In vitro ATPase activity assay

The ATPase activity of NisT was determined with the malachite green assay as described previously with experimental alterations⁶³. In this assay the release of inorganic orthophosphate after ATP hydrolysis was colorimetrically quantified based on a Na₂HPO₄ standard curve.

All reactions were performed at 30 °C in a total volume of 30 μl in activity assay buffer containing 0.4% CYMAL5 and 10 mM MgCl₂.

In each reaction ~ 2 μg of detergent-solubilized and purified NisT was used and the reaction was started by adding ATP (0–5 mM). The background of the reaction was a sample without MgCl₂. After 30 min the reaction was stopped by transferring 25 μl of each reaction into a 96-well plate containing 175 μl stop-solution (20 mM sulphuric acid). Consecutively, 50 μl of a staining solution (0.096% (w/v) malachite green, 1.48% (w/v) ammonium heptamolybdate and 0.173% (w/v) Tween-20 in 2.36 M sulphuric acid) was added. After 10 min the amount of free inorganic orthophosphate was quantified by measuring the absorption at 595 nm using an iMark microplate reader (Bio-Rad).

The specific ATPase activity of NisT was plotted against ATP concentrations and fitted using the Michaelis–Menten Eq. (³). Note that y is the reaction velocity, V_max the maximal reaction velocity, x is the substrate concentration and K_m the Michaelis–Menten constant. The analysis was performed using Prism 7.0c (GraphPad).

For the reactions with substrates (NisA variants) or interaction partner (NisB and NisC) NisT was pre-incubated at 30 °C for 10 min before ATP was added to start the reaction. All reactions were performed at 30 °C in a total volume of 30 μl in activity assay buffer containing 0.4% CYMAL5, 400 mM glutamate and 10 mM MgCl₂. In each reaction ~ 2 μg of detergent-solubilized and purified NisT was used and the reaction was started by adding 5 mM ATP and stopped after 15 min following the procedure described above. In this reaction the concentration of the different substrates (0–40 µM) and/or interaction partner was varied and the ATPase activity was normalized to the specific ATPase activity of NisT without substrate/interaction partner. In these cases, the background was subtracted prior to normalization.