Regulation of Amino Acid Biosynthesis

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The enzyme aspartokinase plays a central role in bacterial amino acid biosynthesis. It catalyzes the first step in the aspartate pathway, whose products include four of the eight amino acids that are essential for the nutrition of the adult human. The control of the aspartate pathway depends largely on the presence of isozymes of aspartokinase, which differ in the regulation of their synthesis and activity. Our major research accomplishments in the last two years include the general definition at the genetic, enzymatic, and physiological level of the complete aspartokinase isozyme pattern of Bacillus subtilis and the detailed characterization of the operon that encodes one of these isozymes, aspartokinase II (AKII). The primary aim of the proposed research is to study the molecular mechanisms controlling the expression of the AKII operon, the mode of allosteric regulation of AKII by its feedback inhibitor L- lysine, and the physiological function of the two non-identical subunits of AKII. A parallel line of investigation, to be begun while the studies on AKII are in progress, will focus on the other aspartokinase isozymes of B. subtilis, of which one (AKI) is inhibited by the cell wall constitute diaminopmelate and the other (AKIII) by threonine and lysine. AKI functions primarily in the synthesis of diaminopimelate during growth in rich media and in the production of diaminopimelate and dipicolinate during sporulation, whereas AKII and AKIII serve in the biosynthesis of lysine, threonine, and methionine in growth cells. Initial experiments will involve the cloning and sequencing of the genes encoding AKI and AKIII and will be following by the study of the mechanisms that regulate the synthesis and activity of these enzymes. The in-depth study of the control of these important regulatory enzymes will contribute fundamentally to our understanding of the principles and strategies of metabolic regulation.