Since the discovery of thiamine diphosphate (ThDP, active form of vitamin B1) as active cofactor in 1937 by Lohmann and Schuster, its catalytic mechanism has become an interest of many enzymologist. The ThDP-dependent enzymes catalyze a variety of enzymatic reactions including decarboxylation, condensation, ligation etc. Here is reported a mechanistic study carried out with multiple ThDP enzymes: Escherichia coli 1-deoxy- D-xylulose-5-phosphate (DXP) synthase, E. coli E1 component (E1o) of the 2-oxoglutarate dehydrogenase complex (OGDHc), yeast pyruvate decarboxylase (YPDC) and benzaldehyde lyase (BAL) by using state-of-the art instruments: the circular dichroism (CD) method can monitor events on the enzyme itself followed by pre-steady state rate determination by stopped-flow (SF) CD, the NMR method monitors ThDP-bound intermediates after release from the enzyme (fortuitously all the major ones are stable in acid), and is also useful to provide positive identification of the species seen in the CD spectrum. Several of these enzymes have in common initial pyruvate decarboxylation to form aldehyde or carboligation products. Steady state and pre-steady state analysis with the enzyme DXP synthase, enabled observation of remarkable stabilization of ThDP-bound pre-decarboxylation intermediate C2α-lactylThDP (LThDP) on the enzyme with pyruvate, and subsequent LThDP decarboxylation greatly accelerated by the second substrate D-glyceraldehyde-3-phosphate (GAP) by at least 600-fold. Further stabilization of LThDP was observed on DXP synthase variants Y392F, R478A and R420A which are shown to be essential for binding of D-GAP, without affecting the LThDP decarboxylation rates in the presence of GAP. While DXP synthase stabilizes pre-decarboxylation intermediate LThDP, the enzyme E1o from E. coli on decarboxlation of 2-oxoglutarate stabilizes the resulting enamine intermediate followed by hydroxy-carboxypropylidene-ThDP radical formation (with both substrates 2-oxoglutarate and 2-oxoadipate), characterized by CD, SFCD, NMR and electron paramagnetic resonance (EPR) spectroscopy. The E. coli E1o was also used in the chiral synthesis of α-hydroxy ketones with good yield and high enantiomeric excess, by varying both the 2-oxoacid substrates and exogenous aldehydes. Next, The conjugated 2-oxoacid substrates acetyl pyruvate, its methyl ester and (E)-4-(4 chlorophenyl)-2-oxo-3-butenoic acid (CPB) with thiamin enzymes YPDC, BAL and E1o led to formation of charge transfer bands on the enzymes which are attributed to transitions between the thiazolium ring of thiamin and a C2-β, γ double bond.Ph. D.Includes bibliographical referencesIncludes vitaby Hetalben Pate
