Structural Superimposition

Abstact

Noncanonical metabolite 5' caps have recently been identified on RNAs, and the DXO/Rai1 family of enzymes can remove these caps in eukaryotes. While the binding modes of NAD, FAD and dephospho-CoA (dpCoA) caps in the active site of mouse DXO have been determined, how DXO recognizes the UDP-glucose (UDP-Glc) and UDP-N-acetylglucosamine (UDP-GlcNAc) caps is not known. In addition, the molecular mechanism by which DXO catalyzes the decapping reactions is still poorly understood, especially the location of the water/hydroxide that attacks the scissile phosphate to initiate the decapping. Here we report the crystal structure of mouse DXO in complex with UDP-GlcNAc at 1.8 A resolution. The binding mode of the compound explains why DXO removes the entire cap from RNA. We have also determined the structures of mouse DXO in complex with purine oligonucleotides, pA5 and pGGGUU. Most importantly, we have produced a model of DXO in a catalytically competent complex with substrates, revealing that a water/hydroxide coordinated to the first metal ion is the nucleophile that attacks the scissile phosphate. The conformation of the scissile phosphate is similar to an alternate conformer of the 5' phosphate in pA5, which provides experimental support for the modeled substrate complex.