Thiamin Biosynthesis in Eukaryotes:  Characterization of the Enzyme-Bound Product of Thiazole Synthase from Saccharomyces cerevisiae and Its Implications in Thiazole Biosynthesis

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.

THI1, a protein involved in the biosynthesis of thiamin in Arabidopsis thaliana: Structural analysis of THI1(A140V) mutant

“…The following gradient was used at 1 mL·min −1 flow rate: solvent A is water, solvent B is 100 mM KPi, pH 6.6, solvent C is Methanol; 0 min 100% B; 5 min 10% A and 90% B; 8 min 25% A, 60% B and 15% C; 14 min 25% A, 60% B and 15% C; 19 min 30% A, 40% B and 30% C; 21 min 100% B; 30 min 100% B [14]. The absorbance at 258 nm was monitored.…”

“…Many intermediates of the thiamine pathway are predicted by extrapolations from the characteristics of the prokaryotic thiamine biosynthesis [14]. Labeling studies in S. cerevisiae have demonstrated that the thiamine-thiazole is synthesized from an unidentified five-carbon carbohydrate, glycine, and cysteine [12].…”

“…Labeling studies in S. cerevisiae have demonstrated that the thiamine-thiazole is synthesized from an unidentified five-carbon carbohydrate, glycine, and cysteine [12]. This biosynthesis has not yet been reproduced in vitro, but a metabolomic study has recently proposed NAD + (nicotinamide adenine dinucleotide) as the early source of a five-carbon carbohydrate and the advanced intermediate as an ADP adduct of 5-(2-hydroxyethyl)-4-methylthiazole-2-carboxylic acid [4,14]. The gene encoding THI1 protein was originally cloned due to its capacity to complement Escherichia coli mutant line BW535 (xth, nfo and nth genes), which is defective in DNA base excision repair pathways [13].…”

“…Its overall folding is similar to the dinucleotide binding domains with a higher similarity to FAD binding enzymes. Nevertheless several studies suggest that THI1 binds NAD + , whereas FAD binding remains unknown [10,14]. THI1 shares some structural similarity with an orthologue from Bacillus subtilis, ThiO, a FAD-dependent enzyme required for the thiazole biosynthesis, showing a well conserved dinucleotide binding architecture [10].…”

Cofactor biosynthesis—still yielding fascinating new biological chemistry

“…While the reaction catalyzed by the thiazole synthase (THI4) has not yet been fully reconstituted, the enzyme copurified with three tightly bound metabolites (35, 40 and 41) and the structure of the enzyme complexed to 41 has been determined. [12][13][14][15] The identity of these metabolites suggested that NAD is the precursor to the thiazole. This was confirmed by the identification of three partial reactions catalyzed by the C204A mutant of THI4 (28 to 29, 29 to 30 and 29 to 35 in Figure 5).…”