Function And Catalytic Activity Sites On Adenylosuccinate Lyase From Mycobacterium Tuberculosis

Mycobacterium tuberculosis is a bacteria of chronic infectious tuberculosis(TB), which can violate many organs, the most common in pulmonary tuberculosis.Each year the number of deaths from TB is up to 1.5 million.With the completion of whole genome sequencing in M.tuberculosis and urgent demand of looking for a new anti-TB drugs target,it is of great importance to elucidate the gene function of Mycobacterium tuberculosis, especially the structure and function of potential drug targets.Purine biosynthetic pathway is an important biosynthetic pathway of Mycobacterium tuberculosis which makes a potential target of new anti-TB drugs.The research in metabolism of purine synthesis gene is not only beneficial to get a deeper understanding of M.tuberculosis, but also can provide theoretical guidance for research and development of new anti-TB drugs.We cloned the adenylosuccinate lyase gene(pur B) of Mycobacterium tuberculosis. Then Arg10 Glu,His76Glu,Thr104 Lys,His147Ala,Gln229 Ala, Ser277 Ala,Ser278Ala, Lys283 Glu, Arg291 Glu, Arg326 Glu, His76Glu/Lys283 Glu and Arg10Glu/Arg291Glu/Arg326 Glu point mutations in Mtpur B were obtained using gene site-directed mutagenesis. They were ligated with complementary p BAD24 series carrier respectively.The p BAD24 series plasmid vectors were transformed into SE1888, the pur B gene inserted mutant of Escherichia coli W3110.The genetic complementation results show that Mtpur B and Mt Pur B S278 A point mutation genes are able to complement the SE1888 were grown in MG minimal medium,while the other can't. It preliminary evidences that the proteins Mt Pur B and Mt Pur B S278 A have adenylosuccinate lyase function,and demonstrated that Arg10,His76,Thr104,His147,Gln229,Ser277,Lys283,Arg291 and Arg326 were theessential structural residues.In order to further understand the function of Mt Pur B,the enzyme activities of Mt Pur B, Mt Pur B R10 E, Mt Pur B S278 A and Mt Pur B K283 E were analyzed by isothermal titration calorimetry(ITC). The results show that the enzyme activity and substrate affinity of Mt Pur B were the highest in the reaction conditions at 37 ? and p H7.0. Under this condition, the Kcat of Mt Pur B was 1.62±0.0092 S-1 and Km was0.104±0.0031 m M with the substrate SAMP, while the Kcat was 0.784±0.0085 S-1and Km was 0.0233±0.0013 m M with substrate SAICAR.Compared with Mt Pur B,the catalytic efficiency of Mt Pur B R10 E and Mt Pur B S278 A was significantly lower. The catalytic efficiency of Mt Pur B S278 A for both two substrates was reduced by about 2 times. The catalytic efficiency of Mt Pur B R10 E for substrate SAMP decreases by about 8 times, while the catalytic efficiency of Mt Pur B R10 E for SAICAR substrate is reduced more than 40 times. In addition, the Mt Pur B K283 E was completely determined not catalytic activity in vitro, which is consistent with the growth of the genetic complementation results.