Mechanistic studies on thiamin phosphate synthase from Bacillus subtilis.

摘要

Thiamin phosphate synthase catalyzes the penultimate step of thiamin pyrophosphate biosynthesis in Bacillus subtilis. The goal of much of the work described in this thesis was to determine the chemical mechanism of this enzyme. To facilitate much of the work needed for these mechanistic studies, we have overexpressed, purified and characterized pyridoxine kinase and hydroxymethylpyrimidine phosphate (HMP-P) kinase. We have confirmed the biochemical activities of both of the genes for these enzymes. In addition, we discovered that HMP-P kinase also catalyzed the phosphorylation of HMP. These kinases were used to generate pyrimidine substrates and 18O-labeled HMP-PP for the mechanistic studies on thiamin phosphate synthase.; The bioorganic approaches for these mechanistic studies included a substituent effect and positional isotope exchange (PIX) experiment. We found that an electron withdrawing trifluoromethyl substituent on the pyrimidine substrate caused a rate reduction of at least 7800-fold below the rate of conversion for natural HMP-PP. In addition, a substrate analog harboring an electron-donating methoxy substituent at the same position caused a rate acceleration of 2.8-fold. We also demonstrated that thiamin phosphate synthase caused a positional isotope exchange with 18O-labeled HMP-PP, which was prepared in situ using HMP-P kinase. Both of these bioorganic approaches suggested that thiamin phosphate synthase catalyzed a dissociative substitution reaction, where the pyrophosphate leaving group dissociated in a step prior to attack by the thiazole phosphate. However, these methods did not allow us to conclude whether the intermediate attacked by the thiazole was a carbocation or a deprotonated form of the carbocation.; We attempted to directly detect the enzyme intermediate by X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy. We found that substrate-soaked crystals of the Ser130Ala mutant of thiamin phosphate synthase afforded a structure containing an intermediate at the active site. Because of the resolution of protein crystallography, we were unable to determine the charge of this intermediate. All attempts using NMR spectroscopy on the native and Ser130Ala thiamin phosphate synthase failed in the direct detection of this intermediate. We have described the syntheses of 13C-labeled HMP-PP and 13C-labeled 2-methoxy-HMP-PP, which were used in these NMR experiments.; Finally, we have described the antibiotic mode of action of the thiamin antagonist bacimethrin. We found that bacimethrin was easily converted to 2'-methoxy-thiamin pyrophosphate by the thiamin biosynthetic enzymes from Escherichia coli. Despite this, bacimethrin and its metabolites did not inhibit the enzymatic production of natural thiamin pyrophosphate. Instead, we found that 2'-methoxy-thiamin pyrophosphate likely inhibited alpha-ketoglutarate dehydrogenase, transketolase and deoxyxyulose-5-phosphate synthase.