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. 2010 Nov 8;49(46):8653-6.
doi: 10.1002/anie.201003419.

A "radical dance" in thiamin biosynthesis: mechanistic analysis of the bacterial hydroxymethylpyrimidine phosphate synthase

Abhishek Chatterjee  1 , Amrita B Hazra, Sameh Abdelwahed, David G Hilmey, Tadhg P Begley
Affiliations

A "radical dance" in thiamin biosynthesis: mechanistic analysis of the bacterial hydroxymethylpyrimidine phosphate synthase

Abhishek Chatterjee et al. Angew Chem Int Ed Engl. .
No abstract available

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Figures

Figure 1
Figure 1
A) The pyrimidine synthase-catalyzed conversion of AIR 1 to HMP-P 2. Reduction of SAM 3 generates the 5-deoxyadenosyl radical 4, which triggers the rearrangement reaction by hydrogen atom abstraction from AIR 1. The colors in structures 1 and 2 show the origin of the atoms of HMP-P in the AIR structure. B) AIR showing the numbering of the ribose ring.
Figure 2
Figure 2
Determining the fate of C1′ and C3′ of AIR A) NMR analysis of the pyrimidine synthase reaction mixture using AIR labeled with 13C at the 1 position of the substrate shows: (i) 13C signal for C1 of AIR (88 ppm) in the SAM free control reaction. (ii) Full reaction mixture showing a new signal at 171 ppm along with the C1 of unreacted AIR. (iii) Addition of 30 mM sodium formate to the reaction mixture enhances the intensity of the new peak at 171 ppm. B) An expanded view of the NMR spectra around the formate peak at 171 ppm for samples (ii) and (iii). C) UV-Vis spectrum of hemoglobin (green) and carboxyhemoglobin (black) and the resulting difference spectrum. D) The ThiC catalyzed reaction run in the presence of hemoglobin shows that carboxyhemoglobin production is dependent on the AIR concentration.
Figure 2
Figure 2
Determining the fate of C1′ and C3′ of AIR A) NMR analysis of the pyrimidine synthase reaction mixture using AIR labeled with 13C at the 1 position of the substrate shows: (i) 13C signal for C1 of AIR (88 ppm) in the SAM free control reaction. (ii) Full reaction mixture showing a new signal at 171 ppm along with the C1 of unreacted AIR. (iii) Addition of 30 mM sodium formate to the reaction mixture enhances the intensity of the new peak at 171 ppm. B) An expanded view of the NMR spectra around the formate peak at 171 ppm for samples (ii) and (iii). C) UV-Vis spectrum of hemoglobin (green) and carboxyhemoglobin (black) and the resulting difference spectrum. D) The ThiC catalyzed reaction run in the presence of hemoglobin shows that carboxyhemoglobin production is dependent on the AIR concentration.
Figure 3
Figure 3
Hydrogen atom abstraction from AIR occurs at C4′ and at C5′. A) AIR isotopomers synthesized for this study. B) 1H NMR signal for the 5′-methyl group of 5-deoxyadenosine isolated from the ThiC reactions with the AIR isotopomers. Incorporation of D in the methyl group for 4D and 5,5′D2 AIR is noted by the presence of the additional, upfield shifted broader doublet. C) Rate of formation of 5-deoxyadenosine and HMP-P measured over 5 hours reveal a 1:1 product ratio. D) MS analysis of 5-deoxyadenosine produced in the early phase of the reaction reveal predominant mono-deuteration (m/z=253) with 4D and 5D AIR and 50% di-deuteration (254 Da) with UD-AIR.
Figure 4
Figure 4
Mechanistic proposal for the rearrangement catalyzed by ThiC (R■ = 5′deoxyadenosyl radical, R-H = 5′deoxyadenosine).
See this image and copyright information in PMC

References

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