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J. Biol. Chem., Vol. 281, Issue 43, 32534-32539, October 27, 2006

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Crystal Structure and Desulfurization Mechanism of 2'-Hydroxybiphenyl-2-sulfinic Acid Desulfinase^*

Woo Cheol Lee, Takashi Ohshiro, Toshiyuki Matsubara, Yoshikazu Izumi, and Masaru Tanokura¹

From the Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1Yayoi, Bunkyoku, Tokyo 113-8657 and the Department of Biotechnology, Tottori University, 4-101 Koyama-Minami, Tottori 680-8552, Japan

The desulfurization of dibenzothiophene in Rhodococcus erythropolis is catalyzed by two monooxygenases, DszA and DszC, and a desulfinase, DszB. In the last step of this pathway, DszB hydrolyzes 2'-hydroxybiphenyl-2-sulfinic acid into 2-hydroxybiphenyl and sulfite. We report on the crystal structures of DszB and an inactive mutant of DszB in complex with substrates at resolutions of 1.8Å or better. The overall fold of DszB is similar to those of periplasmic substrate-binding proteins. In the substrate complexes, biphenyl rings of substrates are recognized by extensive hydrophobic interactions with the active site residues. Binding of substrates accompanies structural changes of the active site loops and recruits His⁶⁰ to the active site. The sulfinate group of bound substrates forms hydrogen bonds with side chains of Ser²⁷, His⁶⁰, and Arg⁷⁰, each of which is shown by site-directed mutagenesis to be essential for the activity. In our proposed reaction mechanism, Cys²⁷ functions as a nucleophile and seems to be activated by the sulfinate group of substrates, whereas His⁶⁰ and Arg⁷⁰ orient the syn orbital of sulfinate oxygen to the sulfhydryl hydrogen of Cys²⁷ and stabilize the negatively charged reaction intermediate. Cys, His, and Arg residues are conserved in putative proteins homologous to DszB, which are presumed to constitute a new family of desulfinases.

Received for publication, March 29, 2006 , and in revised form, July 21, 2006.

The atomic coordinates and structure factors (code 2DE2, 2DE3, and 2DE4) 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. 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.

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