MOLECULAR BASIS AND ENZYMOLOGY OF MICROBIAL BIOSYNTHESIS

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The pentose phosphate pathway is an important metabolic scheme required for the metabolism of virtually all living organisms. The addition of two unique enzymes, ribulose bisphosphate carboxylase/oxygenase (RubisCO) and phosphoribulokinase (PRK), allows this pathway to function 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 found in both prokaryotes and eukaryotes, function as biosynthetic enzymes, in the opposite direction form their usual role in vivo. Recent work from our laboratory has shown that the structural genes for the above enzymes, along with genes encoding fructose bisphosphatase (FBPase), glyceraldehyde phosphate dehydrogenase, and transketolase, are found in two distinct chromosomal operons in the facultatively anaerobic purple nonsulfur bacterium Rhodobacter sphaeroides. Both recombinant proteins in Escherichia coli, and many of the recombinant proteins purified to homogeneity. Thus, one of the major goals of this project is to study structure-function relationships of PRK, FBPase and transketolase, using the techniques of protein chemistry and molecular biology, the first time that this dual approach has become feasible for these important proteins. The second major thrust of this project will be to study the molecular basis for differential regulation of the two operons. Control is manifested at both the transcriptional and posttranscriptional levels. Since exposure to increasing or decreasing levels of carbon and oxygen has a major effect on gene expression, we will probe the mechanism of control and its relation to intracellular regulatory systems known to be mechanism of control and its relation to intracellular regulatory systems known to be responsive to external stimuli. This research also focuses on the role of nucleolytic processing of polycistronic transcripts and its relation to differential gene expression, a key factor important for regulating expression of both prokaryotic and eukaryotic genes. These studies thus present an excellent opportunity to relate control at both the enzymic and molecular levels, to increase our general understanding of intermediary metabolism.