1 - Deoxy-d-xylulose -5 - Phosphate Synthase Preparation And Preliminary Exploration Of The Mechanism Of Action

Natural isoprenoid compounds are a class of widely distributing secondary metabolites with most complex structures. Two different pathways of isopronoid biosynthesis have been established to date, one is the classical mevalonate (MVA) pathway, and the other is the methylerythritol phosphate (MEP) pathway. Studies have shown that MEP pathway, which is present in many eubacteria (including human pathogens), green algae, lichens, and higher plants, has not yet been found in human beings, so enzymes of this pathway have been regarded as potential targets for screening of antibiotics, antimalarial drugs and herbicides. 1-deoxy-D-xylulose 5-phosphate synthase (DXS) is the first rate-limiting enzyme of the pathway. It is generally accepted that only glyceraldehyde-3-phosphate (D-GAP) can be utilized as natural substrate by DXS to synthesize DXP in the presence of pyruvate and thiamine pyrophosphate (TPP), whereas dihydroxyacetone phosphate (DHAP), the natural isomer of D-GAP, is not convertable without Triosephosphate Isomerase (TIM).But in our initial investigation on the performance of the enzyme, we found that DHAP is also a direct substrate of DXS of Rhodobacter capsulatus (RcDXS) and DXS of Escherichia coli (EcDXS) in the production of DXP and the enzyme possesses similar function to TIM. To further verify the unexpected result, RcDXS and EcDXS were repurified on a gel filtration column after Ni-affinity chromatography. Isomerization of DHAP or D-GAP were catalyzed by the pure enzymes and detected by pre-column derivatization HPLC and ultraviolet spectrophotometry. Proton transfer and proton exchange of substrates with bulky solvent during the interconversion catalyzed by DXSs were determined by 1H-NMR spectroscopy. Based on all above, a hypothesis for the catalytic mechanism of the DXS-mediated triosephosphate isomerization reaction was put forth, and in-depth research is being carried out.DXP is the natural substrate of DXR (1-deoxy-D-xylulose-5-phosphate reduetoisomerase), the rate-limiting enzyme of MEP pathway. Therefore, synthesis of DXP is of high importance in the study of the mechanism of DXR and screening of new antibiotics. A tandem and an "one pot" enzymatic synthesis of the compound was established in this dissertation in which dihydroxyacetone (DHA), sodium pyrophosphate, and sodium pyruvate were used as substrates, and acid phosphatase, DXS, TIM were used as catalysts. The advantage of this procedure is that the starting materials are stable, cheap and easily available.