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 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.
Berglez
Janette
J
RMIT University, Bundoora, Vic., Australia.
Pilling
Patricia
P
Macreadie
Ian
I
Fernley
Ross T
RT
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
C105176:6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase-7,8-dihydropteroate synthase; D009097:Multienzyme Complexes; D029701:Saccharomyces cerevisiae Proteins; D005492:Folic Acid