.0-18 Mark 1 - Deoxy-d-xylulose 5 - Phosphate Synthesis

With the discovery and establishment of the 2-methyl-D-erythritol-4-phosphate (MEP) pathway for isoprenoid biosynthesis, the enzymes of it have already been useful tools for screening potential antibiotics and anti-malaria drugs. Hence, the study of key enzymes and key intermediate 1-deoxy-D-xylulose 5-phosphate (DXP) are attracting increased interest nowadays. DXR (1-Deoxy-D-xylulose 5-phosphate reductoisomerase) is the rate-limiting enzyme of this pathway and the most promising target for new antibiotics discovery, but up to date, the mechanism of its catalysis is not really clear. DXP is the natural substrate of DXR. Therefore, the synthesis of isotope-labeled and unlabeled DXP is of practical significance in the study of the mechanism of DXR and form new drug screening. In this study, we explored the preparation of 0-18 labeled DXP with enzymatic method. And also, we tried to find a more practical and feasible way to synthesis unlabeled DXP.Firstly, we prepared Rhodobacter capsulatus DXS by using E. coli BL21 (DE3)-DXS cells. And then we used affinity chromatography and gel exclusion chromatography to purify the product. The function of DXS was subsequently tested. As a result, we obtained DXS with up to 90% purity and high activity. In the course of the investigation, we ran into a new aspect of catalytic mechanism of DXS which hasn't been reported yet and have got some preliminary results. Further study is being carryed out.Secondly, we prepared 0-18 labeled DXP enzymatically and purified the product by ion-exchange chromatography. Both the yield and the purity of the target are satisfactory. By analyzing the reaction procedures and the labeling patterns of 0-18 labeled DXP, we provided more evidence for the newly discovered catalytic activity of DXS.Furthermore, we established a new chemo-enzymatic route to synthesize unlabeled DXP. In this new, "one pot" method, (D/L)-glycidol or (L)-glycidol which is cheap, stable and commercially avaible was used as starting material and DXP was obtained in a relatively big scale and high yield. And now, some experiments are being carried out to optimize it.