A missing enzyme in thiamin thiazole biosynthesis: identification of TenI as a thiazole tautomerase

Abstract

In many bacteria tenI is found clustered with genes involved in thiamin thiazole biosynthesis. However, while TenI shows high sequence similarity with thiamin phosphate synthase, the purified protein has no thiamin phosphate synthase activity, and the role of this enzyme in thiamin biosynthesis remains unknown. In this contribution, we identify the function of TenI as a thiazole tautomerase, describe the structure of the enzyme complexed with its reaction product, identify the substrates phosphate and histidine 122 as the acid/base residues involved in catalysis, and propose a mechanism for the reaction. The identification of the function of TenI completes the identification of all of the enzymes needed for thiamin biosynthesis by the major bacterial pathway.

Figure 1

(a) Mechanistic proposal for the formation of the thiamin thiazole in B. subtilis. The thiazole synthase (ThiG) reaction product 14 is coupled to HMP-PP 17 to form thiamin phosphate 18. Thz-P 15 and cThz-P 16 are possible intermediates in this coupling reaction. (b) Reactions showing conversion of ThiS by ThiF and NifS into ThiS-COSH 7, glycine oxidase ThiO converting glycine 19 to glycine imine 12 and the oxidation of thiamin phosphate 18 to thiochrome 20.

Figure 2

Time course for the thiazole reconstitution reaction run in the presence and absence of TenI.

Figure 3

TenI does not affect glycine oxidase, thiamin phosphate synthase or ThiS-COSH formation. The blue trace is the reaction in the absence of TenI and the pink trace is the reaction in the presence of TenI. (a) The presence of TenI does not affect the rate of glycine oxidase (ThiO). (b) The presence of TenI does not affect the rate of the HMP-PP/Thz-P coupling reaction catalyzed by thiamin phosphate synthase. (c) The rate of the thiazole synthase catalyzed reaction, using preformed ThiS-COSH, is enhanced by TenI.

Figure 4

Substrate selectivity of thiamin phosphate synthase. The plot shows the time course for the alkylation of cThz*-P 14 (black trace), Thz-P 15 (blue trace) and cThz-P 16 (red trace) by HMP-PP 17.

Figure 5

Routes for the conversion of cThz*-P 14 and cThz-P 16 to thiamin phosphate 18. In bacteria lacking TenI, ThiE can catalyze the coupling of the thiazole tautomer 14 and HMP-PP 17 to give 21, which is converted to 18 directly or via intermediate 22.

Figure 6

Reverse-phase HPLC analysis of the degradation of cThz*-P 14 to carboxy vinylthiazole 23. The blue trace shows pure cThz*-P, and the green trace shows the formation of the carboxy vinylthiazole as a degradation product after 5 days. Chromatograms are offset by 2 minutes.

Figure 7

HPLC analysis demonstrating that TenI catalyzes the conversion of cThz*-P 14 (blue trace, Peak A) to a new compound (green trace) that comigrates with cThz-P 16 (Peak B).

Figure 8

Reconstitution of thiazole biosynthesis in the presence and absence of TenI. (a) Comigration of the product of thiazole biosynthesis in the absence of TenI (green) with cThz*-P (pink) and comigration of the product of thiazole biosynthesis in the presence of TenI (purple) with cThz-P (orange). (b) Comigration of the alkaline phosphatase treated product of thiazole biosynthesis in the absence of TenI (green) with cThz* (pink) and comigration of the alkaline phosphatase treated product of thiazole biosynthesis in the presence of TenI (purple) with cThz (orange).

Figure 9

a) Stereoview of the active site of the TenI with bound cThz-P. b) Stereoview of a model of the active site of TenI with bound cThz*-P.

Figure 10

Mechanistic proposals for the TenI-catalyzed thiazole aromatization reaction.