GC-MS Study DXS Triosephosphate Isomerization Activity Mechanism

2-Methyl-D-erythritol4-phosphate (MEP) isoprenoid biosynthetic pathway has stimulated widespread interest in recent years. This biosynthetic pathway, comprised of seven enzymatic steps, is an alternative biosynthetic route for natural isoprenoids present in many bacteria, higher plants, and the malarial parasites except human beings. Thus it has become an attractive target for the development of new antimalarial and antibacterial compounds. But the mechanism studies of the pathway specific enzymes are not incisive, which seriously hindered the searching of new antimicrobial compounds.1-Deoxy-D-xylulose-5-phosphate synthase (DXS) is the first key enzyme of the MEP pathway that catalizes the condensation between D-glyceraldehyde3-phosphate (D-GAP) and pyruvate to form1-deoxy-D-xylulose-5-phosphate (DXP) in the presence of thiamine diphosphate and a divalent metal ion. In the previous studies of this group on E. coli DXS (EcDXS), it was found that DXS can also catalyze the interconcersion between the two natural triosephosphate, namely D-GAP and dihydroxyacetone phosphate (DHAP), an specific reaction mediated by triosephosphate isomerase (TPI).In this thesis, paeonol, a natural phenolic compound with antibacterial nature and its derivatives were assayed to determine if they possess the inhibitory activity on DXS. The results showed that one compound exhibited fairly good inhibition on DXS at a concentration of0.1mg/mL.Furthermore, we also tried to explore the mechanism of the triosephosphate isomerization reaction catalyzed by DXS by using silanization-GC-MS method. Because D-GAP and DHAP are two sugar phosphates, they do not have volatility. Thus they can not be analyzed directly by GC-MS process. In this thesis, we planned to establish proper derivatization methods, such as silanization and acylation so that both the compounds could be measured on GC-MS. Based on this method, we could characterize the protolysis process during the isomerization of the two triosephosphates in the reaction catalyzed by DXS with in-situ deuterium labeling technique. Silanization of DHAP and D-GAP were conducted with the addition of bis(trimethylsilyl)trifluoroacetamide (BSTFA).We also primarily confirmed the detective method on GC-MS based on optimizing the reaction time and temperature. Using this method,We detected siliconalkylation products in samples of D-GAP isomerization.We also found that the resolution of D-GAP and DHAP was higher by LC-MS,at losting phosphate groups and derivatived into phenylhydrazone.So we intend to decode DHA and D-GA to study siliconalkylation and acylation of them, further research is in progress.