2.5 Analysis of the overall reaction and the individual reaction steps of lipoylation
  1. Overall reaction activity. The overall reaction activity was determined at constant concentrations of R-(+)-lipoic acid (2 mM), ATP (2 mM), MgCl2 (2 mM), Hapo (0.5 -400 μM), and LplA (10 μM) The reaction was started with the addition of ATP. The amount of lipoylated H protein in the mixture was determined using HPLC as described in our previous study (Zhang et al., 2019).
  2. Activity assay of the lipoate adenylation reaction (Step 1).The reaction activity was determined in a reaction mixture of 0.25 mL containing 10 μM LplA, 2 mM ATP, 2 mM MgCl2, and 2 mM R-(+)-lipoic acid. The final volume was made up with 50 mM Tris-HCl (pH 7.5). The reaction was initiated by adding ATP, incubated at 30 ºC for 15 min (unless stated otherwise), and terminated by heating for 90s in boiling water to completely denature and precipitate LplA. Lip-AMP was chromatographically separated and quantified as described previously (Fujiwara et al., 2010), using a gradient HPLC on a Shimadzu Shim pack GIST C18 column (5 µm, 4.5 × 150 mm), with the mobile phase A being acetonitrile containing 0.1% trifluoroacetic acid, and the mobile phase B being 50 mM PBS buffer (pH 6.6) containing 5 mM tetrabutylammonium hydrogen sulfate (TBAHS). The formation of Lip-AMP can be observed as a single, clearly resolved peak. Because no chemical standard of Lip-AMP is commercially available, Lip-AMP was quantified indirectly as follows: after ATP, ADP and AMP were quantitatively determined, Lip-AMP is calculated as the difference between the initial amount of ATP and the total amount of the ATP remained and the ADP and AMP formed.
  3. Activity assay of the lipoate transfer reaction (Step 2). The determination of the second step activity was similar to that in the first step, in which lipoic acid, ATP and MgCl2 were replaced with Lip-AMP and Hapo. Lip-AMP was obtained through an overnight reaction of the first step and after a heat treatment of the reaction mixture in boiling water for 1 minute to denature LplA completely. Then, the sample was centrifuged at 10,000 x g and 4 °C for 1 minute. The supernatant was collected and used as the substrate of the second step. The lipoate transfer reaction was initiated by adding LplA, incubated at 30 ºC for 15 min, and terminated by heating 1 minute in boiling water. The method for the detection of Hlip was the same as that used for the overall reaction.