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J. Biol. Chem., Vol. 280, Issue 46, 38803-38813, November 18, 2005

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Molecular Basis of Cysteine Biosynthesis in Plants

STRUCTURAL AND FUNCTIONAL ANALYSIS OF O-ACETYLSERINE SULFHYDRYLASE FROM ARABIDOPSIS THALIANA^*

From the Donald Danforth Plant Science Center, St. Louis, Missouri 63132

In plants, cysteine biosynthesis plays a central role in fixing inorganic sulfur from the environment and provides the only metabolic sulfide donor for the generation of methionine, glutathione, phytochelatins, iron-sulfur clusters, vitamin cofactors, and multiple secondary metabolites. O-Acetylserine sulfhydrylase (OASS) catalyzes the final step of cysteine biosynthesis, the pyridoxal 5'-phosphate (PLP)-dependent conversion of O-acetylserine into cysteine. Here we describe the 2.2 Å resolution crystal structure of OASS from Arabidopsis thaliana (AtOASS) and the 2.7 Å resolution structure of the AtOASS K46A mutant with PLP and methionine covalently linked as an external aldimine in the active site. Although the plant and bacterial OASS share a conserved set of amino acids for PLP binding, the structure of AtOASS reveals a difference from the bacterial enzyme in the positioning of an active site loop formed by residues 74-78 when methionine is bound. Site-directed mutagenesis, kinetic analysis, and ligand binding titrations probed the functional roles of active site residues. These experiments indicate that Asn⁷⁷ and Gln¹⁴⁷ are key amino acids for O-acetylserine binding and that Thr⁷⁴ and Ser⁷⁵ are involved in sulfur incorporation into cysteine. In addition, examination of the AtOASS structure and nearly 300 plant and bacterial OASS sequences suggest that the highly conserved 8A-9A surface loop may be important for interaction with serine acetyltransferase, the other enzyme in cysteine biosynthesis. Initial protein-protein interaction experiments using AtOASS mutants targeted to this loop support this hypothesis.

Received for publication, May 15, 2005 , and in revised form, September 1, 2005.

The atomic coordinates and structure factors (code 1Z7W and 1Z7Y) 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 by funds from the Donald Danforth Plant Science Center and United States Department of Agriculture Grant NRI-2005-02518 (to J. M. J.). 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 Present address: Pfizer, Inc., St. Louis, MO 63124.

² Supported by National Science Foundation-Research Experiences for Undergraduates Grant DBI-0244155. Present address: Dept. of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907.

³ To whom correspondence should be addressed: Donald Danforth Plant Science Center, 975 N. Warson Rd., St. Louis, MO 63132. Tel.: 314-587-1450; Fax: 314-587-1550; E-mail: jjez{at}danforthcenter.org.

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