Deoxyribonucleotide biosynthesis in green algae: characterization of thymidylate synthase-dihydrofolate reductase in Scenedesmus obliquus |
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| Authors: | B Bachmann H Follmann |
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| Institution: | 1. Department of Food Science and Experimental Nutrition, School of Pharmaceutical Sciences, University of São Paulo, Av. Prof. Lineu Prestes, 580, Bl. 14, São Paulo, SP, Brazil;2. Department of Exact Science—Statistical Analysis Laboratory, Luiz de Queiroz College of Agriculture, University of São Paulo, Av. Padua Dias, 11, Piracicaba, SP, Brazil;3. University of São Paulo—NAPAN—Food and Nutrition Research Center, Av. Prof. Lineu Prestes, 580, Bl. 14, São Paulo, Brazil;4. Food Research Center (FoRC), CEPID-FAPESP (Research, Innovation and Dissemination Centers, São Paulo Research Foundation), Av. Prof. Lineu Prestes, 580, Bl. 13A, São Paulo, Brazil;1. Dipartimento di Scienze del Farmaco, Università del Piemonte Orientale, Via Bovio 6, 28100, Novara, Italy;2. IXTAL srl, Via Bovio 6, 28100, Novara, Italy;3. Department of Bioscience, Graduate School of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama, Ehime, 790-8566, Japan;4. Faculty of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama, Ehime, 790-8566, Japan;5. Center for Marine Environmental Studies (CMES), Ehime University, 2-5 Bunkyo-cho, Matsuyama, Ehime, 790-8577, Japan;1. Federal University of Santa Catarina, Department of Chemical Engineering, 88034-001, Florianópolis, Santa Catarina, Brazil;2. West University of Santa Catarina, Bioenergy Engineering, 89820-000, Xanxerê, Santa Catarina, Brazil;3. Embrapa Swine and Poultry, 89700-000, Concórdia, Santa Catarina, Brazil;4. West University of Santa Catarina, Biotechnology and Sciences Program, 89560-000, Videira, Santa Catarina, Brazil;5. Embrapa Soybean, 86001-970, Londrina, Parana, Brazil;1. University of Cologne, Department of Chemistry, Institute of Biochemistry, Zülpicher Str. 47, D-50674 Cologne, Germany;2. Department of Plant-Microbe Interactions, Max-Planck Institute for Plant Breeding Research, Carl-von-Linné-Weg 10, D-50829 Cologne, Germany;1. Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot 76100, Israel;2. Structural Proteomic Unit, Weizmann Institute of Science, Rehovot 76100, Israel;3. Department of Plant Molecular Biology and Ecology, Tel-Aviv University, Tel-Aviv 69978, Israel |
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| Abstract: | Thymidylate synthase and dihydrofolate reductase are peak enzymes that accompany the S phase of the unicellular green algae, Scenedesmus obliquus, and are both overproduced in the presence of 5-fluorodeoxyuridine. Such overproducing cultures have served for enzyme isolation and characterization. It has not been possible to separate the two enzyme activities by several methods of protein fractionation, including affinity chromatography on specific immobilized ligands (fluorodeoxyuridylate or N10-formylfolate); both were enriched in parallel approximately 400-fold from algal extracts. The most highly purified samples are of low stability in solution. Enzyme activities are inhibited by methotrexate, 5-fluorodeoxyuridylate, and arabinouridylate but not by hydroxyurea; FdUMP inhibition is fully reversed after removal of the nucleotide. Sedimentation in sucrose gradients (Mr 100,000) and electrophoresis in denaturing polyacrylamide gels (Mr 50,000) suggest that the protein structure resembles more the dimeric, bifunctional thymidylate synthase-dihydrofolate reductase of protozoan species than the separate enzymes found in bacteria and animal cells. |
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