title: Characterization of glycerophosphoethanolamine ethanolaminephosphodiesterase from Streptomyces sanglieri.
abstract: Streptomyces sanglieri extracellularly produces a glycerophosphoethanolamine ethanolaminephosphodiesterase (GPE-EP). The gene encoding the enzyme was found to consist of a 2124-bp ORF, which codes for an N-terminal 48 residue signal peptide required for secretion and a 660 amino acid mature protein with a calculated molecular mass of 72,918 Da. The maximum activity for sn-glycero-3-phosphoethanolamine (GPE) was found at pH 8.4 and 65°C in the presence of 0.1% (w/v) Triton X-100. The enzyme was activated in the presence of 2 mM EDTA; however, Zn(2+) remarkably inhibited activity. During the hydrolysis of GPE at 65°C and pH 8.4, the apparent Vmax, turnover number (kcat) and Km were determined to be 0.430 mmol min(-1) mg-protein(-1), 522 s(-1) and 0.785 mM, respectively. The enzyme exhibited specificity toward GPE and hydrolyzed ethanolamine-type substrates such as 1,2-dihexanoyl-sn-glycero-3-phosphoethanolamine, lysophosphatidylethanolamine and ethanolamine lysoplasmalogen, but not 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine. Moreover, the enzyme showed no activity toward other phospholipids, such as glycerophospholipids and plasmalogens, and sn-glycero-3-phosphodiesters except for sn-glycero-3-phosphoglycerol, suggesting that GPE-EP is not a phospholipase C (PLC). However, the amino acid sequence of GPE-EP shows 86% identity to that of PLC from Streptomyces sp. SirexAA-E (UniProt accession no. G2NFN1). Recombinant GPE-EP was functionally expressed in Escherichia coli using pET-24a(+). GPE hydrolysis by GPE-EP may represent a new pathway for phosphatidylethanolamine metabolism.
authors.affiliation: Department of Symbiotic Systems Science and Technology, Graduate School of Symbiotic Systems Science and Technology, Fukushima University, 1 Kanayagawa, Fukushima 960-1296, Japan.
authors.forename: Shingo
authors.initials: S
authors.lastname: Mineta
authors.affiliation: Division of Biomedical Measurements and Diagnostics, Graduate School of Biomedical Engineering, Tohoku University, 2-1 Seiryo, Aoba, Sendai 980-8575, Japan.
authors.forename: Kazutaka
authors.initials: K
authors.lastname: Murayama
authors.affiliation: Department of Symbiotic Systems Science and Technology, Graduate School of Symbiotic Systems Science and Technology, Fukushima University, 1 Kanayagawa, Fukushima 960-1296, Japan. Electronic address: sugimori@sss.fukushima-u.ac.jp.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Sugimori
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010714:Phosphatidylethanolamines / Q000378:metabolism*; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D021382:Protein Sorting Signals / Q000235:genetics / Q000502:physiology; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D010714:Phosphatidylethanolamines; D021382:Protein Sorting Signals; C002449:glycerophosphoethanolamine; D010727:Phosphoric Diester Hydrolases
keywords: Characterization; Expression; Glycerophosphoethanolamine ethanolaminephosphodiesterase; Molecular cloning; Purification; Streptomyces sanglieri