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J. Biol. Chem., Vol. 281, Issue 52, 40076-40088, December 29, 2006

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Mechanistic and Structural Studies of Apoform, Binary, and Ternary Complexes of the Arabidopsis Alkenal Double Bond Reductase At5g16970^*

Buhyun Youn, Sung-Jin Kim, Syed G. A. Moinuddin, Choonseok Lee, Diana L. Bedgar, Athena R. Harper, Laurence B. Davin, Norman G. Lewis¹, and ChulHee Kang²

From the School of Molecular Biosciences, Washington State University, Pullman, Washington 99164-4660 and the Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164-6340

In this study, we determined the crystal structures of the apoform, binary, and ternary complexes of the Arabidopsis alkenal double bond reductase encoded by At5g16970. This protein, one of 11 homologues in Arabidopsis thaliana, is most closely related to the Pinus taeda phenylpropenal double bond reductase, involved in, for example, heartwood formation. Both enzymes also have essential roles in plant defense, and can function by catalyzing the reduction of the 7-8-double bond of phenylpropanal substrates, such as p-coumaryl and coniferyl aldehydes in vitro. At5g16970 is also capable of reducing toxic substrates with the same alkenal functionality, such as 4-hydroxy-(2E)-nonenal. The overall fold of At5g16970 is similar to that of the zinc-independent medium chain dehydrogenase/reductase superfamily, the members of which have two domains and are dimeric in nature, i.e. in contrast to their original classification as being zinc-containing oxidoreductases. As provisionally anticipated from the kinetic data, the shape of the binding pocket can readily accommodate p-coumaryl aldehyde, coniferyl aldehyde, 4-hydroxy-(2E)-nonenal, and 2-alkenals. However, the enzyme kinetic data among these potential substrates differ, favoring p-coumaryl aldehyde. Tyr-260 is provisionally proposed to function as a general acid/base for hydride transfer. A catalytic mechanism for this reduction, and its applicability to related important detoxification mammalian proteins, is also proposed.

Received for publication, June 20, 2006 , and in revised form, August 25, 2006.

The atomic coordinates and structure factors (codes 2J3H, 2J3I, 2J3J, and 2J3K) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This research was supported by grants from the National Institutes of Health, NIGMS (to C.-H. K. and N. G. L.); Grants MCB-9976684 and MCB-0417291 from the National Science Foundation; Agricultural Plant Biochemistry Grant 2006-03339 from the United States Department of Agriculture; and by grants from McIntire-Stennis, the Murdock Charitable Trust, and the G. Thomas and Anita Hargrove Center for Plant Genomic Research. 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 may be addressed: Inst. of Biological Chemistry, Washington State University, Pullman, WA 99164-6340. Tel.: 509-335-2605; Fax: 509-335-8206; E-mail: lewisn{at}wsu.edu. ² To whom correspondence may be addressed: School of Molecular Biosciences, Washington State University, Pullman, WA 99164-4660. Tel.: 509-335-1409; Fax: 509-335-9688; E-mail: chkang{at}wsunix.wsu.edu.