d-Serine dehydratase from Saccharomyces cerevisiae (DsdSC) is a fold-type III pyridoxal 5'-phosphate-dependent enzyme catalyzing d-serine dehydration. The enzyme contains 1 mol Zn(2+) in its active site and shows a unique zinc dependence. The Zn(2+) is essential for the d-serine dehydration, but not for the α,β-elimination of β-Cl-d-alanine catalyzed as a side-reaction. The fact that dehydration of d-threonine and d-allo-threonine, also catalyzed by DsdSC, is likewise Zn(2+) dependent indicates that Zn(2+) is indispensable for the elimination of hydroxyl group, regardless of the stereochemistry of C(β) . Removal of Zn(2+) results in a less polar active site without changing the gross conformation of DsdSC. (1) H NMR determined the rates of α-hydrogen abstraction and hydroxyl group elimination of d-serine in (2) H(2) O to be 9.7 and 8.5 s(-1) , respectively, while the removal of Zn(2+) abolished both reactions. Mutation of Cys400 or His398 within the Zn(2+) binding sites to Ala endowed DsdSC with similar properties to those of the Zn(2+) -depleted wild-type enzyme: the mutants lost the reactivity toward d-serine and d-threonine but retained that toward β-Cl-d-alanine. (1) H NMR analysis also revealed that both α-hydrogen abstraction and hydroxyl group elimination from d-serine were severely hampered in the C400A mutant. Our data suggest that DsdSC catalyzes the α-hydrogen abstraction and hydroxyl group elimination in a concerted fashion.