The long term objective is to understand the role of 5- aminolevulinate synthase (ALAS), both at the enzyme and gene levels, in the regulation of heme biosynthesis in differentiating erythrocytes. ALAS catalyzes the first and rate limiting step in heme biosynthesis, the PLP-dependent reaction of glycine and succinyl-CoA to yield aminolevulinic acid, CoA and CO2. ALAS synthesis, although ubiquitous, occurs predominately in erythrocytes and hepatocytes, where demands are greater because of the synthesis of hemoglobin and cytochrome P-450, respectively. The erythroid-specific isoform of ALAS (ALAS-E) is expressed concomitantly with the differentiation and maturation of the erythroid cells. Recently a heterozygous group of point mutations in the catalytic domain of the ALAS-E enzyme has been found to cause the human genetic disorder X-linked sideroblastic anemia (XLSA). Characterization of the molecular mechanisms of the ALAS- catalyzed reaction and of the regulation of the expression of the ALAS-E gene are essential to design improved therapies for XLSA patients and/or for improved diagnosis for at-risk family members. Studies projected for the next four years will utilize chemical, biochemical, physical and molecular biological approaches to address three major specific aims: i) to define the role of K313 in the mammalian erythroid ALAS-catalyzed reaction; ii) to identify the functionally important residues of the glycine loop in the PLP cofactor binding site of ALAS; and iii) to evaluate transcriptional regulation of the mammalian erythroid ALAS (ALAS) gene. Results from these studies will provide the first characterization of the function and mechanism of ALAS at the molecular level and will elucidate the regulatory mechanism(s) of expression of the ALAS-E in differentiation erythrocytes. Significantly, they will provide the framework for the interpretation of the already identified ALAS-E genetic mutations associated with XLSA.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK052053-02
Application #
2634300
Study Section
Biochemistry Study Section (BIO)
Project Start
1997-01-01
Project End
2000-12-31
Budget Start
1997-12-31
Budget End
1998-12-30
Support Year
2
Fiscal Year
1998
Total Cost
Indirect Cost
Name
University of South Florida
Department
Biochemistry
Type
Schools of Medicine
DUNS #
City
Tampa
State
FL
Country
United States
Zip Code
33612
Ferreira, Gloria C; Cheltsov, Anton V (2002) Circular permutation of 5-aminolevulinate synthase as a tool to evaluate folding, structure and function. Cell Mol Biol (Noisy-le-grand) 48:11-6
Ferreira, G C; Zhang, J S (2002) Mechanism of 5-aminolevulinate synthase and the role of the protein environment in controlling the cofactor chemistry. Cell Mol Biol (Noisy-le-grand) 48:827-33
Goodfellow, B J; Dias, J S; Ferreira, G C et al. (2001) The solution structure and heme binding of the presequence of murine 5-aminolevulinate synthase. FEBS Lett 505:325-31
Cheltsov, A V; Barber, M J; Ferreira, G C (2001) Circular permutation of 5-aminolevulinate synthase. Mapping the polypeptide chain to its function. J Biol Chem 276:19141-9
Kramer, M F; Gunaratne, P; Ferreira, G C (2000) Transcriptional regulation of the murine erythroid-specific 5-aminolevulinate synthase gene. Gene 247:153-66
Hunter, G A; Ferreira, G C (1999) Pre-steady-state reaction of 5-aminolevulinate synthase. Evidence for a rate-determining product release. J Biol Chem 274:12222-8
Hunter, G A; Ferreira, G C (1999) Lysine-313 of 5-aminolevulinate synthase acts as a general base during formation of the quinonoid reaction intermediates. Biochemistry 38:3711-8
Tan, D; Barber, M J; Ferreira, G C (1998) The role of tyrosine 121 in cofactor binding of 5-aminolevulinate synthase. Protein Sci 7:1208-13
Gong, J; Hunter, G A; Ferreira, G C (1998) Aspartate-279 in aminolevulinate synthase affects enzyme catalysis through enhancing the function of the pyridoxal 5'-phosphate cofactor. Biochemistry 37:3509-17
Tan, D; Harrison, T; Hunter, G A et al. (1998) Role of arginine 439 in substrate binding of 5-aminolevulinate synthase. Biochemistry 37:1478-84