?. Studies On DXR Inhibition Of Tea Polyphenol GCG ?. Studies On The Key Amino Acid Residues Of DXS

The 2-methyl-D-erythritol-4-phosphate(MEP)pathway for the biosynthesis of terpenoids has become a new target for people to find antibacterial drugs,because this pathway is lacking in animals and human beings.In the MEP pathway,the first two reactions are considered to be key rate-limiting reactions: the condensation of D-glyceraldehyde-3-phosphate(D-GAP)with pyruvate catalyzed by 1-deoxy-D-xylulose-5-phosphate synthase(DXS)and the conversion of 1-deoxy-D-xylulose-5-phosphate(DXP)to MEP mediated by DXP reductoisomerase(DXR).Therefore,the studies of DXS and DXR are extremely important for screening new antibacterial drugs.In the first part of the thesis we investigated the inhibitory effect of the polyphenolic compound gallocatechin gallate(GCG)at cellular level.Early research has shown that tea extracts can inhibit the growth of various bacteria and the active components are catechins.In our previous research we found that GCG isolated from tea exhibits specific inhibition against DXR in vitro.In order to further disclose whether the antibacterial activity of tea is due to the suppression of DXR by catechins at the cellular level,we selected in this thesis four bacterial strains,including wide type Rhodobacter sphaeroides and other three engeneered E.coli strains such as E.coli-BW-Lycopene,E.coli-BL21DE(3)-PET15 b,and E.coli-BL21DE(3)-PET15b-EcDXR)to carry out the experiments.We added GCG to the culture media of these four strains,respectively and after incubation we found that GCG could not effectively inhibit the growth of these four bacteria.Further determination indicated that the addition of this compound could not decrease the production of lycopene(a terpene compound)in both R.sphaeroides and E.coli-BW-Lycopene strain either.The results showed that although GCG has good DXR inhibitory activity in vitro,it can not exert corresponding inhibitory effect at cellular level.In the next part,we carried out experiments to study the catalytic details of DXS which is another rate-limiting enzyme of the MEP pathway.At present,there is not much research on the catalytic mechanism of DXS.Thus,it is quite necessary to study the amino acid residues of the catalytic center of DXS to further understand its catalytic mechanism.Some researchers have found that EcDXS mutant EcDXS-H49 Q totally loses its catalytic activity.Based on this mutant,we further mutated its aspartic acid residue at position 427 into 8 different amino acids by site-directed mutagenesis,respectively.Subsequent measurements of the activities of these mutants showed that three double-point mutants,namely EcDXS-H49Q/D427 H,EcDXS-H49Q/D427 K,and EcDXS-H49Q/D427 R which were mutated separately from aspartic acid to basic amino acids exhibied different degrees of activity recovery.The best mutant obtained was EcDXS-H49Q/D427 H whose activity was 104.13% of that of wild-type EcDXS(WT-EcDXS).The molecular docking study on EcDXS-H49Q/D427 H with substrates showed that histidine played an important role not only in helping proton transfer,but also in the process of the binding of the substrates to the catalytic center of the mutant enzyme.There seems to be a special relationship called “backup” between the sites 49 and 427 of WT-EcDXS.The change in the distribution of the basic amino acids in the active center caused by the mutation may result in a change of the binding mode of substrates to amino acid residues in the active site,and a decrease in the tightness of the binding between substrates and the enzyme,leading to a decrease in the catalytic efficiency of EcDXS-H49Q/D427 H.To further test the activities of the obtained mutants,we used glyceraldehyde as the acceptor substrate instead of D-GAP to carry out the reaction.It was found that the mutant EcDXS-H49Q/D427 H showed similar catalytic activity as WT-EcDXS,but the rest of the mutant enzymes could not catalyze the reaction.This data indicated when glyceraldehyde and D-GAP are used as acceptor substrates for DXS,their binding patterns are likely to be consistent.When replacing D-GAP with benzaldehyde or nitrosobenzene as an acceptor substrate,none of the eight mutant enzymes showed catalytic activity.