Normal and healthy cellular activity is dependent on a concerted interplay of genetic regulation and protein function. Our primary interest and longterm goal is to understand genetic and biochemical factors which influence the function of an important metabolic route, the pentose phosphate pathway, required for the metabolism of virtually all organizms. With the addition of two unique enzymes, this pathway functions in a purely biosynthetic mode, such that organizms gain the capacity to use carbon dioxide as a carbon source. Uner these conditions, other enzymes of the pathway, including transketolase (TK) and epimerase (found in virtually all organism) function as biosynthetic catalysts, differen from their usual role in vivo. Structural genes of this pathway have been isolated and are associated in distinct operons on two different chromosomes in the bacterium Rhodobacter sphaeroides, all controlled by a single transcriptional activator protein. The """"""""regulon"""""""" is influenced by other regulators as well, including a global signal transduction system that also controls nitrogen and energy metabolism. This study will thus seek to understand how such regulatory determinants influence the totality of interdependent metabolism required for cellular function, involving molecular signals from more than one chromosome. Regulatory events that signal a cascade of responses influenced by external stimuli will be established. The above studies also present opportunities to investigate the detailed biochemistry of regulator molecules recently discovered, such tha th function of these proteins may be related to their structure, and to the regulatory processes they influence. Included in the structure-function studies of thie project are enzymes that catalyze important pentose phosphate interconversions, each of which plays a significant role during metabolism. For example, TK is required fro thiamine metabolism in all cells and alteration of its functional properties leads to severe pathologies including nutritional deficiency, alcoholism, Wernicke-Korsakoff encephalopathy, and Alzheimer's disease. For each enzyme, recombinant systems will be used to facilitate efforts to learn how these proteins function specifically in biosynthesis and how control of their activity influences normal cellular metabolism.
