Clone

I(460) from time drives I(460) from titration 1.86 1.9 1.86 1.91 3.72 2.71 3.72 2.93 4.65 2.98 9.3 5.83 18.6 11.78 18.6 10.81 27.9 18.22 37.2 22.14 37.2 21.65 55.8 33.33 74.4 44.5 74.4 44.9 74.4 47

I(460)

NADPH ADDED ul [NADPH] uM 1 1 2 2 2.5 5 10 10 15 20 20 30 40 40 40

47.5 45 42.5 40 37.5 35 32.5 30 27.5 25 22.5 20 17.5 15 12.5 10 7.5 5 2.5 0 0

NADPH stock Cuvette volume

930 uM 500 ul SLOPE

10

20

I(460)

47.5 45 42.5 40 37.5 35 32.5 30 27.5 25 22.5 20 17.5 15 12.5 10 7.5 5 2.5 0

I(460) from time drives I(460) from titration Linear regression, I(460) from time drives Linear regression, I(460) from titration

0

10

20

30

40 [NADPH] uM

0.6 I(460)/uM

50

60

70

80

[NADPH] ul [NADPH] uM 40 40 40 10 10 2 2 1 1 2.5 5 15 20 30 20

20 1.86 3.72 3.72 4.65 9.3 18.6 18.6 27.9 37.2 37.2 55.8 74.4 74.4 74.4

uL NTR added NTR dilution Undiluted NTR uL/ml NTR ug NTR uM 0.5 0.1 0.1 0.0500 0.5 0.1 0.1 0.0500 0.5 0.1 0.1 0.0500 0.5 0.1 0.1 0.0500 0.5 0.1 0.1 0.0500 0.5 0.1 0.1 0.0500 0.5 0.1 0.1 0.0500 0.5 0.1 0.1 0.0500 0.5 0.1 0.1 0.0500 0.5 0.1 0.1 0.0500 0.5 0.1 0.1 0.0500 0.5 0.1 0.1 0.0500 0.5 0.1 0.1 0.0500 0.5 0.1 0.1 0.0500 0.5 0.1 0.1 0.0500 0.5 0.1 0.1 0.0500 0.5 0.1 0.1 0.0500 0.5 0.1 0.1 0.0500 0.5 0.1 0.1 0.0500 0.5 0.1 0.1 0.0500 0.5 0.1 0.1 0.0500

CB1954 NADPH nfsA cuvette

32 mM 1000 mM 0.5 mg/ml 2000 ul

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

I(460)/s v uM s-1 v/E 0.170000 0.0196 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000

9.39 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

9.50 9.00 8.50 8.00 7.50 7.00 6.50 6.00 5.50 5.00 Column J

4.50 4.00 3.50 3.00 2.50 2.00 1.50 1.00 0.50 0.00 0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

Column J