Cloning, Expression And Characterization Of Glucosamine-6-phosphate Synthase From Mycobacterium Tuberculosis

Tuberculosis is one of the leading infectious disease.Multidrug-resistant tuberculosis (MDR-TB)and extensively drug-resistant TB(XDR-TB)was reported in all regions of the world and classified rapidly by WHO as a serious emerging threat to public health,especially,but not only, in countries with a high prevalence of the human immunodeficiency virus(HIV).There is urgent need for promoting research and development of new drugs target.Bacterial infection and fungous infection incidence also rised.So there is urgent need for developing low virulence which could treat multidrug-resistant and extensively drug-resistant pathogenic bacterium antibiotics.Standard of life improved,various kind of disease,such as hyperglycemia evocable diabetes and chronic syndrome obesity,hyperinsulinemia,hyperlipoidemia,etc,insulin resistance syndrome.Because these diseases can cause people die,so they become one of three recalcitrant disease.Glucosamine-6-phosphate Synthase,This enzyme catalyses reaction:L-glutamine+ D-fructose-6-phosphate→D-glucosamine-6-phosphate+L-glutamate,the reaction involving ammonia transfer and sugar phosphate isomerisation.This irreversible reaction is the first committed step in hexosamine biosynthesis,leading to the formation of uridine 5'-diphospho-N-acetyl-D-glucosamine, an activated precursor of numerous amino sugar-containing biomacromolecules, including chitin and mannoproteins in fungi,peptidoglycan and lipopolysaccharides in bacteria,and glycoproteins in mammals.GlcN-6-P synthase is an important point of metabolic control of amino sugar biosynthesis,the mammalian enzyme is supposed to be involved in phenomenon of hexosamine-induced insulin resistance in diabetes.So Glucosamine-6-phosphate Synthase has been proposed as a new molecular target for antibacterial,antifungals and treating diabetes.The genes encoding glmS were amplified from Mycobacterium tuberculosis genomic DNA,the PCR products were cloned into pET32a(+)vector,pET32-glmS were constructed successfully.The recombinant plasmids were transformed into competent E.coli BL21(DE3).We study Overexpression glucosamine 6-phosphate synthase effect host strain.Including:①Overexpression glucosamine 6-phosphate synthase effect host strain's growth;②Overexpression glucosamine 6-phosphate synthase effect host strain's cell wall;③Overexpression glucosamine 6-phosphate synthase effect host strain's vancomycin-resistant;④Overexpression glucosamine 6-phosphate synthase effect host strain's Cell wall integrity.As a result,Overexpression glucosamine 6-phosphate synthase accelerate host strain's growth,effect host strain's cell wall forming and integrity,we added detergent sodium dodecyl sulphate(SDS)for help to sonicated recombinant BL21(DE3)cells,and the purified fusion proteins.Because SDS can make proteins denaturalization, so we not detect enzymatic activity in vitro.But before-mentioned experimental result prove overexpression glucosamine 6-phosphate synthase take on enzymatic activity in vivo.The results provide a basis for further validation its drug target value for drug discovery.