Reconstitution of ThiC in thiamine pyrimidine biosynthesis expands the radical SAM superfamily

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Abstract
4-Amino-5-hydroxymethyl-2-methylpyrimidine phosphate (HMP-P) synthase catalyzes a complex rearrangement of 5-aminoimidazole ribonucleotide (AIR) to form HMP-P, the pyrimidine moiety of thiamine phosphate. We determined the three-dimensional structures of HMP-P synthase and its complexes with the product HMP-P and a substrate analog imidazole ribotide. The structure of HMP-P synthase reveals a homodimer in which each protomer comprises three domains: an N-terminal domain with a novel fold, a central (βα)8 barrel and a disordered C-terminal domain that contains a conserved CX2CX4C motif, which is suggestive of a [4Fe-4S] cluster. Biochemical studies have confirmed that HMP-P synthase is iron sulfur cluster–dependent, that it is a new member of the radical SAM superfamily and that HMP-P and 5′-deoxyadenosine are products of the reaction. Mössbauer and EPR spectroscopy confirm the presence of one [4Fe-4S] cluster. Structural comparisons reveal that HMP-P synthase is homologous to a group of adenosylcobalamin radical enzymes. This similarity supports an evolutionary relationship between these two superfamilies.