title: Human CTP:phosphoethanolamine cytidylyltransferase: enzymatic properties and unequal catalytic roles of CTP-binding motifs in two cytidylyltransferase domains.
abstract: CTP:phosphoethanolamine cytidylyltransferase (ECT) is a key enzyme in the CDP-ethanolamine branch of the Kennedy pathway, which is the primary pathway of phosphatidylethanolamine (PE) synthesis in mammalian cells. Here, the enzymatic properties of recombinant human ECT (hECT) were characterized. The catalytic reaction of hECT obeyed Michaelis-Menten kinetics with respect to both CTP and phosphoethanolamine. hECT is composed of two tandem cytidylyltransferase (CT) domains as ECTs of other organisms. The histidines, especially the first histidine, in the CTP-binding motif HxGH in the N-terminal CT domain were critical for its catalytic activity in vitro, while those in the C-terminal CT domain were not. Overexpression of the wild-type hECT and hECT mutants containing amino acid substitutions in the HxGH motif in the C-terminal CT domain suppressed the growth defect of the Saccharomyces cerevisiae mutant of ECT1 encoding ECT in the absence of a PE supply via the decarboxylation of phosphatidylserine, but overexpression of hECT mutants of the N-terminal CT domain did not. These results suggest that the N-terminal CT domain of hECT contributes to its catalytic reaction, but C-terminal CT domain does not.
authors.affiliation: Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: Siqi
authors.initials: S
authors.lastname: Tian
authors.affiliation: Department of Applied Biological Chemistry, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: Jun
authors.initials: J
authors.lastname: Ohtsuka
authors.affiliation: Department of Applied Biological Chemistry, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: Shipeng
authors.initials: S
authors.lastname: Wang
authors.affiliation: Department of Applied Biological Chemistry, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: Koji
authors.initials: K
authors.lastname: Nagata
authors.affiliation: Department of Applied Biological Chemistry, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: Masaru
authors.initials: M
authors.lastname: Tanokura
authors.affiliation: Department of Biological Chemistry, College of Bioscience and Biotechnology, Chubu University, 1200 Matsumoto-cho, Kasugai, Aichi 487-8501, Japan.
authors.forename: Akinori
authors.initials: A
authors.lastname: Ohta
authors.affiliation: Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Horiuchi
authors.affiliation: Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. Electronic address: afukuda@mail.ecc.u-tokyo.ac.jp.
authors.forename: Ryouichi
authors.initials: R
authors.lastname: Fukuda
mesh_terms: D020816:Amino Acid Motifs; D001665:Binding Sites; D002384:Catalysis; D004789:Enzyme Activation; D006801:Humans; D010714:Phosphatidylethanolamines / Q000737:chemistry*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012316:RNA Nucleotidyltransferases / Q000737:chemistry* / Q000378:metabolism*; D013329:Structure-Activity Relationship
chemical_list: D010714:Phosphatidylethanolamines; C483858:phosphatidylethanolamine; D012316:RNA Nucleotidyltransferases; C020126:Ethanolamine-phosphate cytidylyltransferase
keywords: CTP:phosphoethanolamine cytidylyltransferase; Human; Kennedy pathway; Phosphatidylethanolamine