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J. Biol. Chem., Vol. 278, Issue 40, 39133-39142, October 3, 2003

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Crystal Structure of a Mini-intein Reveals a Conserved Catalytic Module Involved in Side Chain Cyclization of Asparagine during Protein Splicing^*,

Yi Ding  , Ming-Qun Xu ¶ ||, Inca Ghosh ¶, Xuehui Chen , Sebastien Ferrandon ¶, Guillaume Lesage ¶ and Zihe Rao   **

From the Laboratory of Structural Biology and the Ministry of Education Laboratory of Protein Science, School of Life Science and Engineering, Tsinghua University, Beijing 100084, People's Republic of China, ^¶New England Biolabs, Inc., Beverly, Massachusetts 01915, and Institute of Biophysics, Chinese Academy of Science, Beijing 100101, People's Republic of China

We have determined the crystal structure of a 154-residue intein derived from the dnaB gene of Synechocystis sp. strain PCC6803 and refined it to a 2.0-Å resolution. The x-ray structure suggests that this intein possesses two catalytic sites that appear to be separately responsible for splicing and cleavage of the N- and C-terminal scissile bonds. The conserved intein block F residues are the important components of a catalytic site for side chain cyclization of the last intein residue, Asn-154. The data suggest that the imidazole ring of His-143 is involved in the activation of the side chain N atom of Asn-154, leading to a nucleophilic attack on the carbonyl carbon of Asn-154. Substitution of His-143 with Ala or Gln resulted in the inhibition of C-terminal cleavage. His-153, Asp-136, and a water molecule appear to constitute an oxyanion binding site by contacting the carbonyl oxygen of Asn-154 to stabilize the transition state. The structure and mutagenesis data also support that the close contact between the hydroxyl groups of Thr-138 and Ser-155, whose side chain participates in an S O acyl shift, plays an important role in the nucleophile orientation. Our structural modeling suggests that this catalytic module is conserved in the C-terminal subdomains of inteins from diverse organisms.

Received for publication, June 12, 2003 , and in revised form, July 23, 2003.

The atomic coordinates and structure factors (code 1MI8) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This research was supported by Projects 30128002 and G1999075600 (China). 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1.

^|| To whom correspondence may be addressed: New England Biolabs, Inc., 32 Tozer Rd., Beverly, MA 01915. Tel.: 978-927-5054; Fax: 978-921-1350; E-mail: xum{at}neb.com.

^** To whom correspondence may be addressed. Tel.: 86-10-62771493; Fax: 86-10-62773145; E-mail: raozh{at}xtal.tsinghua.edu.cn.

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