 45.0 ± 0.3 4.4
K341A 3.14 5.33 ± 0.37 610.5 ± 10.6 113.48 ± 7.96 40.0 ± 0.1 45.1 ± 0.5 2.9
Variant Yield, cE
(mg/mL)
KM (mm) kcat
(min−1)
kcat/KM
(mm−1min−1)
T50 (°C) Tm (°C) Rosetta
score
change
F343S
L350I 1.67 5.71 ± 0.48 535.9 ± 11.9 93.83 ± 8.09
I351V 3.95 4.78 ± 0.39 436.1 ± 9.2 91.20 ± 7.74
T352A 0.03 14.26 ± 1.76 49.8 ± 2.2 3.46 ± 0.43 38.1 ± 0.3 39.3 ± 0.0 1.9
T352G 0.43 6.83 ± 0.37 21.1 ± 0.4 3.09 ± 0.18 4.4
T352P 0.24 5.21 ± 0.36 252.6 ± 4.6 48.50 ± 3.42
T352S 0.23 5.11 ± 1.38 234.4 ± 18.2 45.83 ± 12.86 37.5 ± 0.3
T352V 0.05 ± 0.01 2.68 ± 0.45 79.1 ± 3.1 29.52 ± 5.06 50.0 ± 12.1
T352W 71.5
E353A *
E353D 0.45 2.35 ± 0.99 1.6 ± 0.2 0.68 ± 0.29 50.0 ± 10.9
N354A * 5.38 ± 0.67 2.2 ± 0.0 0.43 ± 0.00 40.8 ± 0.4 46.7 ± 0.0
N354S 0.25 2.12 ± 0.39 67.4 ± 2.8 31.70 ± 5.98 34.2 ± 2.1 1.8
G355A
G355N 0.31 8.65 ± 2.96 0.4 ± 0.0 0.04 ± 0.01 -3.6
M358T * 4.83 ± 0.48 357.2 ± 8.7 74.04 ± 7.58 39.5 ± 0.2
L362M * 5.20 ± 0.19 751.2 ± 6.5 144.39 ± 5.41 40.0 ± 0.2
V363N 2.07 4.64 ± 0.32 478.2 ± 8.3 103.16 ± 7.24
V363Q 1.80 ± 0.67 5.67 ± 0.41 202.7 ± 6.4 36.86 ± 4.52 42.0 ± 0.4 1.6
G365N 3.39 4.77 ± 0.12 961.6 ± 8.5 201.64 ± 5.33
H373R * 6.31 ± 0.30 580.2 ± 6.5 92.00 ± 4.55 39.2 ± 0.3 51.4 ± 0.0
Y375H 0.49 5.45 ± 0.36 418.7 ± 7.4 76.84 ± 5.30
E377Q 0.31 4.86 ± 0.69 1,537.4 ± 58.0 316.60 ± 46.56 39.6 ± 0.2
E377S 1.11 4.64 ± 0.69 590.6 ± 22.4 127.35 ± 19.44
H379R *
Variant Yield, cE
(mg/mL)
KM (mm) kcat
(min−1)
kcat/KM
(mm−1min−1)
T50 (°C) Tm (°C) Rosetta
score
change
H379T
H379Y 1.05 5.02 ± 0.49 35.3 ± 0.9 7.03 ± 0.70 -1.1
H384A 1.09 6.22 ± 0.42 644.9 ± 11.9 103.69 ± 7.30
H384R 0.43 6.00 ± 0.39 31.4 ± 0.5 5.24 ± 0.35
I387K 0.17 9.29 ± 1.08 94.5 ± 3.4 10.18 ± 1.24 43.6 ± 0.4 4.7
E388F 3.62 4.79 ± 0.37 317.7 ± 6.5 66.28 ± 5.31
E388G 5.11 1.62 ± 0.21 177.9 ± 5.0 109.58 ± 14.28 44.6 ± 0.1 -4.4
E388L 3.46 ± 0.33 5.29 ± 0.90 136.5 ± 3.8 30.90 ± 3.17 40.2 ± 0.2
E388R 4.14 3.67 ± 0.80 304.6 ± 16.4 82.96 ± 18.62
Q392N 0.47 4.84 ± 1.10 331.4 ± 21.1 68.41 ± 16.07
Y396C 0.9
Y396D 9.4
Y396E 11.3
Y396F 1.14 6.40 ± 1.03 155.0 ± 7.0 24.23 ± 4.03 38.0 ± 0.2 1.8
Y396G 14.6
Y396P 283.8
Y396R 9.3
Y396S 8.2
Y396V 21.5
Y396W 1.28 1.96 ± 0.54 14.1 ± 1.1 7.20 ± 2.05 38.4 ± 0.5 3.9
F397A 0.68 2.78 ± 0.61 25.1 ± 1.4 9.03 ± 2.03 39.2 ± 4.4 39.1 ± 2.9 6.7
F397D 7.9
F397E 0.0
F397G 12.0
F397I 2.20 2.02 ± 0.28 79.4 ± 2.5 39.24 ± 5.56 37.5 ± 0.1 37.2 ± 0.8 26.9
Variant Yield, cE
(mg/mL)
KM (mm) kcat
(min−1)
kcat/KM
(mm−1min−1)
T50 (°C) Tm (°C) Rosetta
score
change
F397K 0.0
F397N 0.71 8.68 ± 0.95 86.3 ± 3.1 9.94 ± 1.14 38.5 ± 0.8 38.5 ± 0.8 6.0
F397R 8.6
F397S 1.10 4.81 ± 0.23 88.0 ± 1.1 18.31 ± 0.91 35.7 ± 0.7 35.7 ± 0.7 7.6
F397V 0.50 3.01 ± 0.44 7.1 ± 0.2 2.38 ± 0.36 36.7 ± 0.3 27.7
F397W 1.37 9.02 ± 0.79 105.0 ± 3.3 11.64 ± 1.09 38.2 ± 0.4 38.2 ± 0.4 0.5
F397Y 1.6
V398N 0.41 4.11 ± 0.25 400.5 ± 6.3 97.37 ± 6.16 34.3 ± 0.2 4.6
W399A *
W399C * 70.33 ± 5.89 2.2 ± 0.0 41.7 ± 0.1 44.8 ± 0.0
W399G *
W399R * 12.75 ± 0.87 37.9 ± 0.8 49.1 ± 0.0
W399S *
S400A * 3.22 ± 0.22 435.1 ± 6.5 135.09 ± 9.53 39.9 ± 0.3 47.3 ± 0.0
S400G 2.18 30.88 ± 2.17 287.8 ± 8.3 9.32 ± 0.71
S400W 124.8
L402C 5.89 3.12 ± 0.21 744.3 ± 24.0 238.68 ± 17.68
L402E 0.27 2.58 ± 0.14 43.7 ± 0.6 16.96 ± 0.95
D403A
N404A 3.83 7.75 ± 1.29 2.8 ± 0.1 0.32 ± 0.04 41.9 ± 0.1 54.3 ± 0.0
N404C 1.55 18.77 ± 3.31 78.0 ± 4.5 4.25 ± 0.70 43.2 ± 0.2 46.0 ± 0.0 -1.9
N404D 1.19 49.80 ± 3.48 18.7 ± 0.7 0.38 ± 0.03
N404E 1.75 ± 1.46 5.04 ± 0.94 53.5 ± 3.2 15.11 ± 3.71 40.5 ± 3.0 45.3 ± 0.6 4.2
N404L 1.90 ± 0.75 30.74 ± 2.08 22.8 ± 0.6 0.74 ± 0.05 44.6 ± 0.1 -1.6
N404M 2.17 ± 1.10 21.49 ± 2.36 18.1 ± 1.7 1.54 ± 0.54 40.6 ± 0.3 48.4 ± 0.2 -3.2
Variant Yield, cE
(mg/mL)
KM (mm) kcat
(min−1)
kcat/KM
(mm−1min−1)
T50 (°C) Tm (°C) Rosetta
score
change
N404Q 2.24 ± 1.29 2.48 ± 0.81 0.2 ± 0.0 0.14 ± 0.12 48.3 ± 0.3 0.5
N404S 4.96 23.55 ± 5.86 23.4 ± 2.2 0.99 ± 0.26
E406A * 40.1 ± 0.7 49.0 ± 0.0
E406D 0.49 29.16 ± 4.17 28.4 ± 1.5 0.94 ± 0.17 40.4 ± 0.2 42.2 ± 0.0 -4.4
E406F 0.64
E406H 2.36 0.02 ± 0.04 0.0 ± 0.0 1.80 ± 2.83 4.5
E406Q 3.78 ± 1.32 58.18 ± 5.12 36.9 ± 3.0 0.44 ± 0.08 48.3 ± 1.5 4.3
E406T 3.40 2.57 ± 1.38 15.6 ± 1.9 6.05 ± 3.33
W407A *
W407D 0.14 0.37 ± 0.90 3.0 ± 1.4 8.05 ± 20.19 2.5
W407F 0.98 38.81 ± 4.02 6.1 ± 0.3 0.16 ± 0.02 44.8 ± 0.0 6.1
W407G
W407H 0.97 ± 0.47 5.58 ± 3.04 0.5 ± 0.1 1.02 ± 0.79
W407K 0.09 17.69 ± 3.06 3.6 ± 0.2 0.20 ± 0.04 3.5
W407L * 29.5
W407Q
W407R 8.8
W407S -1.8
W407Y
A408F 0.31 37.97 ± 8.36 341.9 ± 33.2 9.00 ± 2.17
A408K 2.59 6.32 ± 0.92 319.8 ± 12.7 50.56 ± 7.62
A408S 0.23 9.32 ± 2.00 158.5 ± 10.3 17.02 ± 3.81 38.4 ± 0.2 1.9
A408T 0.14 12.90 ± 2.77 153.9 ± 10.7 11.93 ± 2.69 37.3 ± 0.5 4.9
W409A 4.66 4.69 ± 0.72 117.3 ± 4.6 25.02 ± 3.97 39.8 ± 0.3 3.6
W409C 6.00 2.44 ± 0.13 156.2 ± 1.9 64.01 ± 3.39 39.4 ± 2.2 5.0
Variant Yield, cE
(mg/mL)
KM (mm) kcat
(min−1)
kcat/KM
(mm−1min−1)
T50 (°C) Tm (°C) Rosetta
score
change
W409D 3.60 2.29 ± 0.14 532.9 ± 7.4 233.16 ± 14.49
W409E 1.63 ± 0.85 1.81 ± 0.15 503.2 ± 8.3 251.30 ± 16.39 39.6 ± 0.4 46.8 ± 0.1 4.9
W409F 0.78 3.65 ± 0.29 615.2 ± 12.3 168.53 ± 13.79 39.9 ± 0.1
W409G 0.44 3.85 ± 0.82 14.1 ± 0.7 3.66 ± 0.81
W409H 1.73 ± 1.15 2.47 ± 0.29 361.6 ± 12.9 141.45 ± 21.50 38.9 ± 0.3 2.1
W409I 1.13 4.64 ± 0.50 6.1 ± 0.2 1.32 ± 0.15 40.3 ± 0.3 10.7
W409L 6.10 2.05 ± 0.16 87.5 ± 1.6 42.57 ± 3.43 39.5 ± 0.3 9.5
W409N 6.60 ± 1.67 5.91 ± 0.89 184.0 ± 8.7 27.96 ± 4.28 39.1 ± 0.5 38.9 ± 0.1 1.9
W409P 82.7
W409R 2.67 ± 0.72 2.73 ± 0.32 142.5 ± 4.1 52.07 ± 6.36 44.5 ± 0.3 3.7
W409S 6.17 4.52 ± 0.76 143.7 ± 6.3 31.79 ± 5.51 39.4 ± 0.4 4.1
W409T 1.78 2.08 ± 0.51 344.1 ± 19.4 165.50 ± 41.39 37.1 ± 0.3 3.1
W409V 4.17 9.62 ± 1.08 139.5 ± 4.8 14.50 ± 1.71 39.3 ± 0.9 5.0
W409Y 0.26 2.79 ± 0.29 693.8 ± 20.3 248.84 ± 25.30 36.8 ± 0.1 43.5 ± 0.0 0.4
Y411R 0.28 ± 0.13 1.99 ± 0.63 50.3 ± 3.3 32.27 ± 11.15
K413Q 3.02 5.41 ± 0.62 386.6 ± 12.1 71.53 ± 8.55 45.8 ± 1.7 5.0
K413R 0.41 0.85 ± 0.29 470.8 ± 32.0 555.04 ± 196.18 40.1 ± 0.1
F415A * 16.63 ± 4.00 40.5 ± 0.3 44.7 ± 0.0
F415N *
F415Y 2.24 10.01 ± 1.13 288.3 ± 9.8 28.82 ± 3.40
H419Y 3.27 ± 0.04 4.88 ± 0.44 742.6 ± 14.1 143.16 ± 11.99 49.1 ± 1.1 -2.1
Y422F 2.83 30.66 ± 5.28 183.8 ± 13.5 6.00 ± 1.12 47.4 ± 0.2 -0.4
E423S * 6.60 ± 0.42 530.4 ± 34.6 80.31 ± 7.36 39.5 ± 0.1 44.8 ± 0.0
R427H 0.46 1.28 ± 0.41 1.0 ± 0.1 0.80 ± 0.26 11.6
R427L 0.41 4.95 ± 0.40 380.1 ± 8.3 76.73 ± 6.49 45.4 ± 0.3 4.9
Variant Yield, cE
(mg/mL)
KM (mm) kcat
(min−1)
kcat/KM
(mm−1min−1)
T50 (°C) Tm (°C) Rosetta
score
change
R427W 0.31 0.98 ± 0.27 35.6 ± 2.0 36.11 ± 10.00 40.2 ± 0.1
P429A 2.51 9.81 ± 1.03 327.2 ± 10.7 33.35 ± 3.65
S432A 1.12 2.23 ± 0.34 69.0 ± 2.3 30.97 ± 4.86 79.3 ± 0.0 0.5
L434S 2.49 0.57 ± 0.16 239.0 ± 13.1 416.14 ± 121.04 43.2 ± 0.0 1.2
W435Y 0.48 7.41 ± 0.72 823.9 ± 23.2 111.17 ± 11.22
A441V 2.07 4.65 ± 0.10 859.5 ± 4.6 184.98 ± 4.01