Saccharomyces cerevisiae THI4p is a suicide thiamine thiazole synthase

Abstract

Thiamine pyrophosphate 1 is an essential cofactor in all living systems. Its biosynthesis involves the separate syntheses of the pyrimidine 2 and thiazole 3 precursors, which are then coupled. Two biosynthetic routes to the thiamine thiazole have been identified. In prokaryotes, five enzymes act on three substrates to produce the thiazole via a complex oxidative condensation reaction, the mechanistic details of which are now well established. In contrast, only one gene product is involved in thiazole biosynthesis in eukaryotes (THI4p in Saccharomyces cerevisiae). Here we report the preparation of fully active recombinant wild-type THI4p, the identification of an iron-dependent sulphide transfer reaction from a conserved cysteine residue of the protein to a reaction intermediate and the demonstration that THI4p is a suicide enzyme undergoing only a single turnover.

Figure 1

A) The late steps in thiamin pyrophosphate biosynthesis. B) Mechanistic proposal for the biosynthesis of thiamin-thiazole in eukaryotes catalyzed by THI4p.

Figure 2

Identification of the site of the M-34Da modification in wtTHI4p. A) The peptide fragments originating from wtTHI4p and the R301Q mutant containing the site of modification. B) A mechanistic hypothesis to explain the mass loss and lack of reactivity with iodoacetamide of the modified peptide. C) Active site of THI4p with bound ADT. The separation of the sulfur atom of ADT and the C_β atom of the dehydroalanine residue is 5.3 Å. The loop Gln203 – Pro208 is from a fourfold-related monomer and has carbon atoms colored cyan. Water molecules are shown as red spheres. The electron density map (2F_o-F_c contoured at 3σ) clearly shows the loss of sulfur from Cys205 to form the dehydroalanine residue. D) Magnified electron density of residue DHA205 and residue Cys204.

Figure 3

Reconstitution of the biosynthesis of ADT 5. A) HPLC analysis of the metabolites associated with wtTHI4p overexpressed in M9 minimal medium ±100 µM iron. B) ESI-FTMS analysis of wtTHI4p overexpressed in M9 minimal medium ±100 µM iron shows iron-dependent modification (ΔM = −34 Da) of the protein. C) HPLC analysis of wtTHI4p catalyzed partial and full reactions and the relevant control reactions. Incubating THI4p with ADPr 7, glycine and iron(II) results in the production of ADT 5. D) Time course for the reaction showing a 1:1 ratio of protein modification and thiazole production (error bars indicate s.d.) E) MS analysis of wtTHI4p over the time course of the reaction showing the progressive conversion of the enzyme to the M-34 Da species.

Figure 4

Characterization of native THI4p from S. cerevisiae. A) Analysis of THI4p in yeast cell free extract using a western blot (right) and coomassie blue (left). Lanes 1–5 contain increasing concentrations (0.5, 1, 2, 4, and 8 µM) of (His)₆-THI4p. Lane 6 contains yeast crude lysate. B) Native THI4p, isolated from yeast cell free extract, analyzed by SDS-PAGE/coomassie blue (1) and western-blot (2). C) In gel chymotrypsin digestion/MALDI-TOF analysis of isolated native THI4p demonstrates that the peptide containing the C204-C205 region has the same modification as observed with THI4p expressed in E. coli. D) Quantitation of THI4p and thiamin produced in a culture of yeast, growing in vitamin free defined medium, demonstrates that THI4p is a substrate rather than a catalyst. E) Proposed mechanism for the iron-mediated sulfur transfer reaction involved in the formation of intermediate 14 (Figure 1).