Steve Ealick's Research Group
4-Amino-5-hydroxymethyl-2-methylpyrimidine Phosphate Synthase
3EPM, ThiC with HMP
3EPN, ThiC with IMR
3EPO, ThiC with HMP-P
*4S2A with [4Fe-4S]
The biosynthesis of thiamin pyrophosphate (TPP) in yeast utilizes a completely different biochemical pathway than that in prokaryotes. 4-Amino-5-hydroxymethyl-2-methylpyrimidine phosphate (HMP-P) synthase (ThiC) catalyzes a complex rearrangement of 5-aminoimidazole ribotide (AIR) to form HMP-P, the pyrimidine moiety thiamin phosphate. We determined the structures of HMP-P synthase from Caulobacter crescentus (CcThiC) in complex with HMP, the product HMP-P, an intermediate analog 4-(1,2-dihydroxyethyl)-2-methyl-imidazole (DHEMI), and a substrate analog imidazole ribonucleotide (IMR). These structures helped characterize the active site and elucidate the mechanism of the reaction.
Each CcThiC protomer consists of three domains. Domain 1 contains a novel fold consisting of seven α-helices and five β-strands. The secondary structural elements form a thin blanket-like structure that folds over domains 2 and 3. Domain 2 has a (βα)8 barrel fold and Domain 3 has residues that form an antiparallel three helix bundle followed by a loop that extends into the top of the (βα)8 barrel from the adjacent protomer. Each protomer contains a bound metal ion, identified as Zn2+, and an HMP molecule.
|CcThiC exists as a dimer formed by joining the two (βα)8 barrels in an approximately antiparallel arrangement. The dimer interface is predominantly nonpolar.|
The active site is within a cavity at the C-terminal end of the (βα)8 barrel. A representative picture of the active site shows the product HPM-P bound.
Chatterjee A, Li Y, Zhang Y, Grove TL, Lee M, Krebs C, Booker SJ, Begley TP, and Ealick SE. Reconstitution of ThiC in thiamine pyrimidine biosynthesis expands the radical SAM superfamily. Nat. Chem. Biol. 4, 758-765 (2008).
*Fenwick MK, Mehta AP, Zhang Y, Abdelwahed SH, Begley TP, and Ealick SE. Noncanonical Active Site Architecture of the Radical SAM Thiamin Pyrimidine Synthase. Nat. Commun. 6:6480 (2015).