Regulation of quinolinic acid phosphoribosyltransferase in root cultures of Nicotiana alata Link and Otto

Quinolinic acid phosphoribosyltransferase (QAPRTase) is the anaplerotic enzyme responsible for regulating flux of the pyridine ring into the pyridine nucleotide pathway in higher plants as well as into alkaloid biosynthesis pathways of pyridine alkaloid producing species such as Nicotiana alata. The regulation of this enzyme was examined with respect to its ability to influence both a major primary metabolic pathway (pyridine nucleotide) as well as a secondary metabolite pathway (pyridine alkaloid) simultaneously and in response to widely divergent environmental stimuli.;Untransformed Nicotiana alata root cultures were used as an experimental system to examine the effect of N-methyl nicotinic acid (trigonelline) and (;Putrescine methyl transferase (PMT) is a key regulatory enzyme in the pathway leading to the synthesis of N-methyl pyrrolinium which provides the pyrrolinium ring of the major alkaloids produced by Nicotiana alata, nicotine and nornicotine. Since both the pyridine ring and the pyrrolinium ring are required for the production of nicotine and nornicotine, the effect of (;The relevance of these results is discussed in relation to the interaction of primary metabolism and the metabolic response to both herbivory and microbial pathogen attack in plants.;In addition, several strategies were used in an attempt to clone the gene for QAPRTase in order to examine regulation of this enzyme at the level of transcription. Direct genetic selection for functional rescue of an Escherichia coli, nadC (bacterial gene for QAPRTase) deletion mutant was the first strategy used. The nicotinic acid auxotroph was transformed with two cDNA libraries constructed from actively growing tissues. No functional rescue resulted.;The second cloning method involved purifying the QAPRTase protein from endosperm tissue of Ricinus communis. Results indicate that several forms of QAPRTase may exist and that the native structure of the enzyme is heterodimeric, providing a possible reason for the negative results in the attempted functional rescue of the E. coli nadC deletion mutant. Purified protein was used for polyclonal antibody production. No binding to the antigen was observed in western blots.