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J. Biol. Chem., Vol. 281, Issue 21, 14939-14947, May 26, 2006

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Membrane Association, Mechanism of Action, and Structure of Arabidopsis Embryonic Factor 1 (FAC1)^*

Byung Woo Han, Craig A. Bingman, Donna K. Mahnke^¶, Ryan M. Bannen¹, Sebastian Y. Bednarek², Richard L. Sabina^¶3, and George N. Phillips, Jr.⁴

From the Department of Biochemistry and the Center for Eukaryotic Structural Genomics, University of Wisconsin, Madison, Wisconsin 53706 and the ^¶Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226

Embryonic factor 1 (FAC1) is one of the earliest expressed plant genes and encodes an AMP deaminase (AMPD), which is also an identified herbicide target. This report identifies an N-terminal transmembrane domain in Arabidopsis FAC1, explores subcellular fractionation, and presents a 3.3-Å globular catalytic domain x-ray crystal structure with a bound herbicide-based transition state inhibitor that provides the first glimpse of a complete AMPD active site. FAC1 contains an (/)₈-barrel characterized by loops in place of strands 5 and 6 that places it in a small subset of the amidohydrolase superfamily with imperfect folds. Unlike tetrameric animal orthologs, FAC1 is a dimer and each subunit contains an exposed Walker A motif that may be involved in the dramatic combined K_m (25-80-fold lower) and V_max (5-6-fold higher) activation by ATP. Normal mode analysis predicts a hinge motion that flattens basic surfaces on each monomer that flank the dimer interface, which suggests a reversible association between the FAC1 globular catalytic domain and intracellular membranes, with N-terminal transmembrane and disordered linker regions serving as the anchor and attachment to the globular catalytic domain, respectively.

Received for publication, December 6, 2005 , and in revised form, March 13, 2006.

^* This work was supported in part National Institutes of Health NIGMS Grants P50 GM64598 and 1 U54 GM074901-01 to the Center for Eukaryotic Structural Genomics, work performed at the Medical College of Wisconsin was supported by a grant from the Research Affairs Committee (RAC) and through a cooperative agreement with Bayer CropScience GmbH, Frankfurt am Main, Germany. Use of the Advanced Photon Source was supported by the United States Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract W-31-109-Eng-38. 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Materials and Methods, Results, Figs. S1-S4, Table 1, and Movie 1.

¹ Supported by a grant from the Department of Energy, Genomes to Life project DE-FG2-04ER25627 and by National Library of Medicine Grant 5T15LM007359.

² Supported by a grant from the Department of Energy, Division of Energy Biosciences project DE-FG02-99ER20332.

³ To whom correspondence may be addressed. Tel.: 414-456-4697; Fax: 414-456-6510; E-mail: sabinar{at}mcw.edu. ⁴ To whom correspondence may be addressed. Tel.: 608-263-6142; Fax: 608-262-3453; E-mail: phillips{at}biochem.wisc.edu.

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