Mycobacterium Tuberculosis NAD Synthase NaMNAT (Rv2421c) Moonlights As Chaperone

Tuberculosis is one of the infectious diseases seriously threatening human health with global significance. About 1/3 of the global population was infected by TB etiological agent Mycobacterium tuberculosis. Moreover, the drug-resistant bacteria emerge in endlessly, they not only resist to first line durg (Isoniazidum,Rifampin,Sstreptomycin. Eethambatal) but also Second-line drugs (such as fluoroquinolones, paminosalicylic acid, kanamycin, cycloserine, ethionamide, amikacin, capreomycin, thiacetazone). So, novel drug targets are urgently needed.Cofactor biosynthesis is a rich source of potential drug targets due to the essential nature of these coenzymes throughout metabolism. Nicotinamide adenine dinucleotide (NADP) is essential for a variety of organisms, and it is both a coenzyme for hydride-transfer enzymes and a substrate for NADP-consuming enzymes, such as ADP-ribose transferases, poly (ADP-ribose) polymerases, cADP-ribose synthases, and sirtuins. A detailed reconstruction of the NAD(P) metabolic subsystem using the SEED genomic platform (http://theseed.uchicago.edu/FIG/index.cgi) helped us accurately annotate respective genes in the entire organism with completely sequenced genomes. It have revealed six NAD(P) biosynthesis pathways:de novo pathway I (Asp), de novo pathway II (Try), NmR pathway I (RNK-dependent), NmR pathway II (RNK-independent), Niacin salvage and Niacin recycling.The conserved and indispensable enzymes in both pathways of NAD biosynthesis in M. tuberculosis, such as NMN/NaMN adenylyltransferase (NMNAT), NAD synthetase (NADS), and NAD kinase (NADK) are the most promising drug targets. Here, NaMNAT gene nadD (Rv2421c) in M. tuberculosis H37Rv was cloned into the prokaryotic vector pET28(+).And the fusion protein was expressed and its chaperone activity was assayed. Structure and function of the gene encoding protein were predicted through bioinformatics tools. The nucleotide sequence of NaNMAT gene in M. tuberculosis H37Rv was obtained from the GenBank database, and a pair of primers was designed. M. tuberculosis H37Rv genome was used as a template, nadD gene was amplified by PCR. The PCR product was ligated to the pMD19-T Simple Vector, and then subcloned into vector pET28a(+). The recombinant plasmid pET28-nadD was identified through colony PCR, plasmid restriction enzyme digestion and sequencing. The recombinant plasmid was transformed into Escherichia coli BL21(DE3). The fusion protein obtained under the optimial expression culture conditions was purified by Ni+ affinity chromatography. The strain M. smegmatis mc2155 was transformed by electroporation with the shuttle plasmid pMV261. We used the reconstructed plasmid pMV261-Rv2421c and pMV261 to test the chaperone function of Rv2421 under the pressure of oxidation,acid,alkali,SDS,CUSO4 ect in M. smegmatis mc2155. And then the insulin agglutination assy was seted up to prove the chaperone function of Rv2421c. The result indicated the NaNMAT in bacteria help the bacteria resist harmfully ambient pressure.We also used bioinformatics to give a research on NaMNAT of M. tuberculosis H37Rv. The results showed that NaMNAT had no transmembrane structure, pi 5.16, and it also had a far relationship with human and Drosophila melanogaster NMANT at evolution and primary structure.