Molecular basis of cysteine biosynthesis in plants: Structural and functional analysis of O-acetylserine sulfhydrylase from Arabidopsis thaliana

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 β8A-β9A 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.