Cloning And Expression Of HMGS And HMGR From Streptococcus Pneumoniae And Their Function Study

Streptococcus pneumoniae is the major pathogenic bacteria causing severe infections such as meningitis,septicemia and pneumonia.In recent years,S.pneumoniae has acquired resistance to penicillin and macrolides antibiotics and has an increasing trend year-to-year due to the environmental pollution and the usage or un-appropriate usage of large quantity on clinical treatment,while the multi-drug resistance of the other antibiotics emerges.Therefore,it is essential to seek for new target in organism metabolism and exploit new antibiotics with homology modeling.The mevalonate pathway is essential for the survival of S.pneumoniae and,by inference,for other gram-positive bacteria.The key enzymes of the mevalonate pathway may therefore represent potential targets for the development of new antibiotics.In this study,HMG-CoA synthase(HMGS) and HMG-CoA reductase (HMGR) of S.pneumoniae were chosen as targets in mevalonate pathway.The mvaS and the mvaA genes were cloned from S.pneumoniae and expressed in E.coli BL21.The recombinant HMGS and HMGR were conducted analysis of dynamics and research of function.Meanwhile,the 3D models about HMGS and HMGR were established,and the candidate inhibitors are acquired according to the HMGR homology modeling of S.pneumoniae.We had succeeded in screening some candidate inhibitors using the recombinant protein of S.pneumoniae.The main results are listed as follows:1.The mvaS was cloned from S.pneumoniae.In comparison with the mvaS sequence of S.pneumoniae published in NCBI(AF290098),there were 98.5%homology and eighteen nucleotides have been changed,resulting in two changed amino acids(Asn259→Ser, Ala349→Val).The recombinant expression plasmid pET-HMGS contained mvaS was not expressed.DNA sequencing analysis showed that a termination codon was occurred because of the nucleotide mutated(A226→T).The fragments of the mvaS gene were amplified to conduct back mutation.The recombinant expression plasmid pET-HMGSm was constructed and expressed successfully.The HMGS of S.pneumoniae was consisted of 398 amino acid residues.2.The recombinant plasmid pET-HMGSm was expressed with 0.4 mM IPTG induction for 4h at 18℃,the product analyzed by SDS-PAGE showed that a specific protein band appeared with a molecular weight about 46 kDa.Optimal conditions for recombinant HMGS were pH 9.75 and 10 mM MgCl₂ at 37℃.The specific activity of the soluble crude extract was 0.76μmol/min/mg, while it increased to 3.24umol/min/mg after purified by HisTrapTM FF crude 1ml column.The Vmax and Km were 4.69μmol/min/mg and 213μM,respectively,under pH 9.75 and 5 mM MgCl₂ at 37℃.3.The mvaA gene was amplified by PCR from S.pneumoniae.In comparison with the mvaA sequence of S.pneumoniae in NCBI(AF290098),there were 99.9%homology and seven nucleotides have been changed resulting in three changed amino acids(Glu44→Val, Asn46→Asp,Gly72→Glu).The HMGR of S.pneumoniae was consisted of 424 amino acids.The recombinant expression vectors pET-HMGR was established and expressed with 1mM IPTG induction at 30℃.The recombinant protein was purified by Ni-NTA affinity chromatography,and the high activity HMGR(about 47kDa) was obtained.Activity was optimal at pH 6.5 and approximately 37℃.The specific activity of the crude extracts and the purified by the Ni²⁺-NTA were 11.22μmol/min/mg and 31.98μmol/min/mg,respectively.The Vmax and Km value determined by the reaction of HMG-CoA to mevalonate were 62.1μmol/min/mg and 260μM, respectively.New Zealand rabbits were immunized with the purify HMGR protein.Antibodies against purified HMGR were raised in rabbit.ELISA assay showed that the titer of these antibodies was up to 1:320,000,and western blotting further confirmed the immunology characteristic of HMGR.4.The homology models of the S.pneumoniae HMGS and HMGR were established based on structure templates of Enterococcus faecalis HMGS and Pseudomonas mevalonii HMGR by composer module of SYBYL7.0 program.Currently,it is a new research area using computer to analyze three-dimensional structure and using homology modeling to search for structural analogues,and then develop new drugs.In this study,some candidate inhibitors were acquired according to the three-dimensional dimmer structure from S.pneumoniae HMGR using Grid Computing Technology and Molecule Docking.5.It was established that the biological activity of novel hits come from virtual screening was test by the expressed HMG-CoA reductase of S.pneumoniae.According to the above method,we have screened out one better inhibitor with the K_i of 76μM from 30 kinds of candidate inhibitors, while Lovastatin was K_i 353μM.New drug may be obtained successfully based on further study.6.Lovastatin was a kind of competing inhibitor targeted for HMG-CoA reductase,but it did not inhibit HMG-CoA synthase.The virtual screening was limited because of the high cost of the substrate HMG-CoA.It was more effective in the virtual screening with the reacting product catalyzed by the recombined protein HMGS instead of HMG-CoA.Therefore,the product can screen preliminaryly plentiful candidate inhibitors and develop new inhibitors with more specific and stronger function.There will be of a broad prospect and enormous economy benefit once novel antibiotics for the HMGS and HMGR are obtained.