# Application of Mammalian Nudix Enzymes to Capped RNA Analysis
## 4 NUDIX Protein Family  
NUDIX enzymes are evolutionary conserved family of pyrophosphhydrolases found across all domains of life, including viruses. NUDIX (namely NUcleoside DIphosphates linked to a moiety X) possess a broad range of substrate specificity, including canonical (d)NTPs, oxidized nucleotides, dinucleotide polyphosphates, nucleotide sugars, capped RNAs, cap structures, and dinucleotide coenzymes (NAD, FAD, dpCoA) and their derivatives [111; 112]. The _E. coli_ MutT protein is referred to as a founding member of this superfamily, that is characterized by a highly conserved NUDIX motif (GX\({}_{5}\)EX\({}_{5}\)[UA]XREX\({}_{2}\)EXKGU, where U is hydrophobic and X any amino acid) [111; 113]. The glutamic acid residues within the NUDIX motif (REUXEE) play role in the binding of divalent metal ions required for the catalytic activity of NUDIX enzymes [111]. The mechanism of substrate hydrolysis by NUDIX enzymes proceeds, in most cases, through nucleophilic substitution by water molecule at specific phosphorus of a di-(or polyphosphate) chain, but hydrolysis can occur also by nucleophilic substitution at carbon as it is in the case of the GDP-mannose hydrolase NUDIX subfamily [114]. Available data (based on crystal structures, mechanistic and kinetic studies) have indicated that NUDIX hydrolases usually require two or three metal ions for catalysis [114; 115]. A recent study that used time-resolved X-ray crystallography of MutT, with its substrate (8-oxo-dGTP) and Mn\({}^{2+}\), allowed for detailed visualization of the reaction process that proceed through nucleophilic substitution by water molecule with three Mn\({}^{2+}\) ions with captured structures of intermediate reaction states [115].  
Among eukaryotic NUDIX hydrolases, Dcp2 (Nudt20) was the first identified enzyme involved in removing the cap structure present at the 5\({}^{\prime}\) end of mRNA [11; 12; 13; 14]. An additional set of seven NUDIX mammalian enzymes possess mRNA cap hydrolysis activity in vitro (Nudt2, Nudt3, Nudt12, Nudt15, Nudt16, Nudt17 and Nudt19 [116; 117]) and in vivo (Nudt3 and Nudt16 [118; 119; 15]). Aforementioned NUDIX proteins cleave within the triphosphate bridge of a cap structure, between \(\alpha\)-\(\beta\), \(\beta\)-\(\gamma\) or both \(\alpha\)-\(\beta\)/\(\beta\)-\(\gamma\) phosphates (Figure 1) (e.g., [117]). Enzymatic activity of these NUDIX-decapping enzymes is influenced by the presence of specific modifications in the cap structure (e.g., by the presence of 7-methylganoise or methylation of adenosine adjacent to m\({}^{7}\)G for hDcp2 activity [14; 19]; by the nucleobase type, as caps with adenosine (or 2\({}^{\prime}\)-O adenosine) are not subject to effective hydrolysis with hNudt16 [120; 121]; or by the preference of hNutd16 for unmethylated cap [120]. As all these sites of the mRNA cap structure are subject of intensive/plethora chemical modifications in order to optimize its effect on mRNA stability, translation efficiency, and immunogenicity (Figure 4), the NUDIX proteins with different specificities towards cap structure(s) emerge as a powerful tool in their analysis.