5-Aminolevulinate Synthase and Heme Biosynthesis

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DESCRIPTION (provided by applicant): 5-Aminolevulinate synthase (ALAS), a pyridoxal 5'-phosphate (PLP)-dependent enzyme of the alpha-oxoaminesynthase family, catalyzes the first and regulatory step of the mammalian heme biosynthetic pathway. Mutations in the gene encoding the erythroid ALAS isoform cause X-linked sideroblastic anemia (XLSA), an erythropoietic disorder resulting in increased tissue iron levels. The prospect of developing pyridoxine-based therapies of universal efficacy for XLSA relies on the knowledge of the mechanism of ALAS, the cofactor-binding pocket (PLP-fold) and their relationship. It is the design of the active site, entailing the PLP cofactor-protein interaction, that discriminates one type of reaction among the wide gamut of PLP-dependent enzyme-catalyzed reactions. Recent studies in the P.I.'s laboratory on the architecture of the active site and mechanism of ALAS (1996-2002)set the stage for the following 3 hypotheses to be tested: 1. Modulation of the PLP cofactor chemistry controls the enzymatic mechanism of ALAS. 2. Substrate specificity of ALAS can be acquired through minor modifications of the protein scaffold for the ctoxoaminesynthase family of PLP-dependent enzymes, which possesses the same general PLP-binding fold and chemistry of catalysis. 3. The distinct catalytic chemistries of ALAS and glutamate l-semialdehyde aminomutase (GSA-AT), two PLP-dependent enzymes which both produce 5-aminolevulinate (ALA) and play crucial roles in the two natural ALA biosynthetic pathways (e.g., in animals and plants), can be generated by "evolution" of a primary protein scaffold. The proposed studies provide a new strategy to establish structure/function relationships within ALAS and other alpha-oxoamine synthase enzymes. Further, the proposed studies provide a novel approach to understanding how shuffling protein structural domains changes function and how enzyme active sites evolve; this strategy promises to be a key particularly for determining the function of unidentified genes in the human genome.