Thiamin Pyrophosphate Depend On The Mechanism Of Enzyme Catalysis Research

Thiamin pyrophosphate (ThDP) could act as a cofactor to mediate a variety of reactions in the metabolism of living organisms. These reactions mainly involves the making and breaking of carbon-carbon bonds adjacent to a carbonyl group. Since this class of enzymes all need ThDP as a cofactor, they are called ThDP-dependent enzymes. This family includes many key enzymes in various biological pathways, such as pyruvate decarboxylase (PDC),1-deoxy-D-xylulose5-phosphate synthase (DXS), acetolactate synthase (ALS) and transketolase (TK).DXS is the first key enzyme of the newly found2-methyl-D-erythritol4-phosphate (MEP) pathway of terpenoid biosynthese and it is also involved in the biosyntheses of thiamin (vitamin B1) and pyridoxol phosphate (vitamin B6). Under the catalysis of DXS, the first intermediate of MEP pathway,1-deoxy-D-xylulose5-phosphate (DXP), is biosynthesized via the condensation of pyruvate and D-glyceraldehyde3-phosphate (D-GAP). The catalytic mechanism of this reaction has not been fully elucidated, which becomes a major barrier for screening new antibiotics and antimalarial drugs targeting DXS. Therefore, we explored the reaction nature of the condensation of pyruvate and D-GAP using isotope-labeling experiments.PDC, also a member of ThDP-dependent enzymes, catalizes the non-oxidative decarboxylation of pyruvate and gives acetaldehyde and CO2, and this step is crucial for the production of ethanol from pyruvate. We also investigated whether PDC employs the similar mechanism as DXS.Firstly, we carried out large-scale preparation of DXS, then we take the protein as a tool to study its catalytic mechanism by using isotope-labeling experiments and analyzing the final product DXP by1H-NMR and LC-ESI-MS. Based on the data, we deduce that the reaction intermediate hydroxyethyl thiamin pyrophosphate (HEThDP) ought to exist in three different resonance forms, namely the so-called C2a-carbanion, enamine and what we found, the enol. Furthermore, since all ThDP-dependent enzymes share a common model of catalytic mechanism, we testified whether the intermediary of DXS reaction is applicable to other ThDP enzymes. Thus, we chose PDC as target and by the preliminary data, we may conclude that the new mechanism we found in DXS reaction could also be suitable for PDC.