Study Of Serine Acetyltransferase From Mycobacterium On Function And Structure

Although tuberculosis （TB） is an ancient disease resulting from infection withMycobacterium tuberculosis （M. tuberculosis）, it remains a great threat to bothindividual and public health throughout the world. The prevalence of humanimmunodeficiency virus （HIV） has enhanced the spread of multi-drug resistant （MDR）and extensively drug resistant （XDR） tuberculosis strains, and the morbidity andmortality of TB have been rising yearly. Therefore, it is a matter of urgency to discovertargets for new anti-TB drugs. L-cysteine is essential amino acid for the synthesis ofproteins, secondary metabolites, coenzymes, and related compounds. The biosynthesisof L-cysteine is usually synthesized in two steps in microorganisms and plants. The firststep involves the enzyme serine acetyltransferase （SAT/CysE）; which catalyzes theformation of O-acetyl-L-serine （OAS） from L-serine and acetyl CoA. This reactionwith serine acetyltransferase is the first step in the two-step biosynthesis of L-cysteinein both microorganisms and plants. The second step involves O-acetylserinesulfhydrylase which catalyzing the formation of L-cysteine from OAS and sulfide.Because of the differing pathways for cysteine anabolism in humans andmicroorganisms, serine acetyltransferase exists only in microorganisms. Since an idealdrug target should be unique to the pathogen, so M. tuberculosis serine acetyltransferaseis considered as a potential drug target.The objectives of our study are:（1） to determine the function of serineacetyltransferase from Mycobacterium smegmatis （M. smegmatis）. To confirm the geneof M. smegmatis serine acetyltransferase and characterize CysE as an anti-TB drugtarget and look for more potential drug target. To detect on the effects of growth andmetabolism from M. smegmatis CysE knockdown strain.（2） To determine the functionof serine acetyltransferase from M. tuberculosis. Firstly, to understand the functiondomain and structure model. Secondly, to optimize M. tuberculosis CysE and study theenzymatic properties for establishing a molecular model of high throughput screening （HTS） CysE inhibitors. At last, to identify active site of M. tuberculosis CysE forenzyme inhibitors.Followings are the experimental methods and results we got in this study:1. Cloning, expression, purification, and characterization of M. smegmatisMSMEG₅947protein.MSMEG₅947was amplified from M. smegmatis mc2155genomic DNA bypolymerase chain reaction （PCR）. Cloning plasmid of pMD18-MSMEG₅947wasconstructed. After sequencing, the constructed plasmid pET16-MSMEG₅947andpCold-MSMEG₅947were transformed into E.coli BL21（DE3）. The expression ofMSMEG₅947/BL21（DE3） was detected by SDS-PAGE and Western Blotting.Soluble MSMEG₅947protein were purified by Ni-NTA affinity chromatography anddetected by SDS-PAGE and Western Blotting. The serine acetyltransferase activity ofMSMEG₅947protein was identified using DTNB （Ellman）.2. Preparation of M. smegmatis mc2155MSMEG₅947KOT strain and the effect ofgene knockout.（1） Preparation of M. smegmatis mc2155MSMEG₅947KOT strainM. smegmatis MSMEG₅947gene with its upstream region （696bp） was amplifiedfrom mc2155genomic DNA. The PCR product was purified and cloned into pMD18-Tvector to generate a plasmid of pMD18-RP-MSMEG₅947. Kan resistance fragmentwas linked into MSMEG₅947gene to produce mutant gene （MSMEG₅947:: KanR）from plasmid pUC4K. The MSMEG₅947:: KanRgene was ligated into pPR27-xylEplasmid construct a conditional replication plasmid pPR27-xylE-MSMEG₅947:: KanR.The cysE gene was amplified by PCR, and cloned into pMD18-T. After sequencing,it was cloned to the plasmid pET23b. The cysE gene with promoter was cloned intorescue plasmid pCG76to generate plasmid pCG76-Phsp60-cysE.The plasmid of pPR27-xylE-MSMEG₅947::KanRwas electroporated into M.smegmatis. The transformants was forced to occur the first homologous recombinationbetween MSMEG₅947::kanRof the pPR27-xylE-MSMEG₅947::KanRandMSMEG₅947of genom e at42°C. MSMEG₅947::KanRas well as the sacB gene,xylE gene was integrated into the genome. The first homologous recombination strain（mc2155MSMEG₅947SOC-1） was screened by the PCR and southern hybridization.The rescue plasmid of pCG76-Tb MSMEG₅947was electroporated to M.smegmatis mc2155MSMEG₅947SCO-1cells. The genR, sacB, xylE andMSMEG₅947genes will be deleted from the genome of mc2155MSMEG₅947SCO-1 when the second single crossover event occured under selection of10%sucrose. M.smegmatis mc2155MSMEG₅947KOT （SM-ΔM₅947stain） was generated. SM-ΔM₅947stain was screened by the PCR and southern hybridization.（2） The growth curve of M. smegmatis MSMEG₅947knockout strainThe growth curves of the mc2155MSMEG₅947KOT strain were obtained at both30C and42C. The growth of mc2155MSMEG₅947KOT strain is same as that ofwild type strain at30°C. But the growth of mc2155MSMEG₅947KOT strain isslower than that of wild type strain at42°C. Thus the MSMEG₅947gene has the effecton the growth of mycobacteria.（3） Morphological characterization of M. smegmatis MSMEG₅947knockout strainusing electron microscopeThe morphology of mc2155MSMEG₅947KOT strain grown at42°C wasobserved with both scanning electron microscope and transmission electron microscope.Compared to the wild type strain, morphology of mc2155MSMEG₅947KOT strain inlogarithmic phase had obvious changes. The mc2155MSMEG₅947KOT strain hadintumescent termination and had a lot of vacuoles in the intracellular. Thus theMSMEG₅947gene has the effect on the morphology of mycobacteria.（4） Analysis of proteome from M. smegmatis MSMEG₅947knockout strainAnalysis of the proteome of M. smegmatis MSMEG₅947knockout strain using thetwo-dimensional electrophoresis. Detected protein spots were analysised using Image Jsoftware. The spots were identified by MALDI-TOF mass spectrometry and MatrixScience-Mascot database according to each Peptide Mass Fingerprinting （PMF）.The proteins related to Tb CysE were involved to metabolic route by bioinformatics.3. Homology model of Mycobacterium tuberculosis serine acetyltransferaseUsing some bioinformatics software such as ProtParam, Inter-ProScan, PSIPRED,NCBI Conserved Domains database and SWISS-MODEL, the structure of M.tuberculosis serine acetyltransferase was predicted.4. Cloning, expression, purification, and characterization of M. tuberculosis CysEprotein.The expression of M. tuberculosis serine acetyl transferase was using pCold plasmid.The serine acetyltransferase activity was detected by HPLC and DTNB （Ellman）. Thespecific activity of the serine acetyltransferase was10.66±0.44μmol· min-1· mg-1. M.tuberculosis CysE displayed optimal activity at pH7.5and37oC.5. Determination the active site of CysE Mutated plasmids pET29b-cysE-M were constructed and transformed into E. coliBL21（DE3） using inverse-PCR, respectively. Mutated CysE protein was expressed byWestern Blotting. Enzyme assays showed CysE mutants had still acetyltransferaseactivity by HPLC. Enzyme activity of mutant enzyme was decreased using DTNB.Conclusions1. The gene encoded serine acetyltransferase was MSMEG₅947in M. smegmatis andwas not essential gene for M. smegmatis.2. There were some different proteins after serine acetyltransferase gene was knockout.Detected proteins were related to the energy metabolism and protein synthesis ofmycobacteria.3. Serine acetyltransferase of M. tuberculosis predictly had a conservative domain（LβH） related to enzyme activity.4. The aspartate of67positions, the histidine of82and117positions from serineacetyltransferase is related to the activity of enzyme.Future research:1. To further to screen and identify different protein of serine acetyltransferase geneknockout strain using two-dimensional electrophoresis;2. Determinations of differences of serine acetyltransferase gene knockout strain atthe transcriptional level using Real Time PCR.3. The use of high-throughput screening, directional design method for M.tuberculosis inhibitor CysE inhibitors.4. To discover inhibitors of M. tuberculosis CysE by high throughput screening orby directly designing inhibitor method.5. Screening more active sites using site-directed mutagenesis and identifiedkinetic characteristics of serine acetyltransferase mutation to determine theeffect of active site.