Study On Key Amino Acid Residues Of DXS In Escherichia Coli

The 2-methyl-D-erythritol4-phosphate (MEP) pathway is the only terpenoid biosynthetic pathway in most of microorganisms (including human pathogenic bacteria), but do not exist in human. Therefore, it is an ideal target in search for new antibiotics. 1-Deoxy-D-xylulose 5-phosphate synthase (DXS) is one of the key enzymes in the MEP pathway. It catalyzes the rate-limiting step of this alternative route and is also an important enzyme in the biosynthesis of vitamins B1 and B6 in some bacteria. Hence, DXS plays important roles in the three pathways.At present, there is little research about the DXS catalytic mechanism. Thus it is of great importance to study the amino acid residues of the active sites of DXS to unravel its mode of action. Based on the crystallography data of DXS and docking experiments, we chose the following 9 critical residues, namely Ser44, His49, Arg99, Lys101,Val259, Tyr392, Arg420, Asp427, and Arg478 of the enzyme to start out work. Firstly, the mutated genes were obtained through site-directed mutagenesis, then the plasmids carrying the genes were transformed into E. coli BL21 DE(3) competent cells, finally the mutated enzymes S44D-DXS, H49Q-DXS, R99N-DXS, K101D-DXS, V259E-DXS, Y392K-DXS, R420N-DXS, D427K-DXS, D427N-DXS and R478E-DXS were prepared, respectively.We subsequently measured the activity and steady state kinetic parameters of these mutants and compared them with the ones of the wild type enzyme. The results demonstrated that S44D mutation leads to DXS activity loss; H49Q, K101D, Y392K and D427K mutations had key effects on DXS activity, R99N, R420N and D427N mutations also had significant impacts on DXS activity, whereas V259E mutation had little influence. Compared with WT-DXS, the activities of S44D, H49Q, K101D, Y392K, D427K, R99N, R420N, D427N and V259E mutants were lowed to 0.58%,4.98%,8.97%,8.15%,8.73%, 27.78%,19.17%,52.75% and 84.04% respectively. We also evaluated the activities of these mutations in the condensation reaction between pyruvate and nitrosobenzene, an analogue of D-GAP and the data exhibit that the mutants S44D-DXS, H49Q-DXS, R99N-DXS, K101D-DXS, D427N-DXS and R478E-DXS possess almost same activity as that of the wild type enzyme, while the mutants Y392K-DXS and D427K-DXS displayed clearly decreased activity. The study can not only help understand DXS catalytic mechanism but aslo offer reasonable ground for novel antimicrobials and antimalarials screening.