Table 1 Enzyme kinetic constants of native, designed and discovered ketoacid decarboxylases.

From: Integrative genomic mining for enzyme function to enable engineering of a non-natural biosynthetic pathway

kcat/KM(M−1 s−1)

C3

C5

C8

iso C5

 

GEO179

0.9±0.02

100±15

1,200±130

41±1.2

Discovered

GEO195

2.4±0.1

200±14

1,400±160

8±0.3

Discovered

GEO175

0.51±0.02

48±3.7

17,000±2,700

3.3±0.2

Discovered

GEO175 L376T_T240S

0.27±0.02

30±7.2

1,100±160

1.6±0.1

Designed

1OVM

32±0.5

2,100±420

80,000±4,300

1,300±130

Native

2VBI

5,700±1,400

52±1.8

14±0.8

1.8±0.1

Native

3FZN

5.4±0.1

1,700±110

350±30

110±10

Native

1ZPD

8,200±545

140±5.6

19±2

0.33±0.01

Native

1OZF

n.d.

0.53±0.04

n.d.

17±0.9

Native

Native KIVD

42±0.6

9,500±470

32,000±5,500

14,000±1,100

Native

KIVD_VLV

0.71±0.07

1.3±0.2

2,800±860

0.24±0.02

Designed

kcat (s−1)

 GEO179

n.d.

0.47±0.03

0.32±0.01

0.39±0.01

 

 GEO195

n.d.

0.49±0.01

0.56±0.02

0.072±0.001

 

 GEO175

n.d.

0.97±0.04

10.1±0.6

n.d.

 

 GEO175 L376T_T240S

n.d.

0.28±0.04

3.4±0.2

n.d.

 

 1OVM

0.2±0.01

1±0.1

1.7±0.1

7.8±0.4

 

 2VBI

25±2.6

n.d.

0.051±0.01

0.025±0.001

 

 3FZN

n.d.

4.7±0.1

1.3±0.1

0.52±0.02

 

 1ZPD

46.7±1.4

0.95±0.02

0.021±0.001

n.d.

 

 1OZF

n.d.

n.d.

n.d.

0.03±0.001

 

 Native KIVD

n.d.

14.3±0.2

7±0.3

61±2.1

 

 KIVD_VLV

n.d.

0.013±0.001

0.5±0.03

n.d.

 

KM (mM)

 GEO179

n.d.

4.6±0.6

0.27±0.03

9.4±0.2

 

 GEO195

n.d.

2.5±0.2

0.4±0.04

9.1±0.3

 

 GEO175

n.d.

20±1.3

0.58±0.09

n.d.

 

 GEO175 L376T_T240S

n.d.

10±2

3.1±0.4

n.d.

 

 1OVM

7.5±0.1

0.5±0.1

0.021±0.001

6.1±0.6

 

 2VBI

4.3±1.0

n.d.

3.6±0.2

14±0.5

 

 3FZN

n.d.

2.7±0.2

3.8±0.3

4.9±0.3

 

 1ZPD

5.7±0.3

7.1±0.3

1.2±0.1

n.d.

 

 1OZF

n.d.

n.d.

n.d.

1.8±0.1

 

 Native KIVD

n.d.

1.5±0.1

0.21±0.04

4.5±0.3

 

 KIVD_VLV

n.d.

10±1

0.18±0.05

n.d.

 
  1. The kinetic constants were determined, as described in Methods, against 2-ketooctanoate (C8), 2-ketoisovalerate (isoC5) and pyruvate (C3). The curves consisted of at least five points. The limit of detection under the conditions tested was 0.2 M−1 s−1, kinetic constants beyond our detection limit are labeled as n.d. (not determined). Michaelis–Menten curve fits are shown in Supplementary Figs 3–6. Errors are shown as ±one s.d.
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