Study Of S-adenosylhomocysteine Nucleosidase Of Escherichia Coli And Drug Virtual Screening

Drug resistance of pathogenic bacteria has attracted wide broad attention.Because of its prevalence in most bacterial pathogens of humans,attention has turned to the enzyme as a target for the development of drugs to selectively block microbial methyl metabolism.S-adenosylmethionine(SAM)is an important nucleoside that serves as an activated group donor in a broad array of bacterial metabolic and biosynthetic reactions.S-adenosylhomocysteine(SAH)and methylthioadenosine(MTA),byproducts of SAM-dependent methylation reactions and polyamine biosynthesis,both are product inhibitors of SAM-involved reaction.The function of SAHN is to cleave the glycosidic linkage of SAH to generate S-ribosylhomocysteine(SRH).SRH is further cleaved by the enzyme Lux S to yield homocysteine,which can be remethylated to methionine.SAHN functions at two vital steps in bacterial pathways related to polyamine biosynthesis,quorum sensing,methylation,and purine and methionine salvage reactions.Many microbial pathogens utilize a single SAHN to hydrolyze both MTA and SAH to adenine and the corresponding thio-sugar,methylthioribose(MTR)and S-ribosylhomocysteine(SRH),respectively,whereas in mammalian cells,SAH and MTA are catabolized by SAH hydrolase(SAHH)and MTA phosphorylase(MTAP)in reversible reactions.Therefore,SAHN is used to study the potential for this enzyme to serve as a target for chemotherapeutic intervention.Targeted inhibition of the breakdown of these regulatory nucleosides in microbial pathogens would therefore exert selective deleterious effects.In this paper,we construct a deletion mutant of sahn gene of the E.coli MG1655 origin using the Red/ET recombination system.The coding sequence of the sahn gene was replaced with the coding sequence of kanamycin resistance cassette via a double crossover event.To remove the DNA fragment relevant to the kanamycin-resistance,a Cre recombinase-encoding was introduced into the recombinants,which resulted in a single Lox P site within the targeted genomic segment.The mutant strain was named ?sahn.At the same time,we amplified the sahn gene using the PCR method to construct the pET-28a-sahn vector,with the genome of E.coli as a template.Protein expression was induced by IPTG(isopropyl-?-d-thiogalactoside)and purified using Ni–NTA column.The concentration of the purified SAHN was determined to be 368 ?g/mL by coomassie blue staining measurement.And the Vmax and Km values of enzymatic reaction were calculated.Through the studies on different levels of the SAHN expression,the quorum sensing of E.coli including the secretion of AI-2 and the biofilm development are found to be apparently affected.Finally,we simulated the interaction of the small molecule inhibitors and the receptor protein SAHN with the molecular docking method.Interaction between ligand and receptor is a molecular recognition process,including electrostatic interaction,hydrogen bonding,hydrophobic effect,Van der Waals? force,etc.Through calculation,we could predict the affinity for the virtual screening of drugs,which provides us with theoretical and experimental bases for novel SAHN-targeted antibiotics.