The Structure And Function Study Of Acetyltransferase RimI From Mycobacterium Tuberculosis

Tuberculosis?TB?is a highly lethal infectious disease.With the emergence of drug resistant strains,it is urgent to find and develop new drug target proteins.Recent studies have shown that the N-terminal acetylation of proteins may be related to the pathogenesis of tuberculosis,and acetyltransferase plays an important role in cell regulation and control,transcription,translation and other pathways.The development of anti-tuberculosis drug targets for acetyltransferases has important scientific significance and application value.At present,the structure and molecular mechanism of NAT acetyltransferase in eukaryotes have been studied,but the N-terminal acetylation in prokaryotes is little known.RimI is an N?-acetyltransferase from Mycobacterium tuberculosis,which is involved in the N-terminal acetylation of ribosomal protein S18.Therefore,it is of great significance to further study the molecular mechanism of MtRimI acetylation.However,since it is difficult to obtain homogeneous,stable and crystallized MtRiml protein samples,no structural information is yet available for mechanistically understanding the MtRimI acetylation at the molecular level.This paper focused on the preparation,structure and function of protein samples of the acetyltransferase MtRimI in Mycobacterium tuberculosis and have made the following major advances:?1?Using molecular cloning technology,combined with a variety of bioinformatics prediction tool to construct a series of different lengths and single point mutation of MtRimI recombinant proteins,we selected the optimal recombinant protein MtRimIC21A_4-153 through a series of physical and chemical characterization,and get the MtRimIC21A_4-153 protein sample with uniform,stable and suitable for high resolution liquid nuclear magnetic resonance?NMR?detection;?2?CD and NMR techniques were used to analyze and compare the structure of MtRimIC21A_4-153 protein and full-length wild-type MtRimI protein.Firstly,we compared a series of MtRimI recombinant protins,indicating the proteins were well folded,and was similar to MtRiml.Then through 2D¹H-¹⁵N HSQC spectrum analysis,it was found that MtRimIC21A_4-153 had the widest chemical displacement distribution of HSQC spectral and the peak rate of amino acid residues was 100%,which was most suitable for the determination of the three-dimensional solution structure by liquid nuclear magnetic;?3?The biological function of MtRimIC21 A_4-153 was verified to be consistent with the full-length wild-type MtRimI protein by bio-layer interference?BLI?and enzyme kinetics assay.BLI experiments showed the similar affinity for the hexapeptide substrate Ala-Arg-Tyr-Phe-Arg-Arg between MtRimIC2IA_4-153 and MtRiml.Enzyme kinetic assay showed that MtRimIC21A_4-153 had almost the same enzyme activity as MtRiml.These results indicated that MtRimIC21A_4-153 could represent full-length wild-type MtRiml protein;?4?Molecular docking technology was used to predict the binding sites and interaction modes of proteins and polypeptides,and it was found that the substrates were bound to the protein mainly through hydrogen bond and electrostatic interaction,providing a basis for further acetylation research.This paper aims to lay a foundation for the determination of the three-dimensional structure of MtRimI protein and the clarification of its molecular mechanism of acetylation,and it is expected to provide new ideas for the discovery of new anti-tuberculosis drug targets.