Fig. 3.

In vitro ligation assays.

(A) Lipoate ligase activity. Western blot analysis shows that LipL1 is an ATP-dependent lipoate ligase only when H-protein is used as substrate, while LipL2 does not display lipoate ligase activity with any substrate.

(B) HPLC traces for LipL1, LipL1_K160A and LipL2 adenylation reactions. Each enzyme was reacted with lipoate and ATP for 10 minutes prior to HPLC analysis with absorbance monitored at 270 nm. The LipL1 reaction produced a major product eluting at 6.2 minutes with a mass of 534 Da as determined by ESI-MS.

(C) Identification of Lipoyl-AMP produced by LipL1. The major product generated by LipL1 was subjected to collision induced dissociation MS/MS, demonstrating that this product contains AMP.

(D) JEG3 complementation by LipL1 and LipL1_K160A. LipL1 is able to lipoylate endogenous E. coli substrates in lipoylation deficient strain JEG3, whereas LipL1_K160A is unable to catalyze this activity. Expression of both proteins was confirmed by stripping and re-probing the blot with α-MBP (lower panel).

(E) Recombinant LipL1_K160A cannot lipoylate the H-protein in an in vitro lipoylation assay.