Biosynthesis of the thiamin thiazole in Bacillus subtilis: identification of the product of the thiazole synthase-catalyzed reaction

Abstract

In this paper, we describe an optimized reconstitution of the thiamin thiazole synthase (ThiG) catalyzed reaction and demonstrate that the enzymatic product is an unanticipated dearomatized thiazole tautomer.

Figure 1

Currently proposed mechanism for the formation of the thiamin thiazole in B. subtilis.

Figure 2

The thiochrome assay previously used for the detection of thiazole formation.

Figure 3

HPLC analysis of the product of the bacterial thiazole synthase reaction mixture. A - The enzymatic reaction mixture, B - Thiazole phosphate 16, the previously assumed reaction product.

Figure 4

HPLC analysis showing the conversion of the peak A compound to thiochrome phosphate (red trace).

Figure 5

Procedures for the production of reference compounds 14 and 15

Figure 6

(a) - HPLC analysis of the thiazole synthase product (pink) and reference compounds 14 (orange) and 15 (blue). (b) - HPLC analysis of the dephosphorylated product of the thiazole synthase (pink) catalyzed reaction and reference compounds 22 (green) and 23 (orange).

Figure 7

¹H-NMR analysis of the dephosphorylated Peak A compound 22. The DXP sample used was labeled with ¹³C on the methyl group (unrelated reasons) hence the additional splitting of the H₂/H₃/H₄ protons. Additional proton coupling, from the 2D dqf-COSY experiment, are indicated on the structure. (Supplementary Figure 4, 5 and 6).