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. 2006 Jun 7;128(22):7158-9.
doi: 10.1021/ja061413o.

Thiamin biosynthesis in eukaryotes: characterization of the enzyme-bound product of thiazole synthase from Saccharomyces cerevisiae and its implications in thiazole biosynthesis

Abhishek Chatterjee  1 , Christopher T Jurgenson, Frank C Schroeder, Steven E Ealick, Tadhg P Begley
Affiliations

Thiamin biosynthesis in eukaryotes: characterization of the enzyme-bound product of thiazole synthase from Saccharomyces cerevisiae and its implications in thiazole biosynthesis

Abhishek Chatterjee et al. J Am Chem Soc. .

Abstract

The biosynthesis of thiamin pyrophosphate in eukaryotes is different from the prokaryotic biosynthesis and is poorly understood to date. Only one thiazole biosynthetic gene has been identified (Thi4 in Saccharomyces cerevisiae). Here we report the identification and characterization of a Thi4-bound metabolite that consists of the ADP adduct of 5-(2-hydroxyethyl)-4-methylthiazole-2-carboxylic acid. The unexpected structure of this compound yields the first insights into the mechanism of thiamin thiazole biosynthesis in eukaryotes.

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Figures

Figure 1
Figure 1
Identification of Thi4 bound small molecules by HPLC (a). Analysis of the HPLC purified peak A : by UV-Vis spectroscopy (b), by 1H NMR spectroscopy (c) and by negative mode ESI-MS (d).
Figure 2
Figure 2
Confirmation of the structure 3 by its enzymatic conversion to thiamin phosphate 5: (a) Scheme showing the reactions performed. (b) HPLC analysis of the cleavage reaction of 2 to 3 and AMP: The blue trace shows the control reaction where no pyrophosphatase has been added to 2; The purple trace shows the cleavage reaction of 2 by the pyrophosphatase. (c) HPLC detection of the thiochrome phosphate 6 produced from 2: blue trace shows the control reaction with no HMP-PP added; Purple trace shows the actual coupling reaction.
Figure 3
Figure 3
Active site structure of Thi4 (PDB code: 2GJC): (a) The structure of Compound 2 and its interactions with the enzyme. (b) Electron density confirming the thiazole carboxylic acid moiety of 2. The density is a composite omit map contoured at the 1σ level.
Figure 4
Figure 4
A proposed mechanism for thiazole biosynthesis in eukaryotes.
See this image and copyright information in PMC

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