Ransey Elizabeth, Paredes Eduardo, Dey Sourav K, Das Subha R, Heroux Annie, Macbeth Mark R
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA.
Department of Chemistry and Center for Nucleic Acids & Technology, Carnegie Mellon University, Pittsburgh, PA, USA.
FEBS Lett. 2017 Jul;591(13):2003-2010. doi: 10.1002/1873-3468.12677. Epub 2017 Jun 14.
The RNA lariat debranching enzyme, Dbr1, is a metallophosphoesterase that cleaves 2'-5' phosphodiester bonds within intronic lariats. Previous reports have indicated that Dbr1 enzymatic activity is supported by diverse metal ions including Ni , Mn , Mg , Fe , and Zn . While in initial structures of the Entamoeba histolytica Dbr1 only one of the two catalytic metal-binding sites were observed to be occupied (with a Mn ion), recent structures determined a Zn /Fe heterobinucleation. We solved a high-resolution X-ray crystal structure (1.8 Å) of the E. histolytica Dbr1 and determined a Zn /Mn occupancy. ICP-AES corroborate this finding, and in vitro debranching assays with fluorescently labeled branched substrates confirm activity.
RNA套索脱支酶Dbr1是一种金属磷酸二酯酶，可切割内含子套索内的2'-5'磷酸二酯键。先前的报道表明，Dbr1的酶活性受到多种金属离子的支持，包括镍、锰、镁、铁和锌。虽然在溶组织内阿米巴Dbr1的初始结构中，仅观察到两个催化金属结合位点中的一个被占据（有一个锰离子），但最近的结构确定了锌/铁异双核。我们解析了溶组织内阿米巴Dbr1的高分辨率X射线晶体结构（1.8 Å），并确定了锌/锰的占有率。电感耦合等离子体发射光谱法证实了这一发现，并且用荧光标记的分支底物进行的体外脱支测定证实了其活性。
