Department of Pharmaceutical Chemistry, University of California, San Francisco , 1700 Fourth Street, San Francisco, California 94158-2330, United States.
Biochemistry. 2014 Jul 22;53(28):4727-38. doi: 10.1021/bi500595c. Epub 2014 Jul 9.
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 Å. This protein adopts a typical amidohydrolase (β/α)8-fold and contains a binuclear zinc center located at the C-terminal end of the β-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 kcat/Km values of 4.7 × 10(4) and 5.7 × 10(4) M(-1) s(-1) and l-arabino-1,4-lactone-5-phosphate (7) with kcat/Km values of 1.3 × 10(4) and 2.2 × 10(4) M(-1) s(-1), 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.
从滑液支原体 53 株（MS53_0025）和无乳链球菌 PG2 株（MAG_6390）中鉴定到一种新型内酯酶，通过蛋白结构测定、分子对接、基因背景分析和文库筛选进行鉴定。MS53_0025 的晶体结构测定分辨率为 2.06 Å。该蛋白采用典型的酰胺水解酶（β/α）8 折叠结构，包含位于β-桶末端的双核锌中心。一个磷酸分子结合在活性位点，与 Lys217、Lys244、Tyr245、Arg275 和 Tyr278 形成氢键。对接和基因背景分析都被用来缩小酶的理论底物谱，从而指导经验性筛选以确定 MS53_0025 和 MAG_6390 能够催化 d-木酮糖-1,4-内酯-5-磷酸（2）和 l-阿拉伯糖-1,4-内酯-5-磷酸（7）水解，kcat/Km 值分别为 4.7×10(4)和 5.7×10(4) M(-1) s(-1)，1.3×10(4)和 2.2×10(4) M(-1) s(-1)。只有整合所有方法时，才能确定这两种磷酸呋喃糖内酯酶的底物谱，这为未来高度相关的酰胺水解酶超家族成员的底物鉴定提供了蓝图。
