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J. Biol. Chem., Vol. 283, Issue 9, 5849-5865, February 29, 2008

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Structure and Catalytic Mechanism of Eukaryotic Selenocysteine Synthase^*

Oleg M. Ganichkin, Xue-Ming Xu, Bradley A. Carlson, Heiko Mix^¶, Dolph L. Hatfield, Vadim N. Gladyshev^¶, and Markus C. Wahl¹

From the Max-Planck-Institut für Biophysikalische Chemie, Zelluläre Biochemie/Makromolekulare Röntgenkristallographie, Am Fassberg 11, D-37077 Göttingen, Germany, the Molecular Biology of Selenium Section, Laboratory of Cancer Prevention, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892, and the ^¶Department of Biochemistry, University of Nebraska, Lincoln, Nebraska 68588

In eukaryotes and Archaea, selenocysteine synthase (SecS) converts O-phospho-L-seryl-tRNA^[Ser]Sec into selenocysteyl-tRNA^[Ser]Sec using selenophosphate as the selenium donor compound. The molecular mechanisms underlying SecS activity are presently unknown. We have delineated a 450-residue core of mouse SecS, which retained full selenocysteyl-tRNA^[Ser]Sec synthesis activity, and determined its crystal structure at 1.65Å resolution. SecS exhibits three domains that place it in the fold type I family of pyridoxal phosphate (PLP)-dependent enzymes. Two SecS monomers interact intimately and together build up two identical active sites around PLP in a Schiff-base linkage with lysine 284. Two SecS dimers further associate to form a homotetramer. The N terminus, which mediates tetramer formation, and a large insertion that remodels the active site set SecS aside from other members of the family. The active site insertion contributes to PLP binding and positions a glutamate next to the PLP, where it could repel substrates with a free -carboxyl group, suggesting why SecS does not act on free O-phospho-L-serine. Upon soaking crystals in phosphate buffer, a previously disordered loop within the active site insertion contracted to form a phosphate binding site. Residues that are strictly conserved in SecS orthologs but variant in related enzymes coordinate the phosphate and upon mutation corrupt SecS activity. Modeling suggested that the phosphate loop accommodates the -phosphate moiety of O-phospho-L-seryl-tRNA^[Ser]Sec and, after phosphate elimination, binds selenophosphate to initiate attack on the proposed aminoacrylyl-tRNA^[Ser]Sec intermediate. Based on these results and on the activity profiles of mechanism-based inhibitors, we offer a detailed reaction mechanism for the enzyme.

Received for publication, November 14, 2007 , and in revised form, December 11, 2007.

The atomic coordinates and structure factors (code 3BC8, 3BCA, and 3BCB) 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 Deutsche Forschungsgemeinschaft Grant WA1126-2, a grant from the National Institutes of Health, the Intramural Research Program of the National Institutes of Health, NCI, Center for Cancer Research, and the Max-Planck-Society. 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 Fig. S1.

¹ To whom correspondence should be addressed. Tel.: 49-551-201-1046; Fax: 49-551-201-1197; E-mail: mwahl{at}gwdg.de.

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