HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 283, Issue 20, 13889-13896, May 16, 2008

This Article Full Text Full Text (PDF) All Versions of this Article: 283/20/13889    most recent M710070200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Ito, K. Articles by Tanokura, M. Search for Related Content PubMed PubMed Citation Articles by Ito, K. Articles by Tanokura, M. Social Bookmarking                      What's this?

Expansion of Substrate Specificity and Catalytic Mechanism of Azoreductase by X-ray Crystallography and Site-directed Mutagenesis^*

Kosuke Ito, Masayuki Nakanishi, Woo-Cheol Lee, Yuehua Zhi, Hiroshi Sasaki, Shuhei Zenno^¶, Kaoru Saigo^¶, Yukio Kitade, and Masaru Tanokura¹

From the Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan, Department of Biomolecular Science, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan, and ^¶Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

AzoR is an FMN-dependent NADH-azoreductase isolated from Escherichia coli as a protein responsible for the degradation of azo compounds. We previously reported the crystal structure of the enzyme in the oxidized form. In the present study, different structures of AzoR were determined under several conditions to obtain clues to the reaction mechanism of the enzyme. AzoR in its reduced form revealed a twisted butterfly bend of the isoalloxazine ring of the FMN cofactor and a rearrangement of solvent molecules. The crystal structure of oxidized AzoR in a different space group and the structure of the enzyme in complex with the inhibitor dicoumarol were also determined. These structures indicate that the formation of a hydrophobic part around the isoalloxazine ring is important for substrate binding and an electrostatic interaction between Arg-59 and the carboxyl group of the azo compound causes a substrate preference for methyl red over p-methyl red. The substitution of Arg-59 with Ala enhanced the V_max value for p-methyl red 27-fold with a 3.8-fold increase of the K_m value. This result indicates that Arg-59 decides the substrate specificity of AzoR. The V_max value for the p-methyl red reduction of the R59A mutant is comparable with that for the methyl red reduction of the wild-type enzyme, whereas the activity toward methyl red was retained. These findings indicate the expansion of AzoR substrate specificity by a single amino acid substitution. Furthermore, we built an authentic model of the AzoR-methyl red complex based on the results of the study.

Received for publication, December 10, 2007 , and in revised form, February 12, 2008.

The atomic coordinates and structure factors (codes 2Z98, 2Z9B, 2Z9C, and 2Z9D) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported in part by the National Project on Protein Structural and Functional Analyses of the Ministry of Education, Culture, Sports, Science, and Technology of Japan and by Grants-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. The synchrotron-radiation experiments were performed at BL6A and BL18B at Photon Factory and NW12 at Photon Factory Advanced Ring for Pulse X-rays with the approval of KEK (Tsukuba, Japan) (proposal numbers 2000G307 and 2003G127). 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.

¹ To whom correspondence should be addressed. Tel.: 81-3-5841-5165; Fax: 81-3-5841-8023; E-mail: amtanok{at}mail.ecc.u-tokyo.ac.jp.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?