SCANNING ALANINE MUTAGENESIS OF THE CDP-ALCOHOL PHOSPHOTRANSFERASE MOTIF OF SACCHAROMYCES-CEREVISIAE CHOLINEPHOSPHOTRANSFERASE

Cholinephosphotransferase (EC 2.7.8.2) catalyzes the formation of a ph osphoester bond via the transfer of a phosphocholine moiety from CDP-c holine to diacylglycerol forming phosphatidylcholine and releasing GRI P. A motif, )-(X)(2)-Ala(117)-Arg(118)-(X)(8)-Gly(127)-(X)(3)- Asp(131 )-(X)(3)-Asp(135), located within the CDP-choline binding region of Sa ccharomyces cerevisiae cholinephosphotransferase (CPTI ?/Author: Pleas e confirm that a gene is meant here.) is also found in several other p hospholipid synthesizing enzymes that catalyze the formation of a phos phoester bond utilizing a CDP-alcohol and a second alcohol as substrat es, To determine if this motif is diagnostic of the above reaction typ e scanning alanine mutagenesis of the conserved residues within S. cer evisiae cholinephosphotransferase was performed. Enzyme activity was a ssessed in vitro using a mixed micelle enzyme assay and in vivo by det ermining the ability of the mutant enzymes to restore phosphatidylchol ine synthesis from radiolabeled choline in an S. cerevisiae strain dev oid of endogenous cholinephosphotransferase activity. Alanine mutants of Gly(114), Gly(127), Asp(131), and Asp(135) were inactive; mutants, Ala(117) and Arg(118) displayed reduced enzyme activity, and Asp(113) displayed wild type activity. The analysis described is the first mole cular characterization of a CDP-alcohol phosphotransferase motif and r esults predict a catalytic role utilizing a general base reaction proc eeding through Asp(131) or Asp(135) via direct nucleophilic attack of the hydroxyl of diacylglyerol on the phosphoester bond of CDP-choline that does not proceed via an enzyme bound intermediate. Residues Ala(1 17) and Arg(118) do not participate directly in catalysis but are like ly involved in substrate binding or positioning with Arg(118) predicte d to associate with a phosphate moiety of CDP-choline.