Table 1 Apparent initial FabD pupylation rates of PafA and PafA variants

From: Electrostatic interactions guide substrate recognition of the prokaryotic ubiquitin-like protein ligase PafA

Substrate

PafA mutant

App. initial pupylation rate [h−1]

FabD

PafA

10.41 ± 0.57

R137A

3.10 ± 0.21

R207A

No pupylation

R218A

5.05 ± 0.15

R137A, R207A

No pupylation

R137A, R218A

0.17 ± 0.03

R207A, R218A

No pupylation

R137A, R207A, R218A

No pupylation

R442A

2.874 ± 0.20

K444A

2.65 ± 0.28

R442A, K444A

2.64 ± 0.23

  1. Pupylation time courses recorded by SDS-PAGE and Coomassie-staining were quantified by gel densitometric analysis of the FabD and FabD-Pup bands. The apparent initial pupylation rate was determined by linear regression of the first four data points in dependence of the used PafA concentration. Fraction of pupylated substrate is expressed in relation to the total amount of substrate present in the reaction. Assays were carried out in independent triplicates. Representative gels are shown in Supplementary Fig. 6c.
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