Characterization of a novel thermostable O-acetylserine sulfhydrylase from Aeropyrum pernix K1.
An O-acetylserine sulfhydrylase (OASS) from the hyperthermophilic archaeon Aeropyrum pernix K1, which shares the pyridoxal 5'-phosphate binding motif with both OASS and cystathionine beta-synthase (CBS), was cloned and expressed by using Escherichia coli Rosetta(DE3). The purified protein was a dimer and contained pyridoxal 5'-phosphate. It was shown to be an enzyme with CBS activity as well as OASS activity in vitro. The enzyme retained 90% of its activity after a 6-h incubation at 100 degrees C. In the O-acetyl-L-serine sulfhydrylation reaction, it had a pH optimum of 6.7, apparent K(m) values for O-acetyl-L-serine and sulfide of 28 and below 0.2 mM, respectively, and a rate constant of 202 s(-1). In the L-cystathionine synthetic reaction, it showed a broad pH optimum in the range of 8.1 to 8.8, apparent K(m) values for L-serine and L-homocysteine of 8 and 0.51 mM, respectively, and a rate constant of 0.7 s(-1). A. pernix OASS has a high activity in the L-cysteine desulfurization reaction, which produces sulfide and S-(2,3-hydroxy-4-thiobutyl)-L-cysteine from L-cysteine and dithiothreitol.
Mino
Koshiki
K
Special Division for Human Life Technology, National Institute of Advanced Industrial Science and Technology (AIST, Kansai), Ikeda, Osaka 563-8577, Japan.
Ishikawa
Kazuhiko
K
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D003540:Cystathionine / Q000737:chemistry / Q000378:metabolism; D003545:Cysteine / Q000031:analogs & derivatives / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D003547:Cysteine Synthase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019620:Desulfurococcaceae / Q000201:enzymology* / Q000235:genetics; D004229:Dithiothreitol / Q000737:chemistry / Q000378:metabolism; D004789:Enzyme Activation / Q000502:physiology; D004795:Enzyme Stability / Q000502:physiology; D006710:Homocysteine / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013440:Sulfides / Q000737:chemistry; D013696:Temperature
D019843:Archaeal Proteins; C473323:S-(2,3-dihydroxy-4-thiobutyl)cysteine; D013440:Sulfides; D006710:Homocysteine; D003540:Cystathionine; D012694:Serine; D003547:Cysteine Synthase; C043943:O-acetylserine; D003545:Cysteine; D004229:Dithiothreitol