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J. Biol. Chem., Vol. 279, Issue 46, 47619-47625, November 12, 2004

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Identification of Crucial Histidines Involved in Carbon-Nitrogen Triple Bond Synthesis by Aldoxime Dehydratase^*

Kazunobu Konishi, Kyoko Ishida, Ken-Ichi Oinuma, Takehiro Ohta¶, Yoshiteru Hashimoto, Hiroki Higashibata, Teizo Kitagawa¶, and Michihiko Kobayashi||

From the Institute of Applied Biochemistry, and Graduate School of Life and Environmental Sciences, The University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan and ^¶Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, Myodaiji, Okazaki, Aichi 444-8787, Japan

Aldoxime dehydratase (OxdA), which is a novel heme protein, catalyzes the dehydration of an aldoxime to a nitrile even in the presence of water in the reaction mixture. The combination of site-directed mutagenesis of OxdA (mutation of all conserved histidines in the aldoxime dehydratase superfamily), estimation of the heme contents and specific activities of the mutants, and CD and resonance Raman spectroscopic analyses led to the identification of the proximal and distal histidines in this unique enzyme. The heme contents and CD spectra in the far-UV region of all mutants except for the H299A one were almost identical to those of the wild-type OxdA, whereas the H299A mutant lost the ability of binding heme, demonstrating that His²⁹⁹ is the proximal histidine. On the other hand, substitution of alanine for His³²⁰ did not affect the overall structure of OxdA but caused loss of its ability of carbon-nitrogen triple bond synthesis and a lower shift of the Fe-C stretching band in the resonance Raman spectrum for the CO-bound form. Furthermore, the pH dependence of the wild-type OxdA closely followed the His protonation curves observed for other proteins. These findings suggest that His³²⁰ is located in the distal heme pocket of OxdA and would donate a proton to the substrate in the aldoxime dehydration mechanism.

Received for publication, June 28, 2004 , and in revised form, August 9, 2004.

^* This work was supported in part by the 21st Century COE Program (www.tara.tsukuba.ac.jp/~coe21/) from the Ministry of Education, Culture, Sports, Science, and Technology, by a Grant-in-aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan, by an Industrial Technology Research Grant Program in 2002 from the New Energy and Industrial Technology Development Organization (NEDO) of Japan, and by the National Project on Protein Structural and Functional Analyses, by a Research Grant (A) for University Research Projects. This work was also supported by JSPS Research Fellowships for Young Scientists (to T. O.) and by Grant-in-aid for Specifically Promoted Research 14001004 (to T. K.). 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.

These authors contributed equally to this work.

^|| To whom correspondence should be addressed. Fax: 81-29-853-4605.

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