Figure 1. Purification of RimI^Mtb and identification of Nα-acetyltransferase activity of RimI^Mtb.

(a) Gel filtration of RimI^Mtb using superdex 75 10/300GL. Ni-NTA purified RimI^Mtb (as shown in protein gel) eluted as monomer (calibration curve given in inset) (b) Confirmation of intact mass (19.1 kDa) of purified RimI^Mtb monomer using LC-ESI-MS (c) MS analysis of control assay (without enzyme) using DPC peptide (ARYFRR) as substrate (d) MS/MS analysis of precursor ion (868.5 Da) of DPC peptide in control assay (e) MS analysis of enzyme assay confirming acetylation of DPC peptide by RimI^Mtb. Modified peptide was observed (910.5) with an increase of 42.0105 Da as compared to unmodified peptide (868.5 Da) concomitant to the addition of acetyl group (f) MS/MS analysis of modified (910.5 Da) precursor ion of the substrate (DPC) peptide. An increase of 42.0105 Da was observed in all the b-ions (in black) while the y-ions (in grey) remained the same as that of unmodified substrate, confirming the site of acetylation as the N-terminal amino acid i.e. Ala.