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J. Biol. Chem., Vol. 279, Issue 35, 36819-36827, August 27, 2004

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

Alexander F. Yakunin, Michael Proudfoot, Ekaterina Kuznetsova, Alexei Savchenko¶, Greg Brown, Cheryl H. Arrowsmith¶||**, and Aled M. Edwards¶||

From the Banting and Best Department of Medical Research and ^||Structural Genomics Consortium, 112 College St., University of Toronto, Toronto, Ontario M5G 1L6, Canada and the ^¶Department of Medical Biophysics, University of Toronto, and Ontario Centre for Structural Proteomics, Ontario Cancer Institute, 200 Elizabeth St., Max Bell Research Centre 5R407, Toronto, Ontario M5G 2C4, Canada

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 Ni²⁺-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 Mg²⁺, the tRNA nucleotidyltransferase hydrolyzed 2',3'-cyclic substrates with the formation of 2'-nucleotides, whereas in the presence of Ni²⁺, 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 Mg²⁺, Mn²⁺, or Ca²⁺, but not Ni²⁺. 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.

Received for publication, May 7, 2004 , and in revised form, June 16, 2004.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.This work was supported financially by Genome Canada, the Ontario Research and Development Challenge Fund, and the Protein Structure Initiative of the National Institutes of Health (GM62414 and GM62413). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^** A Scientist of the Canadian Institutes of Health Research.

Holder of the Banbury Chair of Medical Research.

To whom correspondence should be addressed. Tel.: 416-946-0075; Fax: 416-978-8528; E-mail: a.iakounine{at}utoronto.ca.

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