Steve Ealick's Research Group


Candida glabrata THI6


PDB files:

3NL2 THI6

3NL3 THI6/ThMP

3NM3 THI6/ThMP/PP

3NL5 THI6/Thz/AMP-PCP

3NL6 THI6/ThMP/AMP-PCP

3NM1 THI6/CF3HMP-PP/carboxy-Thz-P

Description:

THI6 is a bifunctional enzyme found in the thiamin biosynthetic pathway in eukaryotes. The N-terminal domain of THI6 catalyzes the ligation of the thiamin thiazole and pyrimidine moieties to form thiamin phosphate and the C-terminal domain catalyzes the phosphorylation of 4-methyl-5-hydroxyethylthiazole (Thz) in a salvage pathway. In prokaryotes, thiamin phosphate synthase (TPS) and 4-methyl-5-hydroxyethylthiazole kinase (Thz kinase; ThiM, previously annotated as ThiK) are separate gene products. We determined the structure of THI6 from Candida glabrata (CgTHI6) as well as several complexes that help characterize the active sites responsible for the two chemical reactions.

The protomer consists of two domains. The N-terminal domain has a (βα)8 barrel fold as does Bacillus subtilis TPS (BsTPS), while the C-terminal domain has an α/β fold as does ThiM.

 


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CgTHI6 is a hexamer composed of six identical protomers with 32 point symmetry.The cage-like hexamer has the shape of a prolate spheroid with a diameter along the twofold axis of about 100 Å and a length along the threefold axis of about 150 Å. There is a large inner cavity that explains the high solvent content of the crystals. The spheroid's caps are formed by trimeric assemblies of the C-terminal (ThiM) domains and the equator is formed by dimeric assemblies of the N-terminal (TPS) domains. The trimeric caps contact each other only through the TPS dimers; TPS dimers contact the trimers, but not each other.


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The active site of the TPS (N-terminal) domain is located in a cavity surrounded by loops similar to that in the BsTPS structure. The Thz binding site in the CgTHI6/Thz/AMP-PCP structure is located at the interface of two domains and is rich in hydrophobic residues.  The overall active site is similar to that of BsThiM.


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Reference:

Paul D, Chatterjee A, Begley TP and Ealick SE. Domain Organization in Candida glabrata THI6, a Bifunctional Enzyme Required for Thiamin Biosynthesis in Eukaryotes. Biochemistry 49:9922-9934 (2010).



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