Purification, characterization, molecular cloning, and extracellular production of a novel bacterial glycerophosphocholine cholinephosphodiesterase from Streptomyces sanglieri

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• 作者：Daisuke Sugimori¹ ; ^{sugimori@sss.fukushima-u.ac.jp" class="auth_mail} ; Junki Ogasawara¹ ; Koki Okuda¹ ; Kazutaka Murayama²
• 关键词：Glycerophosphocholine cholinephosphodiesterase ; Purification ; Characterization ; Molecular cloning ; Expression ; New metabolic pathway ; Streptomyces
• 刊名：Journal of Bioscience and Bioengineering
• 出版年：April, 2014
• 年：2014
• 卷：117
• 期：4
• 页码：422-430
• 全文大小：1450 K

文摘

A novel metal ion-independent glycerophosphocholine cholinephosphodiesterase (GPC-CP) of Streptomyces sanglieri was purified 53-fold from culture supernatant with 1.1% recovery (583聽U/mg-protein). The enzyme functions as a monomer with a molecular mass of 66聽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聽Da. The maximum activity was found at pH 7.2 and 40掳C. The enzyme hydrolyzed glycerol-3-phosphocholine (GPC) over a broad temperature range (37-60掳C) and within a narrow pH range near pH 7. The enzyme was stable at 50掳C for 30聽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聽mM SDS and Mg²⁺; however, Cu²⁺, Zn²⁺, and Co²⁺ remarkably inhibited activity. Enzyme activity was also slightly enhanced by Ca²⁺, Na⁺, EDTA, DTT, and 2-mercaptoethanol. During the hydrolysis of GPC at 37掳C and pH 7.2, apparent V_max and turnover number (k_cat) were determined to be 24.7聽渭mol聽min^鈭?聽mg-protein^鈭? and 29.0聽s^鈭?, respectively. The apparent K_m and k_cat/K_m values were 1.41聽mM and 20.6聽mM^鈭?聽s^鈭?, respectively. GPC hydrolysis by GPC-CP might represent a new metabolic pathway for acquisition of a phosphorus source in actinomycetes.