A novel O-phospho-L-serine sulfhydrylation reaction catalyzed by O-acetylserine sulfhydrylase from Aeropyrum pernix K1.

O-Acetylserine sulfhydrylase (OASS), a pyridoxal 5'-phosphate (PLP)-dependent enzyme, catalyzes the synthesis of L-cysteine from O-acetyl-L-serine and sulfide. O-Acetyl-L-serine is labile at high temperatures at which hyperthermophilic archaea live. Herein, a study of the substrate specificity of OASS from Aeropyrum pernix K1 with respect to O-acetyl-L-serine in L-cysteine synthesis is described. L-Azaserine, 3-chloro-L-alanine, and O-phospho-L-serine reacted with A. pernix OASS in a PLP-dependent manner. Sulfhydrylation reactions using these substrates reached a maximum in the pH range between 7.3 and 8.1. L-Azaserine and O-phospho-L-serine were found to be heat-stable substrates. The presence of FeCl3 or NiCl2 strongly inhibited the O-acetyl-L-serine sulfhydrylation reaction, whereas the O-phospho-L-serine sulfhydrylation reaction was only slightly inhibited. Kinetic analyses revealed that the O-phospho-L-serine sulfhydrylation reaction as well as the O-acetyl-L-serine sulfhydrylation reaction for A. pernix OASS followed a ping-pong bi-bi mechanism. In the case of the O-phospho-L-serine sulfhydrylation reaction at 85 degrees C, the K(m) values for O-phospho-L-serine and sulfide, and the rate constant were 250 mM, 12.5 mM, and 14000 s(-1), respectively. The reactivity of O-phospho-L-serine in the L-cysteine synthetic reaction provides a key for understanding the biosynthesis of L-cysteine by hyperthermophilic archaea. This is the first report of an enzyme that catalyzes the O-phospho-L-serine sulfhydrylation reaction.