title: Purification, characterization, molecular cloning, and extracellular production of a novel bacterial glycerophosphocholine cholinephosphodiesterase from Streptomyces sanglieri.
abstract: A novel metal ion-independent glycerophosphocholine cholinephosphodiesterase (GPC-CP) of Streptomyces sanglieri was purified 53-fold from culture supernatant with 1.1% recovery (583 U/mg-protein). The enzyme functions as a monomer with a molecular mass of 66 kDa. The gene encoding the enzyme consists of a 1941-bp ORF that produces a signal peptide of 38 amino acids for secretion and a 646 amino acid mature protein with a calculated molecular mass of 70,447 Da. The maximum activity was found at pH 7.2 and 40°C. The enzyme hydrolyzed glycerol-3-phosphocholine (GPC) over a broad temperature range (37-60°C) and within a narrow pH range near pH 7. The enzyme was stable at 50°C for 30 min and between pH 5-10.5. The enzyme exhibited specificity toward GPC and glycerol-3-phosphoethanolamine and hydrolyzed glycerol-3-phosphate and lysophosphatidylcholine. However, the enzyme showed no activity toward any diacylglycerophospholipids and little activity toward other glycerol-3-phosphodiesters and lysophospholipids. The enzyme was not inhibited in the presence of 2 mM SDS and Mg(2+); however, Cu(2+), Zn(2+), and Co(2+) remarkably inhibited activity. Enzyme activity was also slightly enhanced by Ca(2+), Na(+), EDTA, DTT, and 2-mercaptoethanol. During the hydrolysis of GPC at 37°C and pH 7.2, apparent Vmax and turnover number (kcat) were determined to be 24.7 μmol min(-1) mg-protein(-1) and 29.0 s(-1), respectively. The apparent Km and kcat/Km values were 1.41 mM and 20.6 mM(-1) s(-1), respectively. GPC hydrolysis by GPC-CP might represent a new metabolic pathway for acquisition of a phosphorus source in actinomycetes.
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
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: Junki
authors.initials: J
authors.lastname: Ogasawara
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: Koki
authors.initials: K
authors.lastname: Okuda
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
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis / Q000187:drug effects; D007700:Kinetics; D053858:Metabolic Networks and Pathways / Q000235:genetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D021382:Protein Sorting Signals; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013302:Streptomyces / Q000145:classification / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D010727:Phosphoric Diester Hydrolases; C061931:glycerophosphocholine cholinephosphodiesterase
keywords: Characterization; Expression; Glycerophosphocholine cholinephosphodiesterase; Molecular cloning; New metabolic pathway; Purification; Streptomyces