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Nucleic Acids Research 2006 34(13):3811-3818; doi:10.1093/nar/gkl517
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Published online 11 August 2006

Nucleic Acids Research, 2006, Vol. 34, No. 13 3811-3818
© 2006 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commerical use, distribution, and reproduction in any medium, provided the original work is properly cited.

Article

Identification by Mn2+ rescue of two residues essential for the proton transfer of tRNase Z catalysis

Asako Minagawa, Hiroaki Takaku, Ryohei Ishii1, Masamichi Takagi, Shigeyuki Yokoyama1,2,3 and Masayuki Nashimoto*

Department of Applied Life Sciences, Niigata University of Pharmacy and Applied Life Sciences Niigata, Niigata 956-8603, Japan 1 RIKEN Genomic Sciences Center Tsurumi, Yokohama 230-0045, Japan 2 RIKEN Harima Institute at SPring-8 Sayo-gun, Hyogo 679-5148, Japan 3 Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo Bunkyo-ku, Tokyo 113-0033, Japan

*To whom correspondence should be addressed. Tel: +81 250 25 5119; Fax: +81 250 25 5021; Email: mnashimoto{at}niigatayakudai.jp

Received May 17, 2006. Accepted July 6, 2006.

Thermotoga maritima tRNase Z cleaves pre-tRNAs containing the 74CCA76 sequence precisely after the A76 residue to create the mature 3' termini. Its crystal structure has revealed a four-layer {alpha}ß/ß{alpha} sandwich fold that is typically found in the metallo-ß-lactamase superfamily. The well-conserved six histidine and two aspartate residues together with metal ions are assumed to form the tRNase Z catalytic center. Here, we examined tRNase Z variants containing single amino acid substitutions in the catalytic center for pre-tRNA cleavage. Cleavage by each variant in the presence of Mg2+ was hardly detected, although it is bound to pre-tRNA. Surprisingly, however, Mn2+ ions restored the lost Mg2+-dependent activity with two exceptions of the Asp52Ala and His222Ala substitutions, which abolished the activity almost completely. These results provide a piece of evidence that Asp-52 and His-222 directly contribute the proton transfer for the catalysis.


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