Characterization of aminoalcoholphosphotransferases from Arabidopsis thaliana and soybean

Aminoalcoholphosphotransferases (AAPTs, EC 2.7.8.1 and EC 2.7.8.2) catalyze the condensation of 1,2-diacylglycerols with CDP-aminoalcohols to form pho sphatidylaminoalcohols. Using a soybean (Glycine max) AAPT cDNA (GmAAPT1) a s a heterologous hybridization probe, two additional plant AAPT-encoding cD NAs, designated AtAAPT1 and AtAAPT2, were isolated from an Ambidopsis thali ana cDNA library. Southern blot assays suggest that these two cDNAs may rep resent the only AAPT genes in this species. Heterologous expression of AtAA PT1 and AtAAPT2 in a yeast strain deficient in AAPT activities permitted th e determination of substrate specificities of the two Arabidopsis enzymes ( designated AtAAPT1p and AtAAPT2p). Although each AAPT isoform was capable o f incorporating both CDP-ethanolamine and CDP-choline into phosphatidyletha nolamine (PE) and phosphatidylcholine (PC), respectively, AtAAPT2p displaye d a somewhat greater preference for CDP-choline over CDP-ethanolamine in co mparison to AtAAPT1p. The previously characterized soybean AAPT, GmAAPT1p, and AtAAPT1p showed similar degrees of Ca2+ and CMP inhibition; AtAAPT2p, h owever, was inhibited to a lesser degree in the presence of these compounds . All three plant AAPTs are capable of catalyzing the reverse reaction, gen erating CDP-choline and diacylglycerol from PC in the presence of CMP. Fina lly, overexpression of the soybean AAPT cDNA in transgenic tobacco using a strong constitutive promoter resulted in only modest increases in enzymatic activity, suggesting the possibility of post-transcriptional regulation. ( C) Elsevier, Paris.