Characterization of Aminolevulinate Synthase

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The biosynthesis of tetrapyrroles is initiated from simple molecules and comprises few enzymatic steps. Moreover, the pathway for the biosynthesis of tetrapyrroles is remarkably similar in all living systems; it is initiated with the biosynthesis of 5- aminolevulinate (ALA). In animals, fungi and some bacteria, the first step of the pathway is catalyzed by ALA synthase, the enzyme of focus of this proposal. While ALA synthase has been recognized as a key enzyme in heme biosynthesis since its discovery in 1958, there has been limited opportunity to study its structure and function due to the inability to obtain high concentrations of purified, active enzyme. The long term goal of this project is to understand how 5-aminolevulinic acid, the first committed intermediate in the tetrapyrrole biosynthetic pathway, is synthesized in higher animals, and the mechanism by which this process is regulated both in liver and erythroid cells. Future studies projected for the next two years will utilize chemical, biochemical, and molecular biological approaches to overexpress the mammalian 5-aminoevulinate synthase gene in E. coli and purify the overexpressed enzyme in active form; and identify the pyridoxal phosphate binding site and active site of the 5-aminolevulinate synthase. %%% As 5-aminolevulinate synthase is the first enzyme involved in the biosynthesis of tetrapyrroles, and the rate-limiting enzyme of the heme biosynthetic pathway in the liver, these studies are of fundamental importance to the understanding of the metabolism of hemes, chlorophylls, corrins and bile pigments.