The cytidine deaminase signature HxE(x)_nCxxC of DYW1 binds zinc and is necessary for RNA editing of ndhD‐1

Abstract

SummaryIn flowering plants, RNA editing involves deamination of specific cytidines to uridines in both mitochondrial and chloroplast transcripts. Pentatricopeptide repeat (PPR) proteins and multiple organellar RNA editing factor (MORF) proteins have been shown to be involved in RNA editing but none have been shown to possess cytidine deaminase activity.The DYW domain of some PPR proteins contains a highly conserved signature resembling the zinc-binding active site motif of known nucleotide deaminases. We modified these highly conserved amino acids in the DYW motif of DYW1, an editing factor required for editing of the ndhD-1 site in Arabidopsis chloroplasts.We demonstrate that several amino acids of this signature motif are required for RNA editing in vivo and for zinc binding in vitro.We conclude that the DYW domain of DYW1 has features in common with cytidine deaminases, reinforcing the hypothesis that this domain forms part of the active enzyme that carries out RNA editing in plants.

A Conserved Glutamate Residue in the C-terminal Deaminase Domain of Pentatricopeptide Repeat Proteins Is Required for RNA Editing Activity

“…This region has canonical zinc binding motifs (HXE, CXXC) (18 -20), which are conserved in deaminases that act on nucleotides, RNA, and DNA (16,(21)(22)(23)(24)(25)(26). The DYW deaminase domain has recently been shown to bind zinc ions (19,20). In addition, mutagenesis of the zinc binding motifs has been shown to interfere with editing in transgenic plants (20) and through transient expression in protoplasts (27).…”

“…The DYW deaminase domain has recently been shown to bind zinc ions (19,20). In addition, mutagenesis of the zinc binding motifs has been shown to interfere with editing in transgenic plants (20) and through transient expression in protoplasts (27). The glutamate residue of the HXE motif has been shown to be directly involved in the E. coli cytidine deaminase mechanism through deprotonation of the substrate water molecule and transfer of the proton to the product ammonia (21).…”

“…Furthermore, DYW1 has canonical zinc binding motifs and has been shown to bind zinc ions (19,20). Mutagenesis of the zinc binding motifs and the catalytic glutamate residue of DYW1 ablated that ability to edit the ndhD site (20). Thus, the editing of the ndhD site requires both CRR4 and DYW1, and the functions of site recognition and deamination appear to be separated into two proteins in this case.…”

“…DYW1 has an intact DYW deaminase domain; however, the N-terminal PLS-like region is small and composed of degenerate repeats. Furthermore, DYW1 has canonical zinc binding motifs and has been shown to bind zinc ions (19,20). Mutagenesis of the zinc binding motifs and the catalytic glutamate residue of DYW1 ablated that ability to edit the ndhD site (20).…”

RNA Recognition Motif-Containing Protein ORRM4 Broadly Affects Mitochondrial RNA Editing and Impacts Plant Development and Flowering

“…The DYW domain exhibits sequence similarity to known cytidine deaminase motifs (Salone et al, 2007). Mutagenesis of conserved deaminase residues in DYW1, QED1, RARE1, OTP84, and CREF7 leads to loss of editing activity (Boussardon et al, 2014;Wagoner et al, 2015;Hayes et al, 2015), indicating the enzyme activity for catalyzing the C-to-U conversion may reside in the DYW domain.…”