Sébastien Alphonse, Ankan Banerjee, Swathi Dantuluri, Stewart Shuman, and Ranajeet Ghose
Nucleic Acids Research, gkab241, doi:10.1093/nar/gkab241
Fungal tRNA ligases generate a 2’-PO4 splice junction that is converted into a 2’-OH in a two-step reaction by members of the Tpt1 family of NAD+-dependent 2’-phosphotransferases. Tpt1 enzymes are essential components of the fungal tRNA splicing machinery and thus promising targets for antifungal drug discovery. How Tpt1 recognizes its substrates NAD+ and 2’-PO4-modified RNA, and potential conformational changes accompanying substrate binding, are ill-defined. Here, we utilized solution NMR and complementary biophysical approaches to provide new mechanistic insights into NAD+-recognition by a Tpt1 homolog from the Gram-negative bacterium Runella slithyformis, culminating in structures of free Tpt1 and a Tpt1•NAD+ binary complex. The importance of enzymic contacts to NAD+ were validated by mutagenesis.