Publications

• Discovery of a previously unrecognized ribonuclease from Escherichia coli that hydrolyzes 5'-phosphorylated fragments of RNA.

  Ghodge S.V., Raushel F.M.

  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 usin ... >> More

  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. << Less

  Biochemistry 54:2911-2918(2015) [PubMed] [EuropePMC]

• Prospecting for unannotated enzymes: discovery of a 3',5'-nucleotide bisphosphate phosphatase within the amidohydrolase superfamily.

  Cummings J.A., Vetting M., Ghodge S.V., Xu C., Hillerich B., Seidel R.D., Almo S.C., Raushel F.M.

  In bacteria, 3',5'-adenosine bisphosphate (pAp) is generated from 3'-phosphoadenosine 5'-phosphosulfate in the sulfate assimilation pathway, and from coenzyme A by the transfer of the phosphopantetheine group to the acyl-carrier protein. pAp is subsequently hydrolyzed to 5'-AMP and orthophosphate, ... >> More

  In bacteria, 3',5'-adenosine bisphosphate (pAp) is generated from 3'-phosphoadenosine 5'-phosphosulfate in the sulfate assimilation pathway, and from coenzyme A by the transfer of the phosphopantetheine group to the acyl-carrier protein. pAp is subsequently hydrolyzed to 5'-AMP and orthophosphate, and this reaction has been shown to be important for superoxide stress tolerance. Herein, we report the discovery of the first instance of an enzyme from the amidohydrolase superfamily that is capable of hydrolyzing pAp. Crystal structures of Cv1693 from Chromobacterium violaceum have been determined to a resolution of 1.9 Å with AMP and orthophosphate bound in the active site. The enzyme has a trinuclear metal center in the active site with three Mn(2+) ions. This enzyme (Cv1693) belongs to the Cluster of Orthologous Groups cog0613 from the polymerase and histidinol phosphatase family of enzymes. The values of kcat and kcat/Km for the hydrolysis of pAp are 22 s(-1) and 1.4 × 10(6) M(-1) s(-1), respectively. The enzyme is promiscuous and is able to hydrolyze other 3',5'-bisphosphonucleotides (pGp, pCp, pUp, and pIp) and 2'-deoxynucleotides with comparable catalytic efficiency. The enzyme is capable of hydrolyzing short oligonucleotides (pdA)5, albeit at rates much lower than that of pAp. Enzymes from two other enzyme families have previously been found to hydrolyze pAp at physiologically significant rates. These enzymes include CysQ from Escherichia coli (cog1218) and YtqI/NrnA from Bacillus subtilis (cog0618). Identification of the functional homologues to the experimentally verified pAp phosphatases from cog0613, cog1218, and cog0618 suggests that there is relatively little overlap of enzymes with this function in sequenced bacterial genomes. << Less

  Biochemistry 53:591-600(2014) [PubMed] [EuropePMC]