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Metal content and kinetic properties of yeast RNA lariat debranching enzyme.

In eucaryotic cells, intron lariats produced by the spliceosome contain a 2'5' phosphodiester linkage. The RNA lariat debranching enzyme, Dbr1, is the only enzyme known to hydrolyze this bond. Dbr1 is a member of the metallophosphoesterase (MPE) family of enzymes, and recent X-ray crystal structures and biochemistry data demonstrate that Dbr1 from Entamoeba histolytica uses combinations of Mn2+, Zn2+, and Fe2+ as enzymatic co-factors. Here, we examine the kinetic properties and metal dependence of the Dbr1 homolog from Saccharomyces cerevisiae (yDbr1). Elemental analysis measured stoichiometric quantities of Fe and Zn in yDbr1 purified following heterologous expression E. coli. We analyzed the ability of Fe2+, Zn2+, and Mn2+ to reconstitute activity in metal-free apoenzyme. Purified yDbr1 was highly active, turning over substate at 5.6 sec-1, and apo-yDbr1 reconstituted with Fe2+ was the most active species, turning over at 9.2 sec-1. We treated a human lymphoblastoid cells with the iron-chelator deferoxamine and measured a two-fold increase in cellular lariats. These data suggest that Fe is an important biological co-factor for Dbr1 enzymes.