Functional Annotation and Structural Characterization of a Novel Lactonase Hydrolyzing d-Xylono-1,4-lactone-5-phosphate and l-Arabino-1,4-

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• 作者：Magdalena Korczynska ; Dao Feng Xiang ; Zhening Zhang ; Chengfu Xu ; Tamari Narindoshvili ; Siddhesh S. Kamat ; Howard J. Williams ; Shawn S. Chang ; Peter Kolb ; Brandan Hillerich ; J. Michael Sauder ; Stephen K. Burley ; Steven C. Almo ; Subramanyam Swaminathan ; Brian K. Shoichet ; Frank M. Raushel
• 刊名：Biochemistry
• 出版年：2014
• 出版时间：July 22, 2014
• 年：2014
• 卷：53
• 期：28
• 页码：4727-4738
• 全文大小：677K
• ISSN：1520-4995

文摘

A novel lactonase from Mycoplasma synoviae 53 (MS53_0025) and Mycoplasma agalactiae PG2 (MAG_6390) was characterized by protein structure determination, molecular docking, gene context analysis, and library screening. The crystal structure of MS53_0025 was determined to a resolution of 2.06 脜. This protein adopts a typical amidohydrolase (尾/伪)₈-fold and contains a binuclear zinc center located at the C-terminal end of the 尾-barrel. A phosphate molecule was bound in the active site and hydrogen bonds to Lys217, Lys244, Tyr245, Arg275, and Tyr278. Both docking and gene context analysis were used to narrow the theoretical substrate profile of the enzyme, thus directing empirical screening to identify that MS53_0025 and MAG_6390 catalyze the hydrolysis of d-xylono-1,4-lactone-5-phosphate (2) with k_cat/K_m values of 4.7 脳 10⁴ and 5.7 脳 10⁴ M^鈥? s^鈥? and l-arabino-1,4-lactone-5-phosphate (7) with k_cat/K_m values of 1.3 脳 10⁴ and 2.2 脳 10⁴ M^鈥? s^鈥?, respectively. The identification of the substrate profile of these two phospho-furanose lactonases emerged only when all methods were integrated and therefore provides a blueprint for future substrate identification of highly related amidohydrolase superfamily members.