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Guorong Xu

Document Type

Dissertation - University Access Only

Award Date


Degree Name

Doctor of Philosophy (PhD)

Department / School


First Advisor

Thomas P. West


Catabolism of the pyrimidine bases uracil and thymine as well as genie regulation of this catabolism was investigated for Pseudomonas stutzeri ATCC 17588. The ability of this pseudomonad strain to utilize pyrimidine bases and their respective catabolic products as nitrogen sources was examined in a liquid Stanier minimal medium containing the carbon source succinate (0.4%) or glucose (0.4%). Growth of P. stutzeri was observed after 3 days at 30°C on either uracil, dihydrouracil, β-alanine, thymine, dihydrothymine or β-aminoisobutyric acid (0.2%) as a nitrogen source. The pyrimidine reductive catabolic pathway enzymes, namely dihydropyrimidine dehydrogenase, dihydropyrimidinase, and N-carbamoyl-β-alanine amidohydrolase, were assayed in extracts prepared from cells grown in minimal medium as well as in media containing pyrimidines or their catabolic products as a sole nitrogen sources. The results indicated reductive catabolism of pyrimidines was occurring and that pyrimidines or their catabolic products did influence the levels of the reductive pathway enzymes. In addition, reductive catabolism of the pyrimidine bases was found to be inducible by uracil or thymine. Thus, regulation of pyrimidine catabolism in P. stutzeri was shown to exist. The second enzyme of the pyrimidine reductive catabolic pathway is dihydropyrimidinase (4, 5- dihydropyrimidine amidohydrolase, EC or hydantoinase. This enzyme is responsible for the hydrolytic ring-opening reactions of dihydrouracil, dihydrothymine, or hydantoin. In this work, a partial purification of dihydropyrimidinase from P. stutzeri has been performed. After the enzyme was partially purified, the biochemical properties of dihydropyrimidinase were characterized. These properties included the effects of temperature and pH on the enzyme activity, the metal ion requirements for enzyme activity, enzyme substrate specificity as well as its enzyme kinetics. Dihydropyrimidinase showed an optimal activity at 45°C and optimal pH between 7.5 and 9.0. The enzyme can be reactivated by Co2+ and is inhibited by Zn2+. The Km of the enzyme for dihydrouracil, dihydrothymine, and hydantoin was 1.96 x 10-5 M, 2.13 x 10-5 M, and 3.64 x 10-5 M, respectively. The Vmax of the enzyme for the three substrates was 0.836, 0.666, and 2.210 µmol/min, respectively. Using gel filtration, the molecular weight for dihydropyrimidinase was shown to be approximately 240,000 dal tons. Using spectroscopy, it appeared that the enzyme contained non-heme iron and cobalt.

Library of Congress Subject Headings

Pyrimidines -- Metabolism




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