Gene Function Analysis Of Escherichia Coli S-adenosylhomocysteine Nucleosidase

S-adenosine methionine (SAM) is the general methyl donor for methylation modification.SAM could be converted S-Adenosylhomocysteine（SAH）by catalysis of methyltransferase.In eukaryotic cells, S-adenosylhomocysteine hydrolase (SAHH) is used to directly convert SAH tohomocysteine. While in a majority of prokaryote, detoxification of SAH is carried out byMTA/SAH nucleosidase to produce SRH. The enzyme LuxS further cleaves SRH tohomocysteine. SAM could be generated polyamine, N-acyl homoserine lactone andmethylthioadenosine (MTA) under the catalyzing of polyamine synthetase and N-acylhomoserine lactone synthetase respectively. In most prokaryotes such as pathogenicmicroorganism, SAHN can crack the MTA to produce adenine and5′-methylthioribose（MTR）, the later is phosphorylated to form5′-methylthioribose-1-phosphate (MTRP) byMTR kinase subsequently. While in eukaryotic cells such as mammals, pathogenic bacteriahost,5′-methylthioadenosine phosphorylase catalyze MTA to generated adenine and MTRP inone step. Then MTRP form methionine (Met) via multistep reactions and Met is regeneratedinto SAM under the action of SAM synthetase, which makes the whole Activated MethylCycle (AMC) complete. There are significant differences between pathogenic microorganismsand eukaryotic cells in terms of the SAH and MTA node of AMC, which makes the SAHNnucleoside enzymes in pathogenic microorganisms become potential targets of anti-infectiondrugs.We first chose the E.coli genome DNA as template for PCR amplification of gene sahn,then the recombinant expression vector pET-28a-SAHN was constructed and transformed intoE.coli BL21. Subsequent we induced SAHN protein expression with IPTG and purifiedSAHN via Ni-NTA chromatography with His-tag recombinant tagged. After that, we used Pallultrafiltration tube to conduct SAHN desalination and concentration and the SDS-PAGE andCoomassie brilliant blue were also used for qualitative and quantitative determination ofSAHN. Finally, we got considerable concentration pure SAHN product. Meanwhile, base onthe V.harveyi BB170strain the recombinant LuxS Enzyme activity was detected successfully.MTA is broken down by SAH hydrolase, which results in the production ofmethylthioribose and adenosine. Adenine is catalyzed to dihydroxy adenine with absobance in 300nm by coupling of xanthine oxidase (XOD).The xanthine oxidase enzyme couplingdetection technique is established based on the above reaction process. The parameters ofreaction kinetics were also determined. Under the condition of37℃, pH7.0, enzymaticreaction Vmaxand Kmvalues were5.4885M·min-1and105.68M, respectively.Finally, the optimum temperature and pH value were studied by using the coupling assayof xanthine oxidase enzyme. The experiment results show that, the optimal temperature forthe enzymatic reaction is37~42℃, and the optimal pH is5.8~6.3. The establishment of theenzyme coupling method and enzymatic reaction analysis of optimum temperature and pHvalue could be of great benefits to the screening and function study of its inhibitors.Great progress has been made on exploring the MTA/SAH nucleosidase (SAHN)inhibitor and developing new broad spectrum antibiotic with the SAHN as a drug target.