Akram Bani Ahmad and Thomas P. West
Department of Chemistry, Texas A&M University-Commerce, Commerce, Texas, USA

Part of the book: Pyrimidines and their Importance

Abstract

The pyrimidine biosynthetic pathway consists of five enzymes that are unique to the formation of pyrimidine nucleotides. The pyrimidine biosynthetic pathway enzymes are aspartate transcarbamoylase, dihydroorotase, dihydroorotate dehydrogenase, orotate phosphoribosyltransferase and orotidine 5’-monophosphate decarboxylase. The focus of this study was to explore the regulation of pyrimidine biosynthesis in Pseudomonas chlororaphis ATCC 17414. To do so, the effect of supplementing the pyrimidine bases orotic acid or uracil into a glucose or succinate-containing culture medium of P. chlororaphis ATCC 17414 on the pyrimidine biosynthetic enzyme activities was first explored. Transcarbamoylase and decarboxylase activities were repressed by orotic acid or uracil addition independent of carbon source in the ATCC 17414 cells. Next, a pyrimidine auxotrophic mutant of P. chlororaphis was isolated using chemical mutagenesis and 5-fluororotic acid resistance. The isolated mutant utilized either uracil, uridine or cytosine as a pyrimidine source to support their growth. The mutant strain was deficient for orotidine 5’-monophosphate decarboxylase activity. The mutant cells were subjected to pyrimidine limitation to determine if nucleotide depletion influenced the synthesis of the pyrimidine biosynthetic enzyme activities. The pyrimidine limitation of a pyrimidine auxotrophic strain often causes derepression of the synthesis of the pyrimidine pathway enzymes. When the orotidine 5’-monophosphate decarboxylase mutant strain cells were limited for pyrimidines for one hour, transcarbamoylase, dihydroorotase, dehydrogenase and phosphoribosyltransferase activities were all derepressed compared to their activities in the mutant strain grown in excess uracil. To learn if the initial pathway enzyme aspartate transcarbamoylase was subject to regulation of its activity, the effect of pyrophosphate and ribonucleotides in P. chlororaphis ATCC 17414 cells was studied. The transcarbamoylase was highly inhibited by uridine 5’- monophosphate, uridine 5’-diphosphate, cytidine 5’-monophosphate and guanosine 5’-triphosphate in the glucose-grown ATCC 17414 cells. Overall, it was concluded that pyrimidine biosynthetic pathway enzymes in P. chlororaphis ATCC 17414 was regulated at the level of enzyme synthesis and that the initial pathway enzyme aspartate transcarbamoylase activity was controlled at the level of enzyme activity.

Keywords: pyrimidine, biosynthesis, bacterium, pseudomonad, orotic acid, uracil

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