Structural Characterization and Function Determination of a Nonspecific Carboxylate Esterase from the Amidohydrolase Superfamily with a Promiscuous Ability To Hydrolyze Methylphosphonate Esters

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• 作者：Dao Feng Xiang ; Desigan Kumaran ; Subramanyam Swaminathan ; Frank M. Raushel
• 刊名：Biochemistry
• 出版年：2014
• 出版时间：June 3, 2014
• 年：2014
• 卷：53
• 期：21
• 页码：3476-3485
• 全文大小：717K
• 年卷期：v.53,no.21(June 3, 2014)
• ISSN：1520-4995

文摘

The uncharacterized protein Rsp3690 from Rhodobacter sphaeroides is a member of the amidohydrolase superfamily of enzymes. In this investigation the gene for Rsp3690 was expressed in Escherichia coli and purified to homogeneity, and the three-dimensional structure was determined to a resolution of 1.8 脜. The protein folds as a distorted (尾/伪)₈-barrel, and the subunits associate as a homotetramer. The active site is localized to the C-terminal end of the 尾-barrel and is highlighted by the formation of a binuclear metal center with two manganese ions that are bridged by Glu-175 and hydroxide. The remaining ligands to the metal center include His-32, His-34, His-207, His-236, and Asp-302. Rsp3690 was shown to catalyze the hydrolysis of a wide variety of carboxylate esters, in addition to organophosphate and organophosphonate esters. The best carboxylate ester substrates identified for Rsp3690 included 2-naphthyl acetate (k_cat/K_m = 1.0 脳 10⁵ M^鈥? s^鈥?), 2-naphthyl propionate (k_cat/K_m = 1.5 脳 10⁵ M^鈥? s^鈥?), 1-naphthyl acetate (k_cat/K_m = 7.5 脳 10³ M^鈥? s^鈥?), 4-methylumbelliferyl acetate (k_cat/K_m = 2.7 脳 10³ M^鈥? s^鈥?), 4-nitrophenyl acetate (k_cat/K_m = 2.3 脳 10⁵ M^鈥? s^鈥?), and 4-nitrophenyl butyrate (k_cat/K_m = 8.8 脳 10⁵ M^鈥? s^鈥?). The best organophosphonate ester substrates included ethyl 4-nitrophenyl methylphosphonate (k_cat/K_m = 3.8 脳 10⁵ M^鈥? s^鈥?) and isobutyl 4-nitrophenyl methylphosphonate (k_cat/K_m = 1.1 脳 10⁴ M^鈥? s^鈥?). The (S_P)-enantiomer of isobutyl 4-nitrophenyl methylphosphonate was hydrolyzed 10 times faster than the less toxic (R_P)-enantiomer. The high inherent catalytic activity of Rsp3690 for the hydrolysis of the toxic enantiomer of methylphosphonate esters make this enzyme an attractive target for directed evolution investigations.