Nα-acetylation is a universal protein modification related to a wide range of physiological processes in eukaryotes and prokaryotes.Riml,an Nα-acetyltransferase in Mycobacterium tuberculosis,is responsible for the acetylation of the α-amino group of the N-terminal residue in the ribosomal protein S18.Despite growing evidence that protein acetylation may be correlated with the pathogenesis of tuberculosis,no structural information is yet available for mechanistically understanding the MtRiml acetylation.To enable structural studies for MtRiml,we constructed a serial of recombinant MtRiml proteins and assessed their biochemical properties.We then chose an optimal construct MtRimlC21A4-153 and expressed and purified the truncated high-quality protein for further biophysical and functional characterizations.The 2D 1H-15N heteronuclear single quantum coherence spectrum of MtRimlC21A4-153 exhibits wider chemical shift dispersion and favorable peak isolation,indicating that MtRimlC21A4-153 is amendable for further structural determination.Moreover,bio-layer interferometry experiments showed that MtRimlC21A4-153 possessed similar micromolar affinity to full-length MtRiml for binding the hexapeptide substrate Ala-Arg-Tyr-Phe-Arg-Arg.Enzyme kinetic assays also exhibited that MtRimlC21A4-153 had almost identical enzymatic activity to MtRiml,indicating insignificant influence of the recombinant variations on enzymatic functions.Furthermore,binding sites of the peptide were predicted by molecular docking approach,suggesting that this substrate binds to MtRiml primarily through electrostatic and hydrogen bonding interactions.Our results lay a foundation for the further structural determination and dynamics detection of MtRiml.