Domain Organization in Candida glabrata THI6, a Bifunctional Enzyme Required for Thiamin Biosynthesis in Eukaryotes,

Thiamin (vitamin B1) is an essential component of all living systems. The active form of thiamin, thiamin pyrophosphate (ThDP), acts as a cofactor for several important enzymes in carbohydrate and amino acid metabolism. The mechanistic role of the cofactor is stabilization of an acyl carbanion intermediate (1). Thiamin biosynthetic pathways are found in prokaryotes and some eukaryotes (yeast, fungi and plants); however, vertebrates cannot synthesize thiamin. Therefore, thiamin is an essential component of the human diet with a daily requirement of 1.0–1.2 mg (2). Deficiency of thiamin leads to diseases such as beriberi and Wernicke-Korsakoff syndrome (3, 4). The absence of thiamin biosynthetic enzymes in mammals makes them potential targets for new antimicrobial and antifungal drug design. Thiamin is composed of a thiazole and a pyrimidine moiety, which are synthesized separately and joined to form thiamin phosphate (ThMP). Prokaryotes and eukaryotes use different strategies for the production of thiamin. The details of thiamin biosynthesis are well characterized both structurally and mechanistically in prokaryotes (Scheme 1) (5–7). Thiazole formation in bacteria requires six gene products and involves the oxidative condensation of 1-deoxy-D-xylulose-5-phosphate, glycine (in Bacillus subtilis) or tyrosine (in Escherichia coli) and cysteine (8, 9). Formation of 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate (HMP-P) occurs by a complex rearrangement of 5-aminoimidazole ribonucleotide catalyzed by ThiC, a member of the radical SAM superfamily of enzymes (10, 11). HMP-P is further phosphorylated by ThiD to form 4-amino-5-hydroxymethyl-2-methylpyrimidine pyrophosphate (HMP-PP) (12). The thiazole and pyrimidine modules are then coupled by the enzyme thiamine phosphate synthase (TPS) to form ThMP (Scheme 2A) (13–15), which is finally phosphorylated to ThDP by the enzyme ThiL (16, 17). Scheme 1 Scheme 2 In addition to the biosynthetic pathway, many organisms contain salvage pathways. The enzyme 4-methyl-5-hydroxyethylthiazole kinase (ThiM) catalyzes the adenosine 5′-triphosphate ATP-dependent phosphorylation of 4-methyl-5-hydroxyethylthiazole (Thz) to form 4-methyl-5-hydroxyethylthiazole phosphate (Thz-P) (Scheme 2B) (18). ThiD is responsible for the phosphorylation of 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP) (12). In contrast to the prokaryotic system, the mechanistic and structural understanding of thiamin biosynthesis is still at an early stage in the eukaryotes (Scheme 3). Currently, only four enzymes are known to catalyze the biosynthesis of ThMP. THI4 converts nicotinamide adenine dinucleotide, glycine and a yet unknown sulfur source to form the adenosine diphospho-5-β-ethyl-4-methyl-thiazole-2-carboxylic acid (ADT) (19, 20). THI5 is responsible for HMP-P formation; however, the mechanistic and structural details of this process remain unknown (21). THI20 catalyzes the phosphorylation of HMP-P (22). The coupling of Thz-P and HMP-PP in eukaryotes is catalyzed by THI6 (23). This bifunctional enzyme contains a TPS domain and a ThiM domain. Earlier studies on THI6 from Saccharomyces cerevisiae showed that the N-terminal domain is responsible for the TPS activity and the C-terminal domain is responsible for ThiM activity (24). The recent identification of the product of the S. cerevisiae thiazole synthase as ADT (19, 20) suggests that Thz-P utilization is a salvage reaction and that carboxy-Thz-P is the biosynthetic substrate. Scheme 3 Here we report the crystal structures of unliganded Candida glabrata THI6 (CgTHI6) and of several complexes that map out the two active sites. These include ThMP; ThMP and pyrophosphate (PP); Thz and β,γ-methylene adenosine 5′-diphosphate (AMP-PCP); ThMP and AMP-PCP and 4-amino-5-hydroxymethyl-2-trifluoromethylpyrimidine pyrophosphate (CF3HMP-PP) and carboxy-Thz-P. The THI6 structure reveals a cage-like homohexamer in which the two active sites are separated by about 40 A. The two active sites are not connected by an obvious channel. We also report biochemical studies that show that the TPS domain accepts Thz-P as a substrate, but not ADT, the product of THI4.