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.
Mino
Koshiki
K
The Special Division for Human Life Technology, National Institute of Advanced Industrial Science and Technology, 1-8-31 Midorigaoka, Ikeda, 563-8577 Osaka, Japan.
Ishikawa
Kazuhiko
K
D002384:Catalysis; D003545:Cysteine / Q000096:biosynthesis* / Q000737:chemistry; D003547:Cysteine Synthase / Q000378:metabolism*; D019620:Desulfurococcaceae / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D010768:Phosphoserine / Q000378:metabolism*; D013057:Spectrum Analysis; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013696:Temperature
D013438:Sulfhydryl Compounds; D010768:Phosphoserine; D003547:Cysteine Synthase; D003545:Cysteine