Control of the pyrimidine biosynthetic pathway in Pseudomonas pseudoalcaligenes

Abstract

The five de novo enzyme activities unique to the pyrimidine biosynthetic pathway were found to be present in Pseudomonas pseudoalcaligenes ATCC 17440. A mutant strain with 31-fold reduced orotate phosphoribosyltransferase (encoded by pyrE) activity was isolated that exhibited a pyrimidine requirement for uracil or cytosine. Uptake of the nucleosides uridine or cytidine by wild-type or mutant cells was not detectable; explaining the inability of the mutant strain to utilize either nucleoside to satisfy its pyrimidine requirement. When the wild-type strain was grown in the presence of uracil, the activities of the five de novo enzymes were depressed. Pyrimidine limitation of the mutant strain led to the increase in aspartate transcarbamoylase and dihydroorotate dehydrogenase activities by more than 3-fold, and dihydroorotase and orotidine 5'-monophosphate decarboxylase activities about 1.5-fold, as compared to growth with excess uracil. It appeared that the syntheses of the de novo enzymes were regulated by pyrimidines. In vitro regulation of aspartate transcarbamoylase activity in P. pseudoalcaligenes ATCC 17440 was investigated using saturating substrate concentrations; transcarbamoylase activity was inhibited by Pi, PPi, uridine ribonucleotides, ADP, ATP, GDP, GTP, CDP, and CTP.

Comparison of aspartate transcarbamoylase regulation in Pseudomonas alcaligenes and Pseudomonas mendocina

“…The regulation of the transcarbamoylases by the same trinucleotide inhibitors would seem to indicate that the species are related taxonomically. With respect to the (WEST 1994). In this study, the P. alcaligenes transcarbamoylase was highly inhibited by pyrophosphate, UDP, ADP and CTP under saturating substrate concentrations.…”

“…The P. fluorescens enzyme was inhibited by purine and pyrimidine ribonucleotides as well as by phosphate and pyrophos-phate (ADAIR and JONES 1972). In Pseudomonas pseudoalcaligenes, the aspartate transcarbamoylase activity in cell-free extracts was also strongly inhibited by pyrophosphate as well as by purine and pyrimidine ribonucleotides (WEST 1994). Transcarbamoylase activity in Pseudomonas stutzeri was highly inhibited by pyrophosphate, ATP, UTP and GTP (WEST 1997).…”

“…Aspartate transcarbamoylase activity was assayed at 30 °C using a reaction mix (1 ml) that contained 0.1 M Tris-HCl buffer (pH 8.5), 10 mM L-aspartate (pH 8.5), 1 mM dilithium carbamoylphosphate and cell extract (WEST 1994). An effector concentration of 5 mM was present in the reaction mix.…”

“…In P. fluorescens, the K m value of the purified aspartate transcarbamoylase for L-aspartate or carbamoylphosphate was 1.08 mM or 0.08 mM, respectively (ADAIR andJONES 1972, BERGH andEVANS 1993). The K m of the crude P. pseudoalcaligenes transcarbamoylase for L-aspartate was 2.66 mM while its K m for carbamoylphosphate was 0.29 mM (WEST 1994). The K m of the P. stutzeri transcarbamoylase for L-aspartate was 3.30 mM while its K m for carbamoylphosphate was 0.21 mM (WEST 1997).…”

Regulation of pyrimidine synthesis in Pseudomonas mendocina

“…In particular, pyrimidine biosynthesis in P. mendocina has not been investigated. Prior studies have investigated pyrimidine biosynthesis in a number of species of Pseudomonas (ISAAC and HOLLOWAY 1968, CONDON et al 1976, CHU and WEST 1990, WEST 1994, 1997. The pathway enzymes are aspartate transcarbamoylase (EC 2.1.3.2), dihydroorotase (EC 3.5.2.3), dihydroorotate dehydrogenase (EC 1.3.3.1), orotate phosphoribosyltransferase (EC 2.4.2.10) and orotidine 5'monophosphate (OMP) decarboxylase (EC 4.1.1.23) which are encoded by the genes pyrB, pyrC, pyrD, pyrE and pyrF, respectively (O'DONOVAN and NEUHARD 1970).…”