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.
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Acknowledgements
We thank NE-CAT beamline 24-ID-C, supported by US National Institutes of Health grant RR15301, for the use of beam time. We thank C. Kinsland (Cornell University Protein Facility) for the preparation of the HMP-P synthase overexpression plasmid and L. Kinsland for assistance in the preparation of this manuscript. This work was supported by US National Institutes of Health grants DK44083 (T.P.B.), GM63847 (S.J.B.) and DK67081 (S.E.E.), the Beckman Foundation (Young Investigator Award to C.K.) and the Dreyfus Foundation (Camille Dreyfus Teacher Scholar Award to C.K.). S.E.E. is indebted to the W.M. Keck Foundation and the Lucille P. Markey Charitable Trust.
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A.C. performed all biochemical studies. Y.L. and Y.Z. performed all crystallographic studies. T.L.G. assisted in protein purification and EPR spectroscopy. M.L. recorded and analyzed Mössbauer spectra. C.K. and S.J.B. directed the EPR and Mössbauer studies, T.P.B. directed the biochemical studies and S.E.E. directed the crystallographic studies.
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Chatterjee, A., Li, Y., Zhang, Y. et al. Reconstitution of ThiC in thiamine pyrimidine biosynthesis expands the radical SAM superfamily. Nat Chem Biol 4, 758–765 (2008). https://doi.org/10.1038/nchembio.121
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DOI: https://doi.org/10.1038/nchembio.121
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