Crystal structure of the Entamoeba histolytica RNA lariat debranching enzyme EhDbr1 reveals a catalytic Zn2+ /Mn2+ heterobinucleation

Abstract

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 Ni²⁺ , Mn²⁺ , Mg²⁺ , Fe²⁺ , and Zn²⁺ . 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 Mn²⁺ ion), recent structures determined a Zn²⁺ /Fe²⁺ heterobinucleation. We solved a high-resolution X-ray crystal structure (1.8 Å) of the E. histolytica Dbr1 and determined a Zn²⁺ /Mn²⁺ occupancy. ICP-AES corroborate this finding, and in vitro debranching assays with fluorescently labeled branched substrates confirm activity.

Keywords: ICP-AES; heterobinucleation; metallophosphoesterase.