Purification, properties, and crystallization of Saccharomyces cerevisiae dihydropterin pyrophosphokinase-dihydropteroate synthase
The tri-functional enzyme of Saccharomyces cerevisiae dihydroneopterin aldolase (DHNA)-dihydropterin pyrophosphokinase (PPPK)-dihydropteroate synthase (DHPS) catalyzes three sequential steps in folate biosynthesis. A cDNA encoding the PPPK and DHPS domains of the tri-functional enzyme has been clone...
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| Veröffentlicht in: | Protein expression and purification 2005-06, Vol.41 (2), p.355-362 |
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| creator | Berglez, Janette Pilling, Patricia Macreadie, Ian Fernley, Ross T |
| description | The tri-functional enzyme of Saccharomyces cerevisiae dihydroneopterin aldolase (DHNA)-dihydropterin pyrophosphokinase (PPPK)-dihydropteroate synthase (DHPS) catalyzes three sequential steps in folate biosynthesis. A cDNA encoding the PPPK and DHPS domains of the tri-functional enzyme has been cloned. This bi-functional enzyme was expressed as a His(6) fusion protein in Escherichia coli and the protein was purified to apparent homogeneity. The purified protein possesses both PPPK and DHPS activities as measured by the incorporation of [(3)H]p-ABA into the appropriate substrate. The pH optimum of the DHPS activity was determined to be 8.5. Gel filtration measurement indicates that the protein exists as a dimer in solution. A robotic screening method was used to identify crystallization conditions. Bi-pyramidal crystals of the enzyme formed with the protein in the presence of a pterin substrate analog in phosphate buffer (pH 6.3) and these diffracted to 2.3A. Structural information from these crystals could be used to design novel drugs to inhibit folate biosynthesis. |
| doi_str_mv | 10.1016/j.pep.2005.02.003 |
| format | Article |
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A cDNA encoding the PPPK and DHPS domains of the tri-functional enzyme has been cloned. This bi-functional enzyme was expressed as a His(6) fusion protein in Escherichia coli and the protein was purified to apparent homogeneity. The purified protein possesses both PPPK and DHPS activities as measured by the incorporation of [(3)H]p-ABA into the appropriate substrate. The pH optimum of the DHPS activity was determined to be 8.5. Gel filtration measurement indicates that the protein exists as a dimer in solution. A robotic screening method was used to identify crystallization conditions. Bi-pyramidal crystals of the enzyme formed with the protein in the presence of a pterin substrate analog in phosphate buffer (pH 6.3) and these diffracted to 2.3A. 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A cDNA encoding the PPPK and DHPS domains of the tri-functional enzyme has been cloned. This bi-functional enzyme was expressed as a His(6) fusion protein in Escherichia coli and the protein was purified to apparent homogeneity. The purified protein possesses both PPPK and DHPS activities as measured by the incorporation of [(3)H]p-ABA into the appropriate substrate. The pH optimum of the DHPS activity was determined to be 8.5. Gel filtration measurement indicates that the protein exists as a dimer in solution. A robotic screening method was used to identify crystallization conditions. Bi-pyramidal crystals of the enzyme formed with the protein in the presence of a pterin substrate analog in phosphate buffer (pH 6.3) and these diffracted to 2.3A. Structural information from these crystals could be used to design novel drugs to inhibit folate biosynthesis.</description><subject>Amino Acid Sequence</subject><subject>Crystallization</subject><subject>Crystallography, X-Ray</subject><subject>Dimerization</subject><subject>Enzyme Stability</subject><subject>Folic Acid - biosynthesis</subject><subject>Gene Expression Regulation, Enzymologic</subject><subject>Hydrogen-Ion Concentration</subject><subject>Molecular Sequence Data</subject><subject>Multienzyme Complexes - chemistry</subject><subject>Multienzyme Complexes - genetics</subject><subject>Multienzyme Complexes - isolation & purification</subject><subject>Saccharomyces cerevisiae - enzymology</subject><subject>Saccharomyces cerevisiae Proteins - chemistry</subject><subject>Saccharomyces cerevisiae Proteins - genetics</subject><subject>Saccharomyces cerevisiae Proteins - isolation & purification</subject><subject>Sequence Alignment</subject><subject>Sequence Homology, Amino Acid</subject><issn>1046-5928</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2005</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkU1P3DAQhn1oBRT6A3qpfOqJpONv51ghaJGQqAQ9W44z0XqbTVI7Wyk99pdjYCW4cbDG0jzvSDMPIZ8Y1AyY_rqtZ5xrDqBq4DWAeEdOGEhdqYbbY_Ih5y0AYxrUETlmymptOD8h_3_uU-xj8EucxnM6p2nGtETM59SPHQ1pzYsfhvjvCaBTT-98CBufpt0aMNOACf_GHD3SLm7WruQXTHGk81q-mymX9zuOPmP1qj_5BWlex2VTGmfkfe-HjB8P9ZT8urq8v_hR3dx-v774dlMFodlS-QaYtaoXXSP7TljL2kb1UnUs8HIBbrrWcoHo26YNIAxyA8pIJhvNfQAUp-TL89yy5J895sXtYg44DH7EaZ-dNsZaoc2bIG-s1NzKN0FmtJBSQQHZMxjSlHPC3s0p7nxaHQP3qM9tXdHnHvU54K7oK5nPh-H7dofdS-LgTjwA2AKc4g</recordid><startdate>20050601</startdate><enddate>20050601</enddate><creator>Berglez, Janette</creator><creator>Pilling, Patricia</creator><creator>Macreadie, Ian</creator><creator>Fernley, Ross T</creator><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7T7</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>M7N</scope><scope>P64</scope><scope>7SC</scope><scope>7SP</scope><scope>7TB</scope><scope>F28</scope><scope>JQ2</scope><scope>L7M</scope><scope>L~C</scope><scope>L~D</scope><scope>7X8</scope></search><sort><creationdate>20050601</creationdate><title>Purification, properties, and crystallization of Saccharomyces cerevisiae dihydropterin pyrophosphokinase-dihydropteroate synthase</title><author>Berglez, Janette ; 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A cDNA encoding the PPPK and DHPS domains of the tri-functional enzyme has been cloned. This bi-functional enzyme was expressed as a His(6) fusion protein in Escherichia coli and the protein was purified to apparent homogeneity. The purified protein possesses both PPPK and DHPS activities as measured by the incorporation of [(3)H]p-ABA into the appropriate substrate. The pH optimum of the DHPS activity was determined to be 8.5. Gel filtration measurement indicates that the protein exists as a dimer in solution. A robotic screening method was used to identify crystallization conditions. Bi-pyramidal crystals of the enzyme formed with the protein in the presence of a pterin substrate analog in phosphate buffer (pH 6.3) and these diffracted to 2.3A. Structural information from these crystals could be used to design novel drugs to inhibit folate biosynthesis.</abstract><cop>United States</cop><pmid>15866722</pmid><doi>10.1016/j.pep.2005.02.003</doi><tpages>8</tpages></addata></record> |
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| subjects | Amino Acid Sequence Crystallization Crystallography, X-Ray Dimerization Enzyme Stability Folic Acid - biosynthesis Gene Expression Regulation, Enzymologic Hydrogen-Ion Concentration Molecular Sequence Data Multienzyme Complexes - chemistry Multienzyme Complexes - genetics Multienzyme Complexes - isolation & purification Saccharomyces cerevisiae - enzymology Saccharomyces cerevisiae Proteins - chemistry Saccharomyces cerevisiae Proteins - genetics Saccharomyces cerevisiae Proteins - isolation & purification Sequence Alignment Sequence Homology, Amino Acid |
| title | Purification, properties, and crystallization of Saccharomyces cerevisiae dihydropterin pyrophosphokinase-dihydropteroate synthase |
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