title: Purification, properties, and crystallization of Saccharomyces cerevisiae dihydropterin pyrophosphokinase-dihydropteroate synthase.
abstract: 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.
authors.affiliation: RMIT University, Bundoora, Vic., Australia.
authors.forename: Janette
authors.initials: J
authors.lastname: Berglez
authors.forename: Patricia
authors.initials: P
authors.lastname: Pilling
authors.forename: Ian
authors.initials: I
authors.lastname: Macreadie
authors.forename: Ross T
authors.initials: RT
authors.lastname: Fernley
mesh_terms: D000595:Amino Acid Sequence; D003460:Crystallization; D018360:Crystallography, X-Ray; D019281:Dimerization; D004795:Enzyme Stability; D005492:Folic Acid / Q000096:biosynthesis; D015971:Gene Expression Regulation, Enzymologic; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
chemical_list: C105176:6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase-7,8-dihydropteroate synthase; D009097:Multienzyme Complexes; D029701:Saccharomyces cerevisiae Proteins; D005492:Folic Acid