The HD Domain of the Escherichia coli tRNA Nucleotidyltransferase Has 2′,3′-Cyclic Phosphodiesterase, 2′-Nucleotidase, and Phosphatase Activities

Abstract

In all mature tRNAs, the 3-terminal CCA sequence is synthesized or repaired by a template-independent nucleotidyltransferase (ATP(CTP):tRNA nucleotidyltransferase; EC 2.7.7.25). The Escherichia coli enzyme comprises two domains: an N-terminal domain containing the nucleotidyltransferase activity and an uncharacterized C-terminal HD domain. The HD motif defines a superfamily of metal-dependent phosphohydrolases that includes a variety of uncharacterized proteins and domains associated with nucleotidyltransferases and helicases from bacteria, archaea, and eukaryotes. The C-terminal HD domain in E. coli tRNA nucleotidyltransferase demonstrated Ni 2؉ -dependent phosphatase activity toward pyrophosphate, canonical 5-nucleoside tri-and diphosphates, NADP, and 2-AMP. Assays with phosphodiesterase substrates revealed surprising metal-independent phosphodiesterase activity toward 2,3-cAMP, -cGMP, and -cCMP. Without metal or in the presence of Mg 2؉ , the tRNA nucleotidyltransferase hydrolyzed 2,3-cyclic substrates with the formation of 2-nucleotides, whereas in the presence of Ni 2؉ , the protein also produced some 3-nucleotides. Mutations at the conserved His-255 and Asp-256 residues comprising the C-terminal HD domain of this protein inactivated both phosphodiesterase and phosphatase activities, indicating that these activities are associated with the HD domain. Low concentrations of the E. coli tRNA (10 nM) had a strong inhibiting effect on both phosphatase and phosphodiesterase activities. The competitive character of inhibition by tRNA suggests that it might be a natural substrate for these activities. This inhibition was completely abolished by the addition of Mg 2؉ , Mn 2؉ , or Ca 2؉ , but not Ni 2؉ . The data suggest that the phosphohydrolase activities of the HD domain of the E. coli tRNA nucleotidyltransferase are involved in the repair of the 3-CCA end of tRNA.

RETRACTED: Cell–cell signaling in Xanthomonas campestris involves an HD-GYP domain protein that functions in cyclic di-GMP turnover

“…The Regulatory Activity of the HD-GYP Domain Depends on the Enzymatic Activity. The HD residues that form the presumed catalytic diad of the HD superfamily of proteins (14) have been shown to be essential for the enzymatic activity of the one member of the family that has been experimentally investigated (21). To examine the relationship between the regulatory and enzymatic activities of the HD-GYP domain, mutations at the conserved H231 and D232 residues of RpfG (H231A, D232A and H231A, D232A) were introduced by site-directed mutagenic PCR into the construct expressing HD-GYPHis 6 (see Materials and Methods).…”

Nuclease Activity of the Human SAMHD1 Protein Implicated in the Aicardi-Goutières Syndrome and HIV-1 Restriction

“…Most of the characterized HD domain proteins show metaldependent phosphohydrolase activity against various nucleotides and nucleic acids (12,14,22,29). Our results show that, in addition to dNTP hydrolysis, the full-length human SAMHD1 protein possesses nuclease activity toward ssDNAs, ssRNAs, and RNA in DNA/RNA hybrids.…”

Structure of PhnP, a Phosphodiesterase of the Carbon-Phosphorus Lyase Pathway for Phosphonate Degradation

“…Loops 1 and 3 also provide the residues for two of three interprotein salt bridges as well as residues that form the majority of the interprotein hydrogen bonds. As mentioned previously, loop 1 also contains Trp 19 , which forms extensive hydrophobic interactions with the deep pocket created by residues of helices 3 and 4 and loops 6 and 8 of the other monomer. Therefore, the area around the Zn 2ϩ site is responsible for providing the majority of protein-protein interaction, and its integrity is crucial for the overall stability of the dimer.…”

“…PhnP Structure Solution and Refinement-Diffraction data were collected as previously described (19). Data were indexed, integrated, and scaled using DENZO and SCALEPACK (20).…”

“…This metal-binding site is located at the edge of the monomer next to the dimerization interface. A tetrahedral coordination sphere for single Zn 2ϩ ion is formed by Cys 19 , Cys 21 , Cys 23 , and His 225 residues. The cysteine residues are contributed by loop 1 and ␣-helix 1, whereas His 225 is contributed by ␣-helix 8.…”