Structure-Function Studies on Aspartate Transcarbamoylase and Regulation of Pyrimidine Biosynthesis by a Positive Activator Protein, PyrR in Pseudomonas putida

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The regulation of pyrimidine biosynthesis was studied in Pseudomonas putida. The biosynthetic and salvage pathways provide pyrimidine nucleotides for RNA, DNA, cell membrane and cell wall biosynthesis. Pyrimidine metabolism is intensely studied because many of its enzymes are targets for chemotheraphy. Four aspects of pyrimidine regulation are described in this dissertation. Chapter I compares the salvage pathways of Escherichia coli and P. putida. Surprisingly, P. putida lacks several salvage enzymes including nucleoside kinases, uridine phosphorylase and cytidine deaminase. Without a functional nucleoside kinase, it was impossible to feed exogenous uridine to P. putida. To obviate this problem, uridine kinase was ... continued below

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Kumar, Alan P. December 2003.

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  • Kumar, Alan P.

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The regulation of pyrimidine biosynthesis was studied in Pseudomonas putida. The biosynthetic and salvage pathways provide pyrimidine nucleotides for RNA, DNA, cell membrane and cell wall biosynthesis. Pyrimidine metabolism is intensely studied because many of its enzymes are targets for chemotheraphy. Four aspects of pyrimidine regulation are described in this dissertation. Chapter I compares the salvage pathways of Escherichia coli and P. putida. Surprisingly, P. putida lacks several salvage enzymes including nucleoside kinases, uridine phosphorylase and cytidine deaminase. Without a functional nucleoside kinase, it was impossible to feed exogenous uridine to P. putida. To obviate this problem, uridine kinase was transferred to P. putida from E. coli and shown to function in this heterologous host. Chapter II details the enzymology of Pseudomonas aspartate transcarbamoylase (ATCase), its allosteric regulation and how it is assembled. The E. coli ATCase is a dodecamer of two different polypeptides, encoded by pyrBI. Six regulatory (PyrI) and six catalytic (PyrB) polypeptides assemble from two preformed trimers (B3) and three preformed regulatory dimers (I2) in the conserved 2B3:3I2 molecular structure. The Pseudomonas ATCase also assembles from two different polypeptides encoded by pyrBC'. However, a PyrB polypeptide combines with a PyrC. polypeptide to form a PyrB:PyrC. protomer; six of these assemble into a dodecamer of structure 2B3:3C'2. pyrC' encodes an inactive dihydroorotase with pyrB and pyrC' overlapping by 4 bp. Chapter III explores how catabolite repression affects pyrimidine metabolism. The global catabolite repression control protein, Crc, has been shown to affect pyrimidine metabolism in a number of ways. This includes orotate transport for use as pyrimidine, carbon and nitrogen sources. Orotate is important because it interacts with PyrR in repressing the pyr genes. Chapter IV describes PyrR, the positive activator of the pyrimidine pathway. As with other positive activator proteins, when pyrimidine nucleotides are depleted, PyrR binds to DNA thereby enhancing expression of pyrD, pyrE and pyrF genes. When pyrimidine nucleotides are in excess, the PyrR apoprotein binds to orotate, its co-repressor, to shut down all the pyrimidine genes. Like many positive activators, PyrR is subject to autoregulation and has catalytic activity for uracil phosphoribosyltransferase inducible by orotate.

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  • December 2003

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  • Feb. 15, 2008, 3:10 p.m.

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Kumar, Alan P. Structure-Function Studies on Aspartate Transcarbamoylase and Regulation of Pyrimidine Biosynthesis by a Positive Activator Protein, PyrR in Pseudomonas putida, dissertation, December 2003; Denton, Texas. (digital.library.unt.edu/ark:/67531/metadc4362/: accessed November 23, 2017), University of North Texas Libraries, Digital Library, digital.library.unt.edu; .