MOLECULAR BASIS AND ENZYMOLOGY OF MICROBIAL BIOSYNTHESIS

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Normal and healthy cellular activity is dependent on a concerted interplay of genetic regulation and protein function. Our primary interest and long- term goal is to understand genetic and biochemical factors which influence the function of an important metabolic route, the pentose phosphate pathway. This is a metabolic scheme required for the metabolism of virtually all living organisms. With the addition of two unique enzymes, ribulose bisphosphate carboxylase/oxygenase (RubisCO) and phosphoribulokinase (PRK), this pathway functions in a purely biosynthetic mode, such that organisms gain the capacity to use carbon dioxide as the sole source of carbon. Under these conditions, other enzymes of the pathway, including ubiquitous catalysts such as transkelotase (TK) and fructose l ,6-bisphosphatase (FBPase), found in both prokaryotes and eukaryotes, function as biosynthetic enzymes, in the opposite direction from their usual role in vivo. The genes encoding nearly all the enzymes of this pathway have been isolated and found to be associated in distinct chromosomal operons (the cbb regulon) in the nonsulfur purple bacterium Rhodobacter sphaeroides. In addition, a transcriptional activator protein and a gene that encodes a sensor kinase, have been found to regulate the expression of the cbb genes. Other regulatory elements have also been discovered and much of this study is directed at elucidating the regulatory mechanism that mitigates the sensory transduction pathway controlling gene expression and ultimately biosynthetic metabolism. Since exposure to varying levels of carbon and oxygen has a profound effect on gene expression, a concerted effort will focus on relating such external stimuli to the regulatory cascade. The second major thrust of this project will involve a study of structure- function relationships of PRK, FBPase, and TK. Since their genes have been expressed as highly active recombinant proteins that are easily purified, it will be possible to use site-directed mutagenesis procedures in combination with known x-ray structural models to enhance our knowledge of how these proteins function. These are all extremely important metabolic enzymes; for example TK is required for thiamine metabolism in all cells and alteration of its kinetic and chemical properties leads to severe pathological conditions including nutritional deficiency, alcoholism, Wernicke-Korsakoff encephalopathy, and Alzheimer's disease. FBPase is essential for gluconeogenesis and PRO is one of the enzymes unique to the reductive pentose phosphate pathway. In each case, the recombinant systems developed here have the potential to substantially increase available information of these enzymes thus affording an unusual opportunity to relate the control of cellular metabolism to the function and structure of key catalysts.