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J. Biol. Chem., Vol. 282, Issue 45, 33107-33117, November 9, 2007

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Crystal Structure of the Oxygenase Component (HpaB) of the 4-Hydroxyphenylacetate 3-Monooxygenase from Thermus thermophilus HB8^*

Seong-Hoon Kim, Tamao Hisano, Kazuki Takeda, Wakana Iwasaki, Akio Ebihara, and Kunio Miki¹

From the SPring-8 Center, RIKEN Harima Institute, Koto 1-1-1, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan and the Department of Chemistry, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan

The 4-hydroxyphenylacetate (4HPA) 3-monooxygenase is involved in the initial step of the 4HPA degradation pathway and catalyzes 4HPA hydroxylation to 3,4-dihydroxyphenylacetate. This enzyme consists of two components, an oxygenase (HpaB) and a reductase (HpaC). To understand the structural basis of the catalytic mechanism of HpaB, crystal structures of HpaB from Thermus thermophilus HB8 were determined in three states: a ligand-free form, a binary complex with FAD, and a ternary complex with FAD and 4HPA. Structural analysis revealed that the binding and dissociation of flavin are accompanied by conformational changes of the loop between 5 and 6 and of the loop between 8 and 9, leading to preformation of part of the substrate-binding site (Ser-197 and Thr-198). The latter loop further changes its conformation upon binding of 4HPA and obstructs the active site from the bulk solvent. Arg-100 is located adjacent to the putative oxygen-binding site and may be involved in the formation and stabilization of the C4a-hydroperoxyflavin intermediate.

Received for publication, April 25, 2007 , and in revised form, August 15, 2007.

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

^* This study was performed under the "Structurome" Project of RIKEN Harima Institute and was supported in part by a Grant-in-Aid for Young Scientists 17681009 (A) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to T. H.). 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: Dept. of Chemistry, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto, 606-8502, Japan. Tel.: 81-75-753-4029; Fax: 81-75-753-4032; E-mail: miki{at}kuchem.kyoto-u.ac.jp.

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