The overall goal of this research program is to elucidate the mechanisms that regulate membrane lipid production and coordinate this process with growth and protein synthesis. A considerable amount of metabolic energy is expended in the biogenesis of membrane lipid and organisms in general exert a high degree of control over the activity of this pathway. In Escherichia coli, the bulk of the available evidence indicates that the control point is at an early step in fatty acid biosynthesis; however, the identity of the rate-controlling enzyme and an understanding of how it is regulated remains one of the major unanswered questions about bacterial physiology. Comparable concentrations of malonyl- and acetyl-acyl carrier protein (ACP) are present in vivo, indicating that the first condensation reaction in fatty aid biosynthesis is rate-limiting. This reaction is catalyzed by a unique condensing enzyme, acetoacetyl-ACP synthase, that is biochemically and genetically distinct from the other beta-kito-acyl-ACP synthases. Acetoacetyl-ACP synthase is ideally positioned in the biochemical pathway to function as the pacemaker of fatty acid production in organisms and organelles that possess dissociated (Type II) fatty acid synthase systems. Nothing is known about the biochemical mechanism, regulatory properties, or genetics of this new condensing enzyme. The work proposed in this application will test the hypothesis that fatty acid biosynthesis, and hence bulk membrane phospholipid formation, is controlled by modulating the activity of acetoacetyl-ACP synthase. The specific experimental aims of the grant will be (1) to purify acetoacetyl-ACP synthase and characterize its biochemical mechanism and regulatory properties, and (2) to isolate mutants with defective or altered acetoacetyl-ACP synthase activity and examine the metabolic basis for any physiological imbalance promoted by these mutations. The results of this work will provide significant new information on the relationship between regulation at the level of fatty acid chain initiation and the overall rate of membrane phospholipid biogenesis.
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