Regulation of the Pyrimidine Biosynthetic Pathway In Pseudomonas Lemonnieri

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Chemistry

Date of Award

1-10-2024

Abstract

The focus is on the regulation of the pyrimidine biosynthetic pathway in a soil bacterium. The five pathway enzymes unique to pyrimidine formation are aspartate transcarbamoylase, dihydroorotase, dihydroorotate dehydrogenase, orotate phosphoribosyl transferase, and orotidine 5'-monophosphate (OMP) decarboxylase. The impact of 0.4% carbon source and pyrimidine base supplementation on the pyrimidine biosynthetic enzyme activities in the bacterium Pseudomonas lemonnieri ATCC 12983 was determined from enzyme assays. The bacterial cells will be grown in a minimal medium containing 0.4% glucose or sodium succinate as a carbon source and either 50 milligrams/liter of uracil or orotic acid. The late exponential phase-growing cells are collected by centrifugation, ultrasonically disrupted with the cell extract, then centrifuged, followed by dialysis overnight at 4°C. The carbon source impacted the de novo pyrimidine biosynthesis pathway in P. lemonnieri ATCC 12983 by exhibiting higher activities in the glucose medium compared to the succinate medium except for the OPRTase activity. An examination of uracil or orotic acid addition to the culture medium assay was investigated. It was found that pyrimidine addition to the medium affected biosynthetic enzymes differently with carbon source being a factor. After these studies, the isolation of pyrimidine auxotrophic strains using chemical mutagen ethyl methyl sulfonate (EMS) and resistance to 5-fluoorotic acid and 1 milligram/liter uracil. The colonies on the plates were screened for uracil auxotrophy following 96 hours at 300C. Compared to the pyrimidine biosynthetic enzyme activities in uracil-supplemented ATCC 12983 glucose-grown cells, OPRTase or OMP decarboxylase activity was observed to be deficient in the cell extracts of individual mutant strains. Characterization of the uracil requiring mutant strain's growth on solid glucose or succinate minimal medium revealed that either cytosine or uridine could also meet the pyrimidine auxotrophy of the strains. The mutants were limited to pyrimidine for a period of 1 or 2 hours to see the effect of the enzyme activities relative to the carbon source. The starvation experiments in glucose medium have shown increased activities when starved for 1 hour and with their activities increasing further after 2 hours except for ATCase activity. Whereas the starvation in succinate medium decreased the activities for 1 hour of limitation with 2 hours exhibiting a further significant decrease in activities. The regulation of the known regulatory enzyme ATCase activity in vitro in P. lemonnieri was determined with the Km value for L-aspartate being 2.55 (0.15) mM and carbamoyl phosphate being 0.47 (0.30) mM. The study focused on the inhibition of ATCase activity in the presence of nucleotide effectors. The highest inhibition occurred in the presence of 5 mM PPi, AMP, ADP, UMP, GMP, GTP, CMP, and CTP. In conclusion, this investigation demonstrates that the pyrimidine biosynthetic enzyme synthesis and transcarbamoylase activity were regulated in P. lemonnieri. Overall, this study provides new information regarding the regulation of pyrimidine biosynthesis by pyrimidines and related compounds in P. lemonnieri.

Advisor

Thomas West

Subject Categories

Biochemistry | Biochemistry, Biophysics, and Structural Biology | Life Sciences

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