Enzymes of the heme biosynthesis pathway, which are essential for all life and have been conserved throughout evolution, are associated with human diseases and offer potential as targets for antibiotics, fungicides, and herbicides. During the initial funding period we determined four new protein structures and performed associated mutagenic and biochemical analyses. The current proposal emphasizes determination of substrate/product complex structures, with focus on U3S (4th enzyme of heme biosynthesis pathway), URO-D (5th enzyme), PROTO-OX (7th enzyme), and Met8p (branch pathway of siroheme biosynthesis).We have recently co-crystallized URO-D in complex with product by use of a coupled enzyme system that circumvents the difficulty of extreme oxygen sensitivity of pathway intermediates. Preliminary data collected to 2.2A resolution reveal that the product is well ordered in the active site and indicates the location of the catalytic center. Remarkably, an invariant aspartate side chain coordinates all four of the tetrapyrrole amides and may serve as a pivot about which the substrate rotates to allow sequential decarboxylation of all four acetate side chains. Experiments are proposed to test questions of mechanism, including the importance of specific residues for binding and catalysis, the importance of a loop that covers the active site, and the functional significance of dimerization. The approach developed for the URO-D/product complex will be adapted to obtain complexes of inactive URO-D mutants with substrate. This approach will also be adapted to obtain substrate and product complex structures of U3S and Met8p, for which we have already determined the apo structures. Finally, the PROTO-OX structure, towards which we have already collected 2.3A native data, will be of special interest for the development of novel inhibitors.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM056775-06
Application #
6604694
Study Section
Biophysical Chemistry Study Section (BBCB)
Program Officer
Whitmarsh, John
Project Start
1998-07-01
Project End
2006-06-30
Budget Start
2003-07-01
Budget End
2004-06-30
Support Year
6
Fiscal Year
2003
Total Cost
$284,050
Indirect Cost
Name
University of Utah
Department
Biochemistry
Type
Schools of Medicine
DUNS #
009095365
City
Salt Lake City
State
UT
Country
United States
Zip Code
84112
Mabanglo, Mark F; Schubert, Heidi L; Chen, Mo et al. (2010) X-ray structures of isopentenyl phosphate kinase. ACS Chem Biol 5:517-27
Warby, C A; Phillips, J D; Bergonia, H A et al. (2009) Structural and kinetic characterization of mutant human uroporphyrinogen decarboxylases. Cell Mol Biol (Noisy-le-grand) 55:40-5
Phillips, John D; Warby, Christy A; Whitby, Frank G et al. (2009) Substrate shuttling between active sites of uroporphyrinogen decarboxylase is not required to generate coproporphyrinogen. J Mol Biol 389:306-14
Schubert, Heidi L; Phillips, John D; Heroux, Annie et al. (2008) Structure and mechanistic implications of a uroporphyrinogen III synthase-product complex. Biochemistry 47:8648-55
Schubert, Heidi L; Rose, Ruth S; Leech, Helen K et al. (2008) Structure and function of SirC from Bacillus megaterium: a metal-binding precorrin-2 dehydrogenase. Biochem J 415:257-63
Phillips, John D; Whitby, Frank G; Stadtmueller, Beth M et al. (2007) Two novel uroporphyrinogen decarboxylase (URO-D) mutations causing hepatoerythropoietic porphyria (HEP). Transl Res 149:85-91
Schubert, Heidi L; Hill, Christopher P (2006) Structure of ATP-bound human ATP:cobalamin adenosyltransferase. Biochemistry 45:15188-96
Davison, Paul A; Schubert, Heidi L; Reid, James D et al. (2005) Structural and biochemical characterization of Gun4 suggests a mechanism for its role in chlorophyll biosynthesis. Biochemistry 44:7603-12
Macbeth, Mark R; Schubert, Heidi L; Vandemark, Andrew P et al. (2005) Inositol hexakisphosphate is bound in the ADAR2 core and required for RNA editing. Science 309:1534-9
Vevodova, Jitka; Graham, Ross M; Raux, Evelyne et al. (2004) Structure/function studies on a S-adenosyl-L-methionine-dependent uroporphyrinogen III C methyltransferase (SUMT), a key regulatory enzyme of tetrapyrrole biosynthesis. J Mol Biol 344:419-33

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