Nucleic Acids Research Advance Access published online on June 10, 2009
Nucleic Acids Research, doi:10.1093/nar/gkp506
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Nucleic Acid Enzymes |
Functional importance of Crenarchaea-specific extra-loop revealed by an X-ray structure of a heterotetrameric crenarchaeal splicing endonuclease
1Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, 2Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Komaba, Meguro-ku, Tokyo 153-8902, 3Japan Collection of Microorganisms, RIKEN (The Institute of Physical and Chemical Research) BioResource Center, Wako, Saitama 351-0198 and 4Department of Applied Biology, Graduate School of Science and Technology, Kyoto Institute of Technology, Kyoto 606-8585, Japan
*To whom correspondence should be addressed. Tel: +81 3 5841 3528; Fax: +81 3 5841 3444; Email: yoshinas{at}m.u-tokyo.ac.jp Correspondence may also be addressed to Yoh-ichi Watanabe. Tel: +81 3 5841 3528; Fax: +81 3 5841 3444; Email: ywatanab{at}m.u-tokyo.ac.jp
Received March 9, 2009. Accepted May 26, 2009.
Archaeal splicing endonucleases (EndAs) are currently classified into three groups. Two groups require a single subunit protein to form a homodimer or homotetramer. The third group requires two nonidentical protein components for the activity. To elucidate the molecular architecture of the two-subunit EndA system, we studied a crenarchaeal splicing endonuclease from Pyrobaculum aerophilum. In the present study, we solved a crystal structure of the enzyme at 1.7-Å resolution. The enzyme adopts a heterotetrameric form composed of two catalytic and two structural subunits. By connecting the structural and the catalytic subunits of the heterotetrameric EndA, we could convert the enzyme to a homodimer that maintains the broad substrate specificity that is one of the characteristics of heterotetrameric EndA. Meanwhile, a deletion of six amino acids in a Crenarchaea-specific loop abolished the endonuclease activity even on a substrate with canonical BHB motif. These results indicate that the subunit architecture is not a major factor responsible for the difference of substrate specificity between single- and two-subunit EndA systems. Rather, the structural basis for the broad substrate specificity is built into the crenarchaeal splicing endonuclease itself.