title: Discovery of a Previously Unrecognized Ribonuclease from Escherichia coli That Hydrolyzes 5'-Phosphorylated Fragments of RNA.
abstract: TrpH or YciV (locus tag b1266) from Escherichia coli is annotated as a protein of unknown function that belongs to the polymerase and histidinol phosphatase (PHP) family of proteins in the UniProt and NCBI databases. Enzymes from the PHP family have been shown to hydrolyze organophosphoesters using divalent metal ion cofactors at the active site. We found that TrpH is capable of hydrolyzing the 3'-phosphate from 3',5'-bis-phosphonucleotides. The enzyme will also sequentially hydrolyze 5'-phosphomononucleotides from 5'-phosphorylated RNA and DNA oligonucleotides, with no specificity toward the identity of the nucleotide base. The enzyme will not hydrolyze RNA or DNA oligonucleotides that are unphosphorylated at the 5'-end of the substrate, but it makes no difference whether the 3'-end of the oligonucleotide is phosphorylated. These results are consistent with the sequential hydrolysis of 5'-phosphorylated mononucleotides from oligonucleotides in the 5' → 3' direction. The catalytic efficiencies for hydrolysis of 3',5'-pAp, p(Ap)A, p(Ap)4A, and p(dAp)4dA were determined to be 1.8 × 10(5), 9.0 × 10(4), 4.6 × 10(4), and 2.9 × 10(3) M(-1) s(-1), respectively. TrpH was found to be more efficient at hydrolyzing RNA oligonucleotides than DNA oligonucleotides. This enzyme can also hydrolyze annealed DNA duplexes, albeit at a catalytic efficiency approximately 10-fold lower than that of the corresponding single-stranded oligonucleotides. TrpH is the first enzyme from E. coli that has been found to possess 5' → 3' exoribonuclease activity. We propose to name this enzyme RNase AM.
authors: Swapnil V
authors: SV
authors: Ghodge
authors: Frank M
authors: FM
authors: Raushel
mesh_terms: D004277:DNA, Single-Stranded / Q000737:chemistry; D048168:Escherichia coli K12 / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D005095:Exoribonucleases; D006868:Hydrolysis; D007700:Kinetics; D009841:Oligonucleotides / Q000737:chemistry; D010766:Phosphorylation; D012313:RNA / Q000737:chemistry*; D012260:Ribonucleases / Q000737:chemistry*; D013379:Substrate Specificity
chemical_list: D004277:DNA, Single-Stranded; D029968:Escherichia coli Proteins; D009841:Oligonucleotides; D012313:RNA; D005095:Exoribonucleases; D012260:Ribonucleases; C000599517:YciV protein, E coli