Studies On1-deoxy-d-xylulose-5-phosphate Synthase And1-deoxy-D-xylulose-5-phosphate Reductoisomerase, The Two Key Enzymes In MEP Pathway For Terpenoid Biosynthesis

Terpenoids are a class of natural products which are widely present in various organisms and play key roles in all forms of life. Two routes have been found for the biosynthesis of terpenoids:the classic mevalonate (MVA) pathway and the newly established2-methyl-D-erythritol4-phosphate (MEP) pathway. Because the MEP pathway is operative in human pathogens but not in human, all the key enzymes of the pathway are considered good targets for the screening of antibiotics.1-Deoxy-D-xylulose-5-phosphate synthase (DXS) is the first key enzyme in the MEP pathway. It is generally accepted that only D-glyceraldehyde-3-phosphate (D-GAP) can be utilized as a natural substrate by DXS to synthesize1-deoxy-D-xylulose-5-phosphate (DXP) in the presence of thiamine pyrophosphate (ThPP) and pyruate, whereas dihydroxyacetone phosphate (DHAP), the isomer of D-GAP, is not convertible without triosephosphate isomerase (TPI). We have found, however, that DHAP is also a substrate of Escherichia coli DXS (EcDXS) in the production of DXP and that EcDXS exhibits a function similar to TPI.To verify this unusual result and to completely eliminate the possibility that the isomerization activity of DXS was from the co-purified TPI, we reprepared EcDXS by using TPI gene knock-out E. coli strain FB21547(DE3) and tested its TPI activity. The reprepared EcDXS also exhibited TPI activity. Thus, the possible contamination from TPI was totally washed out. We used Michaelis-Menten kinetic analysis to further characterize the TPI activity of EcDXS (Km D-GAP=1.48mM and KmDHAP=6.31mM). We also assessed the influences of pH value, temperature and metal ions on the isomerization activity of EcDXS and the results showed that the highest enzyme activity occurs in the presence of Mg2+at a pH range of7.0-8.5and a temperature of30-40℃. If Mg2+was omitted, EcDXS did not exhibit any TPI activity. We used1H-NMR to trace the isomerization process catalyzed by EcDXS and discussed its possible mechanism.In addition, we prepared two other DXSs, such as Rhodobacter apsulatus DXS and Pseudomonas aeruginosa DXS to further clarify the TPI activity of the protein. The results showed that both DXSs exhibited activity on the interconversion of D-GAP and DHAP. We could definitely conclude that DXS exhibits a function similar to TPI. Because D-GAP and DHAP play important roles in various metabolic pathways, such as the glycolytic pathway, gluconeogenesis and pentose phosphate pathway, it may also imply that DXS serves as the intersection point between the MEP pathway and the sugar metabolism pathways in a large subset of microorganisms.As one of the most promising targets for screening of antibiotics,1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) which catalyzes the rearrangement of DXP to MEP through a divalent cation and NADPH dependent isomerization-reduction sequence has drawn a lot of attention. Up to date, two main catalytic mechanisms of the protein have been proposed and the retroaldol-aldol procedure has been finally accepted. However, some discrepancies of the retro-aldol/aldol mechanism for DXR need further interpretation.18O isotope exchange experiments are one of the effective methods for determination of reaction mechanisms. We prepared18O labeled MEP and after detecting18O labelling pattern and abundance of the product by ESI-MS and13C-NMR, we analyzed the conversion of DXP to MEP mediated by DXR in detail.After conclusive analysis of the experiments, we verified the C3-C4substrate binding mode in which DXP chelates with DXR-bound divalent cation via its hydroxyl groups at C3and C4. Based on this binding mode and early results, a catalytic cycle for the enzyme DXR during the conversion of DXP to MEP is suggested. Taking the new binding fashion of DXP and the catalytic cycle of DXR, the issues on the mechanism of DXR can be well interpreted in the frame of the accepted retroaldol-aldol sequence. Furthermore, the way of L-ribulose-5-phosphate (L-Ru5P) binding to L-ribulose-5-phosphate4-epimerase which mediates the interconversion of L-Ru5P and D-xylulose-5-phosphate through similar retroaldol-aldol mechanism is discussed.The results of this paper will contribute to a comprehensive understanding of the catalytic mechanism of DXS and DXR, and provide a reasonable basis for screening new antibiotics with these two enzymes as targets.