John Phillips
Research Associate Professor of Hematology and Pathology
B.S. University of New Hampshire
Ph.D. Dartmouth College
Research
Heme is essential for life; virtually all organisms are either able to make heme or they have developed a mechanism to parasitize it from other organisms. Heme is a critical component in proteins with diverse functions such as the transport of oxygen (hemoglobin, myoglobin), the production of ATP (cytochromes of the electron transport chain), detoxification reactions in the liver (the cytochrome P450 family of enzymes). In our laboratory we study the synthesis of heme, the pathway consists of eight enzymes that convert glycine and succinyl-CoA into a tetrapyrrole that eventually accepts an iron atom forming heme.
In humans defects in any of the eight steps involved in heme synthesis are termed the porphyrias. The most common of the porphyrias is Porphyria Cutanea Tarda (PCT) that is due to reduced activity of Uroporphyrinogen Decarboxylase (URO-D). We have primarily focused on this fifth step in the heme biosynthetic, using a combination of biochemistry, mouse genetics and structural studies to understand the molecular basis of the disease. There are both environmental and genetic components to the development of PCT. We have constructed a mouse model of the familial form of PCT. We have also developed an environmental model of PCT using environmental toxins such as polychlorinated biphenyls (PCB’s). In all cases, the phenotype of PCT (blisters on sun exposed areas of skin, excess porphyrins in urine and excess iron) is due to an inhibitor of the enzyme. Using these mouse models of PCT we are identifying the changes that occur in the liver that lead to the production of an inhibitor of URO-D.
In collaboration with the structural biochemists here at the University we have crystallized three of the eight enzymes in the heme biosynthetic pathway. We are working to identify the mechanisms for these evolutionarily conserved enzymes to explain the molecular details of tetrapyrrole synthesis. The accompanying figure shows the active site of URO-D with its product, coproporphyrinogen III, bound. Using this molecular model we have identified how the structure of the substrate adopts a domed configuration to fit appropriately into the active site allowing the tetrapyrrole to act as its own cofactor in this unique decarboxylase. We are also employing a structural approach to understand the basis of inhibitor binding to URO-D.
References
1. Lorenzo FP, Phillips JD, Nussenzveig R, Lingam B, Koul PA, Schrier SL, Prchal JT (2011) Molecular basis of two novel mutation found in type I methemoglobinemia. Blood Cells Mol Dis 46(4):277-81
2. Yang Z, Phillips JD, Doty RT, Giraudi P, Ostrow JD, Tiribelli C, Smith A, Abkowitz JL (2010) Kinetics and specificity of FLVCR export function and its dependence on hemopexin. J Biol Chem 285(37):28874-82
3. Crockett DK, Kushnir MM, Phillips JD, Rockwood AL (2010) Time-of-flight mass spectrometry analysis of the ferroportin-hepcidin binding domain complex for accurate mass confirmation of bioactive hepcidin 25. Clin Chim Acta 411(5-6):453-5
4. Barker ML, Hathaway LB, Arch DD, Westbroek ML, Kushner JP, Phillips JD, Franklin MR (2009) Hyper- and hypo-induction of cytochrome P450 activities with Aroclor 1254 and 3-methylcholanthrene in Cyp1a2(-/-) mice. Chem Biol Interact 182(2-3):220-6
5. Arch DD, Bergonia HA, Hathaway L, Kushner JP, Phillips JD, Franklin MR (2009) Longitudinal study of a mouse model of familial porphyria cutanea tarda. Cell Mol Biol 55(2):46-54
6. Warby CA, Phillips JD, Bergonia HA, Whitby FG, Hill CP, Kushner JP (2009) Structural and kinetic characterization of mutant human uroporphyrinogen decarboxylase. Cell Mol Biol 55(2):36-41
7. Spurgon S, Yu M, Phillips JD, Epner EM (2009) Cladribine: not just another purine analogue? Expert Opin Investig Drugs 18(8):1169-81
8. Lin H, Burton D, Li L, Warner DE, Phillips JD, Ward DM, Kaplan J (2009) Gain of function mutations identify amino acids within transmembrane domains of the yeast vacuolar transporter Zrc1 that determine metal specificity. Biochem J 422(2):273-83
9. Badenas C, To-Figueras J, Phillips JD, Warby CA, Muñoz C, Herrero C (2009) Identification and characterization of novel uroporphyrinogen decarboxylase gene mutations in a large series of porphyria cutanea tarda patients and relatives. Clin Genet 75(4):346-53
10. Phillips JD, Warby CA, Whitby FG, Kushner JP, Hill CP (2009) Substrate shuttling between active sites of uroporphyrinogen decarboxylase is not required to generate coproporphyrinogen. J Mol Biol
11. Bergonia HA, Phillips JD, Kushner JP (2009) Reduction of porphyrins to porphyrinogens with palladium on carbon. Anal Biochem 384(1):74-8
12. Ajioka RS, Phillips JD, Weiss RB, Dunn DM, Smit MW, Proll SC, Katze MG, Kushner JP (2008) Down-regulation of hepcidin in porphyria cutanea tarda. Blood 112(12):4723-8 PMC259713
13. De Domenico I, Nemeth E, Nelson JM, Phillips JD, Ajioka RS, Kay MS, Kushner JP, Ganz, T, Ward DM, Kaplan J (2008) The hepcidin-binding site on ferroportin is evolutionarily conserved. Cell Metab 8(2):146-56
14. Schubert HL, Phillips JD, Heroux A, Hill, CP (2008) Structure and mechanistic implications of a uroporphyrinogen III synthase-product complex. Biochemistry 47(33):8648-55
Updated 6/2/2011
