The crystal structure of yeast thiamin pyrophosphokinase

Background: Thiamin pyrophosphokinase (TPK) catalyzes the transfer of a pyr ophosphate group from ATP to vitamin B-1 (thiamin) to form the coenzyme thi amin pyrophosphate (TPP). Thus, TPK is important for the formation of a coe nzyme required for central metabolic functions. TPK has no sequence homolog s in the PDB and functions by an unknown mechanism. The TPK structure has b een determined as a significant step toward elucidating its catalytic actio n. Results: The crystal structure of Saccharomyces cerevisiae TPK complexed wi th thiamin has been determined at 1.8 Angstrom resolution, TPK is a homodim er, and each subunit consists of two domains. One domain resembles a Rossma n fold with four alpha helices on each side of a 6 strand parallel beta she et. The other domain has one 4 strand and one 6 strand antiparallel beta sh eet, which form a flattened sandwich structure containing a jelly-roll topo logy. The active site is located in a cleft at the dimer interface and is f ormed from residues from domains of both subunits. The TPK dimer contains t wo compound active sites at the subunit interface. Conclusions: The structure of TPK with one substrate bound identifies the l ocation of the thiamin binding site and probable catalytic residues. The st ructure also suggests a likely binding site for ATP. These findings are fur ther supported by TPK sequence homologies. Although possessing no significa nt sequence homology with other pyrophospokinases, thiamin pyrophosphokinas e may operate by a mechanism of pyrophosphoryl transfer similar to those de scribed for pyrophosphokinases functioning in nucleotide biosynthesis.