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J. Biol. Chem., Vol. 283, Issue 10, 6459-6466, March 7, 2008

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Crystal Structure of VioE, a Key Player in the Construction of the Molecular Skeleton of Violacein^*

Satoshi Hirano¹, Shumpei Asamizu¹, Hiroyasu Onaka^¶2, Yoshitsugu Shiro, and Shingo Nagano³

From the Biometal Science Laboratory, RIKEN SPring-8 Center, Hyogo 679-5148, Japan and the Department of Biotechnology, Faculty of Engineering and the ^¶Biotechnology Research Center, Toyama Prefectural University, Toyama, 939-0398, Japan

Violacein and the indolocarbazoles are naturally occurring bisindole products with various biological activities, including antitumor activity. Although these compounds have markedly different molecular skeletons, their biosynthetic pathways share the same intermediate "compound X," which is produced from L-tryptophan via indole-3-pyruvic acid imine. Compound X is a short-lived intermediate that is spontaneously converted to chromopyrrolic acid for indolocarbazole biosynthesis, whereas VioE transforms compound X into protodeoxyviolaceinic acid, which is further modified by other enzymes to produce violacein. Thus, VioE plays a key role in the construction of the molecular skeleton of violacein. Here, we present the crystal structure of VioE, which consists of two subunits, each of which forms a structure resembling a baseball glove. Each subunit has a positively charged pocket at the center of the concave surface of the structure. Mutagenesis analysis of the surface pocket and other surface residues showed that the surface pocket serves as an active site. We have also solved the crystal structure of a complex of VioE and phenylpyruvic acid as an analogue of a VioE-substrate complex. A docking simulation with VioE and the IPA imine dimer, which is proposed to be compound X, agreed with the results from the mutational analysis and the VioE-phenylpyruvic acid complex structure. Based on these results, we propose that VioE traps the highly reactive substrate within the surface pocket to suppress CPA formation and promote protodeoxyviolaceinic acid formation caused by proximity and orientation effects.

Received for publication, September 28, 2007 , and in revised form, November 29, 2007.

The atomic coordinates and structure factors (codes 2ZF3, 2ZF4) 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 grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to S. H., S. N., Y. S., and H. O.) and by an Industrial Technology Research Grant in 2007 from the New Energy and Industrial Technology Development Organization (NEDO) of Japan (to H. O.). 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 These authors contributed equally to this work.

² To whom correspondence may be addressed: Dept. of Biotechnology, Faculty of Engineering, Toyama Prefectural University, Toyama, 939-0398, Japan. Tel.: 81-766-56-7500; Fax: 81-766-56-2498; E-mail: onaka{at}putoyama.ac.jp.

³ To whom correspondence may be addressed: RIKEN SPring-8 Center, 1-1-1 Sayo, Hyogo 679-5148, Japan. Tel.: 81-791-58-2817; Fax: 81-791-58-2818; E-mail: snagano{at}riken.jp.

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