M. Tuberculosis H37rv Tryptophan Synthase ¦Á And ¦Â Subunits Of The Enzyme Properties And Structural Analysis

Tuberculosis represents one of the world's greatest sources of mortality and morbidity,with approximately eight million new infections and 2.5-3 million deaths per year.Mycobacterium tuberculosis is main pathogen that leads to tuberculosis in human.Compounding the problem,strains of M.tuberculosis that are resistant to the major drugs used to treat tuberculosis are rapidly emerging worldwide.The eradication of tuberculosis requires the development of novel drugs for therapeutic treatment of M.tuberculosis.The essentiality of mycobacterial aromatic amino acid biosynthesis pathways and their absence from human host indicate that the member enzymes of these pathways promising drug targets for therapeutic agents against pathogen mycobacteria.Trptophan Biosynthesis is continues 5-steps reaction which origins from chorismate.Trptophan Synthase(TSase) is a key enzyme which undergoes last two steps in Trptophan Synthesis and catalyzes the PLP(pyridoxal phosphate)-dependent conversion of indole and L-serine to L-trptophan,making it a potential drug target for antibiotics discovery.TSase has aαββαstructure,αandβsubunit are encoded by trpA and trpB.Rv1613 and Rv1612(namely trpA and trpB) was cloned from M.tuberculosis H37Rv,then expressed in E.coli,Then the recombinant TRPSA and TRPSB with an N-terminal His-tag(His-rMtTRPSA and His-rMtTRPSB) was first purified in E.coli. The molecular weights of His-rMtTRPSA and His-rMtTRPSB,estimated by MALDI-TOF,are 33151.0u and 50224.0u(vector included).The optimize condition ofβreaction is pH7.5,temperature37℃,the cation-[Na+]=0.15M or [Mg2+]=0.18M,[His-rMtTRPSA]/[His-rMtTRPSB]=2.2.Under such condition, Km(indole) is 0.288mM,Km(L-serine) is 0.178mM and Km(PLP) is 20.824mM. Meanwhile,it is identified that a subunit can activateβreaction.Circular Dichroism indicate proteins's secondary structure.The3D structure were modeled by homologous modeling,and the active sites were predicted as Ser390,Ser99,Gln128, Ser249,Asn250,Glu364,Thr204,His100.The active amino acids were mutated to Alanine by site-directed mutagenesis,and the enzyme activity of mutant proteins decrease obviously.In conclusion,we have cloned,expressed and purified the recombinant Trptophan Synthaseαandβsubunit of Mycobacterium tuberculosis,as well as established the method for enzymatic assays and identified catalytic sites.All of these works pave the way for further design of inhibitors against this enzyme and the development of anti-TB drugs with high selectivity and potent efficacy subsequently. It also provides experimental foundation to proteins interaction models.