Enzymes are fundamental players in regulating cell cycle, developmental stages and cellular metabolism inside the living organisms. Targeting enzyme subunits either regulatory or catalytic that bring profound effect on enzyme performance would result in regulation (inhibition or activation) of a particular reaction. It would also render the subsequent pathway to be blocked or initiate. The characterization of essential enzymes from pathogens via recombinant DNA technology lie at the heart of drug discovery process.
In the current study, we focused on characterization of two enzymes phosphoribosylformylglycinamidine (FGAM) synthase (purS) and oxidoreductase. For this, gene for oxidoreductase and phosphoribosylformylglycinamidine synthase (purS) from VRSA (Mu50) were cloned in expression vector pSpeedET and transformed in to E. coli DH5α cells. Positive clones were screened via colony PCR and DNA sequencing for confirmation of in-frame insertion of the desire gene. Recombinant plasmids with 100% correct sequence from positive clones were transformed in E. coli BL21 (DE3) cells for expression, induced by IPTG. Affinty and size exclusion chromatographic techniques were used to purify the expressed proteins. NMR experiments of the purified protein were performed on 800 MHz NMR spectrometer, coupled with cryogenic probe. Circular Dichroism was performed to assess the secondary structure elements of purified proteins. Matrix Assisted Laser Desorption/Ionization technique was used to analyzed the molecular masses of proteins. Oxidoreductase was also functionally analyzed.
Our findings developed an indigenous method of cloning, expression and purification of two essential enzymes with high yield and purity. Both enzymes contain well-folded patterns in solution state NMR, indicating them as good protein targets with structural point of view. The results of CD revealed that the enzymes contain β-sheets and α-helices in their structures. Whereas, results of MALDI experiments confirmed the molecular weight of proteins after His-tag removal.
This work can be helpful in discovering novel small molecules as drug therapy upon its submission to Protein Data Bank.