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J. Biol. Chem., Vol. 283, Issue 40, 27289-27299, October 3, 2008

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Structural Basis for Substrate Recognition and Hydrolysis by Mouse Carnosinase CN2^*

From the Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan

L-Carnosine is a bioactive dipeptide (β-alanyl-L-histidine) present in mammalian tissues, including the central nervous system, and has potential neuroprotective and neurotransmitter functions. In mammals, two types of L-carnosine-hydrolyzing enzymes (CN1 and CN2) have been cloned thus far, and they have been classified as metallopeptidases of the M20 family. The enzymatic activity of CN2 requires Mn²⁺, and CN2 is inhibited by a nonhydrolyzable substrate analog, bestatin. Here, we present the crystal structures of mouse CN2 complexed with bestatin together with Zn²⁺ at a resolution of 1.7Å and that with Mn²⁺ at 2.3Å_. CN2 is a homodimer in a noncrystallographic asymmetric unit, and the Mn²⁺ and Zn²⁺ complexes closely resemble each other in the overall structure. Each subunit is composed of two domains: domain A, which is complexed with bestatin and two metal ions, and domain B, which provides the major interface for dimer formation. The bestatin molecule bound to domain A interacts with several residues of domain B of the other subunit, and these interactions are likely to be essential for enzyme activity. Since the bestatin molecule is not accessible to the bulk water, substrate binding would require conformational flexibility between domains A and B. The active site structure and substrate-binding model provide a structural basis for the enzymatic activity and substrate specificity of CN2 and related enzymes.

Received for publication, February 29, 2008 , and in revised form, May 27, 2008.

The atomic coordinates and structure factors (codes 2ZOF and 2ZOG) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* 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 Figs. S1-S6.

¹ Both authors contributed equally to this work.

² Present address: Dept. of Applied Chemistry, Faculty of Engineering, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki, 852-8521, Japan.

³ Present address: Japan Tobacco Inc., Pharmaceutical Frontier Research Laboratories, 1-13-2, Fukuura, Kanazawa-ku, Yokohama, Kanagawa 236-0004, Japan.

⁴ Present address: P&G K.K. Takasaki Plant, 321 Yawata-machi Takasaki, Gunma 370-0884, Japan.

⁶ Present address: ANBAS Corp., Nakajima Bldg., 4-12-17 Toyosaki, Kita-ku, Osaka, Osaka 531-0072, Japan.

⁵ To whom correspondence may be addressed: Laboratory of Homeostatic Integration, Division of Integrated Protein Functions, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan. Tel.: 81-6-6879-8632; Fax: 81-6-6879-8633; E-mail: nokumura{at}protein.osaka-u.ac.jp.

⁷ To whom correspondence may be addressed: Dept. of Biotechnology, Faculty of Engineering, University of Yamanashi, 4-3-11 Takeda, Kofu City, Yamanashi 400-8511, Japan. Tel./Fax: 81-55-220-8537; E-mail: mkusunoki{at}yamanashi.ac.jp.

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