Clonning, Expression, Characteristics And Inhibitor Screening Of The Hmgr From Streptococcus Pneumoniae

Streptococcus pneumoniae is the major causative organisms of meningitis, bacteremia,and pneumonia.Resently,for the Environmental pollution the excess use of antibiotics,the drug resistance of S.pneumoniae to antibiotics showed ascending tendency.Hydroxymethylglutaryl-coenzyme A reductase(HMGR) is the rate-limiting enzyme in the classical mevalonate pathway,which converts HMG-CoA to mevalonate, the rate-limiting step in isopentenyl diphosphate(IPP) biosynthisis.The present study cloned the full-length HMG-CoA reductase(HMGR) gene of S.pneumoniae,and expressed in E.coli BL21(DE3).The recombinant protein was purified by Ni-NTA affinity chromatography and obtained the high activity HMGR.Activity was optimal at pH6.5 and approximately 37℃The specific activity of the crude extract and the Ni²⁺ -NTA were 11.22 U/mg and 31.48 U/mg respectively.The enzyme catalyzes the normal anabolic reaction using NADPH not NADU as a reductant,the V_maxand K_m of the reductive deacylation of HMG-CoA to mevalonate were 62.1U/mg and 260μM respectively.Lovastatin inhibited competitively with HMG-CoA with the K_i362μM. New Zealand rabbits were immunized with the purify HMGR protein.The antiserum was detected by ELISA and the titer of these antibodies was up to 1:320,000.The specificity of these antibodies against HMGR was further confirmed by western blotting.Comparison of the sequence of HMGR has revealed the existence of two classes of this enzyme,the classⅠenzymes found in eukaryotes and some archaea,and the classⅡfound in certain eubacteria and the archaea.Statins represent a kind of excellent competing inhibitor targeted for the classⅠHMGR,but were relatively poor to the classⅡenzyme of important bacterial pathogens.Potent HMGR inhibitors with Ki values in the nM range to the classⅠenzyme,but in theμM range to the classⅡenzyme,it is high 10³-10⁵ folders.In order to develop new inhibitors with more specific and stronger function targeted for the classⅡHMGR,the 3D model of S.pneumoniae HMGR was built based on structure template of HMGR of Pseudomonas mevalonii by homology modeling using composer module of SYBYL7.0 program.Acoding to the homology modeling of S.pneumoniae,we screen out one better inhibitor with the ki of 76μM from 30 kinds of inhibitors.Currently,using computer to analyze three-dimensional structure and using homology modeling to search for structural analogues and then develop new drugs is a new research area.