Mechanistic Studies on Thiamin Phosphate Synthase:  Evidence for a Dissociative Mechanism

Abstract

Thiamin phosphate synthase catalyzes the coupling of 4-methyl-5-(beta-hydroxyethyl)thiazole phosphate (Thz-P) and 4-amino-5-(hydroxymethyl)-2-methylpyrimidine pyrophosphate (HMP-PP) to give thiamin phosphate. In this paper, we demonstrate that 4-amino-5-(hydroxymethyl)-2-(trifluoromethyl)pyrimidine pyrophosphate (CF(3)-HMP-PP) is a very poor substrate [k(cat)(CH(3)) > 7800k(cat)(CF(3))] and that 4-amino-5-(hydroxymethyl)-2-methoxypyrimidine pyrophosphate (CH(3)O-HMP-PP) is a good substrate [k(cat)(OCH(3)) > 2.8k(cat)(CH(3))] for the enzyme. We also demonstrate that the enzyme catalyzes positional isotope exchange. These observations are consistent with a dissociative mechanism (S(N)1 like) for thiamin phosphate synthase in which the pyrimidine pyrophosphate dissociates to give a reactive pyrimidine intermediate which is then trapped by the thiazole moiety.

Discovery of a SAR11 growth requirement for thiamin’s pyrimidine precursor and its distribution in the Sargasso Sea

“…To minimize unintended traces of vitamins from glassware, all nutrient and vitamin stocks were prepared in combusted glassware (450 1C for 4 h) with nanopure water, 0.1 mm filter sterilized and frozen in amber tubes immediately after preparation. HMP was synthesized as described in Reddick et al (2001). AmMP was synthesized as described in Zhao et al (2012).…”

A Genetic Screen for the Identification of Thiamin Metabolic Genes

“…This suggested the possibility that these proteins might detoxify bacimethrin and CF 3 -HMP by catalyzing hydrolysis of CF 3 -HMP-PP and MeO-TPP, the previously identified toxic forms of the antibiotics (13,16). Synthesis of the necessary substrates to test this hypothesis was carried out as shown in Fig.…”

“…In the presence of low levels of exogenous thiamin, cell growth is not inhibited by bacimethrin (14,15). CF 3 -HMP is converted by HMP-P kinase (ThiD) to CF 3 -HMP pyrophosphate (CF 3 -HMP-PP), which inhibits thiamin phosphate synthase (ThiE) (16). Increased levels of biosynthesized thiamin should overcome growth inhibition caused by CF 3 -HMP.…”

Thiamin Phosphate Synthase: The Rate of Pyrimidine Carbocation Formation

“…The reaction mixture for each time point was quenched by the rapid addition of 0.25 M NaOH, and the amount of product was quantified by the oxidation of TP using K 3 Fe(CN) 6 to yield the intensely fluorescent thiochrome phosphate 7 (Figure 1b). 7 The data were fit by nonlinear regression to the following burst equation:A burst phase rate (λ) of 0.39 ( 0.02 s -1 and a steady-state phase rate (k ss /[E]) of 0.0125 ( 0.0005 s -1 were obtained. The nonzero value for [TP] in the t ) 0 time point results from a fraction of TP-synthase that co-purifies with TP.…”

“…The reaction mixture for each time point was quenched by the rapid addition of 0.25 M NaOH, and the amount of product was quantified by the oxidation of TP using K 3 Fe(CN) 6 to yield the intensely fluorescent thiochrome phosphate 7 (Figure 1b). 7 The data were fit by nonlinear regression to the following burst equation:…”