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Table 1 Kinetic parameters—K m (µM), k cat (s−1), and k cat /K m (s−1 mM−1)—for the oxidation of Mn2+, phenolic (DMP) and nonphenolic (VA) aromatics, and dyes (means and 95% confidence limits) by the resurrected enzymes and extant LiPH8 (a comparison of all catalytic efficiencies is shown in Fig. 4)

From: Experimental recreation of the evolution of lignin-degrading enzymes from the Jurassic to date

 

CaPo

CaCD

AVP

ALiP

LiPH8

Metal ion

 Mn2+

  K m

700 ± 48

275 ± 41

62 ± 10

a

  k cat

185 ± 3

170 ± 6

106 ± 4

  k cat /K m

260 ± 15

617 ± 80

1710 ± 240

Aromatics

 DMPb (low efficiency)

  K m

32,900 ± 2700

66,800 ± 3800

32,500 ± 12,100

  k cat

221 ± 9

109 ± 4

31 ± 5

  k cat /K m

6.7 ± 0.3

1.6 ± 0.03

1.0 ± 0.2

 DMP (high efficiency)

  K m

5.3 ± 1.1

34.0 ± 5.4

4.0 ± 0.07

  k cat

4.5 ± 0.1

18.3 ± 0.7

6.9 ± 0.5

  k cat /K m

837 ± 162

537 ± 55

600 ± 36

 VA

  K m

299 ± 104

773 ± 155

79.3 ± 18

  k cat

7.1 ± 0.5

21.3 ± 1.0

16.2 ± 0.8

  k cat /K m

24 ± 7

28 ± 5

205 ± 4

Dyes

 ABTSb (low efficiency)

  K m

3170 ± 270

1280 ± 350

2150 ± 420

  k cat

539 ± 24

103 ± 10

25 ± 2

  k cat /K m

170 ± 8

80 ± 15

12 ± 1

 ABTS (high efficiency)

  K m

5.4 ± 0.7

13.7 ± 3.5

21.3 ± 2.3

  k cat

2.1 ± 0.1

12.5 ± 0.7

6.5 ± 0.2

  k cat /K m

400 ± 44

911 ± 212

300 ± 25

 RB5

  K m

4.8 ± 0.8

12.6 ± 3.6

  k cat

2.4 ± 0.2

5.4 ± 0.8

  k cat /K m

504 ± 50

428 ± 68

Direction of evolution

  1. The alternative CaPo ancestors oxidized DMP (low efficiency) with K m 455,000 ± 84,000 (CaPo-bis) and 298,000 ± 66,000 μM (CaPo-tris), k cat 129 ± 22 (CaPo-bis) and 108 ± 21 s−1(CaPo-tris), and k cat /K m 0.3 ± 0.05 (CaPo-bis) and 0.4 ± 0.01 s−1mM−1 (CaPo-tris); ABTS (low efficiency) with K m, 1392 ± 345 (CaPo-bis) and 807 ± 104 μM (CaPo-tris), k cat, 241 ± 23 (CaPo-bis) and 128 ± 6 s−1 (CaPo-tris), and k cat /K m, 173 ± 28 (CaPo-bis) and 160 ± 15 s−1  mM−1 (CaPo-tris), while Mn2+ was only oxidized by CaPo-tris with K m 5010 ± 274 µM, k cat 47 ± 1 s−1, and k cat /K m 9.3 ± 0.4 s−1 mM−1
  2. Reactions at 25 °C in 0.1 M tartrate at optimal pH 5.0 (CaPo, and its alternative ancestors, and CaCD) or 5.5 (AVP) for Mn2+, pH 3.5 (AVP) or 3.0 (ALiP and LiPH8) for VA, pH 2 (CaPo) or 3.0 (others) for DMP, pH 3.0 (CaPo) and pH 3.5 (others) for ABTS, and pH 3 for RB5; and saturating H2O2 concentrations of 0.4 mM for CaPo, CaCD, and AVP, 0.2 mM for ALiP, and 0.1 mM for LiPH8
  3. a–Absence of activity
  4. bBiphasic kinetics for DMP and ABTS oxidation by AVP enabled calculation of two sets of constants assigned to two catalytic sites, as reported for extant VP [40], comparable to those present in CaPo/CaCD and ALiP/LiPH8, respectively