Steve Ealick's Research Group


4-Amino-5-hydroxymethyl-2-methylpyrimidine Phosphate Synthase


PDB files:

4S25 [4Fe-4S]/IRN/SAH/zinc trigonal crystal form

4S26 [4Fe-4S]/IRN/SAH/zinc monoclinic crystal form

4S27 [4Fe-4S]/Fe/5-dAdo/Met/AIR

4S28 [4Fe-4S]/Fe/AIR/SAH

4S29 Fe/IRN

Description:

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 had previously published work on the structure of Caulobacter crescentus ThiC (CcThiC) that showed some details of the active site and gave insights into the mechanism. Our recent structures resulting from crystals of Arabidopsis thaliana ThiC (AtThiC) grown under anaerobic condictions showed intact [4Fe-4S] clusters and provided additional information about the active site. he ensemble of ThiC structures demonstrats several unprecedented features of ThiC vis a vis the radical SAM enzyme superfamily and provide further evidence of an evolutional link to the adenosylcobalamin-dependent enzyme superfamily.

Similar to CcThiC, the AtThiC protomer consists of three domains. Domain 1 contains a 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.

 


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AtThiC exists as a dimer formed by joining the two (βα)8 barrels in an approximately antiparallel arrangement. The dimer interface is predominantly nonpolar.


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AtThiC protomer structure with bound [4Fe-4S] cluster, S-adenosylhomocysteine, aminoimidazole ribonucleotide and Fe. Mode of SAH binding in ThiC.  SAH does not anchor to the [4Fe-4S] cluster but instead anchors to a secondary metal via its amino and carboxylate groups.  The differentiated iron binds to chloride in our structures.


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Reference:

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).



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