Structural Determinants for Inhibitor Specificity and Selectivity in PDE2A Using the Wheat Germ in Vitro Translation System

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• 作者：André ; Iffland ; Darcy Kohls ; Simon Low ; Jing Luan ; Yan Zhang ; Michael Kothe ; Qing Cao ; Ajith V. Kamath ; Yuan-Hua Ding ; and Tom Ellenberger
• 刊名：Biochemistry
• 出版年：2005
• 出版时间：June 14, 2005
• 年：2005
• 卷：44
• 期：23
• 页码：8312 - 8325
• 全文大小：847K
• 年卷期：v.44,no.23(June 14, 2005)
• ISSN：1520-4995

文摘

Phosphodiesterases (PDEs) modulate signaling by cyclic nucleotides in diverse processes suchas cardiac contractility, platelet aggregation, lipolysis, glycogenolysis, and smooth muscle contraction.Cyclic guanosine monophosphate (cGMP) stimulated human phosphodiesterase 2 (PDE2) is expressedmainly in brain and heart tissues. PDE2A is involved in the regulation of blood pressure and fluidhomeostasis by the atrial natriuretic peptide (ANP), making PDE2-type enzymes important targets fordrug discovery. The design of more potent and selective inhibitors of PDE2A for the treatment of heartdisease would be greatly aided by the identification of active site residues in PDE2A that determinesubstrate and inhibitor selectivity. The identification of active site residues through traditional mutationalstudies involves the time-consuming and tedious purification of a large number of mutant proteins fromoverexpressing cells. Here we report an alternative approach to rapidly produce active site mutants ofhuman PDE2A and identify their enzymatic properties using a wheat germ in vitro translation (IVT, alsoknown as cell-free translation) system. We also present the crystal structure of the catalytic domain ofhuman PDE2A determined at 1.7 Å resolution, which provided a framework for the rational design ofactive site mutants. Using a rapid IVT approach for expression of human PDE2A mutants, we identifiedthe roles of active site residues Asp811, Gln812, Ile826, and Tyr827 in inhibitor and substrate selectivityfor PDE2A.