Phosphatidylinositol synthase from yeast.
PI is an important precursor for polyphosphoinositides and some sphingolipids and is also involved in the glycolipid anchoring of plasma membrane proteins. This lipid is synthesized from CDP-diacylglycerol and myo-inositol by PI synthase, an enzyme localized in the outer mitochondrial membranes and microsomes in yeast. PI synthase was highly purified from yeast microsomes after solubilization with Triton X-100. The activity is dependent on Mn2+ or Mg2+ and Triton X-100. The reaction follows a sequential Bi-Bi mechanism with binding to CDP-diacylglycerol before myo-inositol and releasing PI prior to CMP. Unlike most of the yeast phospholipid-synthesizing enzymes, PI synthase is a constitutive enzyme. Its expression is insensitive to the addition of myo-inositol and choline to culture medium or the transition of growth phase. The primary translate deduced from the encoding gene, PIS, comprises 220 amino acid residues with a calculated molecular mass of 23,613. The sequence contains several hydrophobic regions and resembles that of the human enzyme. The sequence also contains the local, conserved region found in enzymes catalyzing the transfer of the phosphoalcohol moiety from CDP-alcohol, such as phosphatidylserine synthase, cholinephosphotransferase and phosphatidylglycerolphosphate synthase. Substitution of amino acid at position 114 from His (CAC) to Gln (CAA) results in a 200-fold increase in Km of the enzyme for myo-inositol, making cells auxotrophic for myo-inositol. Disruption of the PIS locus in the genome is lethal, indicating that PI is essential for the survival and growth of yeast cells.
Nikawa
J
J
Department of Biochemical Engineering and Science, Faculty of Computer Science and Systems Engineering, Kyushu Institute of Technology, Iizuka, Fukuoka, Japan.
Yamashita
S
S
D000595:Amino Acid Sequence; D000818:Animals; D051607:CDP-Diacylglycerol-Inositol 3-Phosphatidyltransferase; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D017855:Transferases (Other Substituted Phosphate Groups) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D017855:Transferases (Other Substituted Phosphate Groups); D051607:CDP-Diacylglycerol-Inositol 3-Phosphatidyltransferase