The HD domain of the Escherichia coli tRNA nucleotidyltransferase has 2',3'-cyclic phosphodiesterase, 2'-nucleotidase, and phosphatase activities.
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.
Yakunin
Alexander F
AF
Banting and Best Department of Medical Research and Structural Genomics Consortium, 112 College St., University of Toronto, Toronto, Ontario M5G 1L6, Canada. a.iakounine@utoronto.ca
Proudfoot
Michael
M
Kuznetsova
Ekaterina
E
Savchenko
Alexei
A
Brown
Greg
G
Arrowsmith
Cheryl H
CH
Edwards
Aled M
AM
D000249:Adenosine Monophosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry; D002118:Calcium / Q000737:chemistry; D002855:Chromatography, Thin Layer; D003001:Cloning, Molecular; D017124:Conserved Sequence; D000242:Cyclic AMP / Q000378:metabolism; D003563:Cyclic CMP / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D006868:Hydrolysis; D007477:Ions; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D008345:Manganese / Q000737:chemistry; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009532:Nickel / Q000737:chemistry / Q000378:metabolism; D009708:Nucleotidases / Q000378:metabolism*; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D012316:RNA Nucleotidyltransferases / Q000737:chemistry*; D013379:Substrate Specificity
D007477:Ions; D001224:Aspartic Acid; D003563:Cyclic CMP; D000249:Adenosine Monophosphate; D008345:Manganese; D006639:Histidine; D009532:Nickel; D000242:Cyclic AMP; D012316:RNA Nucleotidyltransferases; C023198:tRNA nucleotidyltransferase; D009708:Nucleotidases; D010727:Phosphoric Diester Hydrolases; D006152:Cyclic GMP; D008274:Magnesium; D002118:Calcium