Defects in heme biosynthesis disrupt iron homeostasis, leading to malfunction in erythropoiesis. Ferrochelatase, the only known human chelatase in the recently recognized chelatase family of enzymes, catalyzes the last step in heme biosynthesis, the insertion of ferrous iron into protoporphyrin. Distortion of porphyrin, following its binding to ferrochelatase, is a crucial step in the catalytic mechanism of this enzyme. Analysis of structural and kinetic data pertinent to the mechanism of chelatases led the P.I. and collaborators to propose that the chelatase-induced distortion of porphyrin substrate not only enhances the reaction rate by decreasing the activation energy of the reaction but also modulates which divalent metal ion is incorporated into the porphyrin ring. In addition, preliminary results show that only a few mutations in the ferrochelatase scaffold are sufficient to alter ferrochelatase towards other metal chelatase activities. The P.I. proposes to use the ferrochelatase scaffold to assess how metal ion selectivity arises within the metal chelatase family. The hypothesis to be addressed is: Chelatases, by differentially distorting the porphyrin substrate, modulate which metal ion is incorporated into the porphyrin ring. To test this hypothesis, the following Specific Aims are proposed: 1. Define the mode and degree of porphyrin distortion induced by murine ferrochelatase and directly- evolved chelatase variants exhibiting different metal ion specificities and enzymatic activities. 2. Assess the molecular and structural determinants for metal ion selectivity and enzymatic activity of ferrochelatase and evolved variants. 3. Optimize metal ion selectivity of evolved chelatase variants. A combination of experimental approaches ranging from construction of focused metal chelatase libraries to the characterization of the mode of porphyrin distortion, metal-ion coordination geometry and kinetic properties will allow us to produce and analyze variants with subtle differences in the scaffold but different metal ion selectivities and chelatase activities. These findings will improve our understanding of heme biosynthesis and iron homeostasis and provide interpretations at a molecular level of erythropoietic disorders, and consequently a rational approach for their prevention, diagnosis and therapy.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM080270-04
Application #
7799727
Study Section
Erythrocyte and Leukocyte Biology Study Section (ELB)
Program Officer
Anderson, Vernon
Project Start
2007-04-01
Project End
2012-03-31
Budget Start
2010-04-01
Budget End
2012-03-31
Support Year
4
Fiscal Year
2010
Total Cost
$230,098
Indirect Cost
Name
University of South Florida
Department
Biochemistry
Type
Schools of Medicine
DUNS #
069687242
City
Tampa
State
FL
Country
United States
Zip Code
33612
Stojanovski, Bosko M; Breydo, Leonid; Uversky, Vladimir N et al. (2016) Murine erythroid 5-aminolevulinate synthase: Truncation of a disordered N-terminal extension is not detrimental for catalysis. Biochim Biophys Acta 1864:441-52
Söderberg, Christopher; Gillam, Mallory E; Ahlgren, Eva-Christina et al. (2016) The Structure of the Complex between Yeast Frataxin and Ferrochelatase: CHARACTERIZATION AND PRE-STEADY STATE REACTION OF FERROUS IRON DELIVERY AND HEME SYNTHESIS. J Biol Chem 291:11887-98
Fratz, Erica J; Clayton, Jerome; Hunter, Gregory A et al. (2015) Human Erythroid 5-Aminolevulinate Synthase Mutations Associated with X-Linked Protoporphyria Disrupt the Conformational Equilibrium and Enhance Product Release. Biochemistry 54:5617-31
Stojanovski, Bosko M; Ferreira, Gloria C (2015) Asn-150 of Murine Erythroid 5-Aminolevulinate Synthase Modulates the Catalytic Balance between the Rates of the Reversible Reaction. J Biol Chem 290:30750-61
Stojanovski, Bosko M; Ferreira, Gloria C (2015) Murine erythroid 5-aminolevulinate synthase: Adenosyl-binding site Lys221 modulates substrate binding and catalysis. FEBS Open Bio 5:824-31
Fratz, Erica J; Hunter, Gregory A; Ferreira, Gloria C (2014) Expression of murine 5-aminolevulinate synthase variants causes protoporphyrin IX accumulation and light-induced mammalian cell death. PLoS One 9:e93078
Stojanovski, Bosko M; Hunter, Gregory A; Jahn, Martina et al. (2014) Unstable reaction intermediates and hysteresis during the catalytic cycle of 5-aminolevulinate synthase: implications from using pseudo and alternate substrates and a promiscuous enzyme variant. J Biol Chem 289:22915-25
Stojanovski, Bosko M; Breydo, Leonid; Hunter, Gregory A et al. (2014) Catalytically active alkaline molten globular enzyme: Effect of pH and temperature on the structural integrity of 5-aminolevulinate synthase. Biochim Biophys Acta 1844:2145-54
Turbeville, Tracy D; Zhang, Junshun; Adams, W Christopher et al. (2011) Functional asymmetry for the active sites of linked 5-aminolevulinate synthase and 8-amino-7-oxononanoate synthase. Arch Biochem Biophys 511:107-17
McIntyre, Neil R; Franco, Ricardo; Shelnutt, John A et al. (2011) Nickel(II) chelatase variants directly evolved from murine ferrochelatase: porphyrin distortion and kinetic mechanism. Biochemistry 50:1535-44

Showing the most recent 10 out of 18 publications