Publications

• Crystallization and preliminary X-ray diffraction analysis of O-acetylserine sulfhydrylase from Aeropyrum pernix K1.

  Mino K., Oda Y., Ataka M., Ishikawa K.

  Crystals of O-acetylserine sulfhydrylase from Aeropyrum pernix K1 were obtained by the hanging-drop vapour-diffusion method at 298 K. An X-ray diffraction data set was collected to 2.25 A resolution at 100 K. The crystal belonged to space group P42(1)2, P4(1)2(1)2, P4(2)2(1)2 or P4(3)2(1)2. The un ... >> More

  Crystals of O-acetylserine sulfhydrylase from Aeropyrum pernix K1 were obtained by the hanging-drop vapour-diffusion method at 298 K. An X-ray diffraction data set was collected to 2.25 A resolution at 100 K. The crystal belonged to space group P42(1)2, P4(1)2(1)2, P4(2)2(1)2 or P4(3)2(1)2. The unit-cell parameters were a = b = 74.5, c = 276.0 A. The presence of two subunits of the enzyme per asymmetric unit gives a crystal Volume per protein mass (V(M)) of 2.28 A(3) Da(-1) and a solvent content of 46%(v/v). << Less

  Acta Crystallogr D Biol Crystallogr 59:338-340(2003) [PubMed] [EuropePMC]

• Characterization of a novel thermostable O-acetylserine sulfhydrylase from Aeropyrum pernix K1.

  Mino K., Ishikawa K.

  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 di ... >> More

  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. << Less

  J. Bacteriol. 185:2277-2284(2003) [PubMed] [EuropePMC]

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

  Mino K., Ishikawa K.

  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 ... >> More

  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. << Less

  FEBS Lett 551:133-138(2003) [PubMed] [EuropePMC]

• Three-dimensional structure of a new enzyme, O-phosphoserine sulfhydrylase, involved in L-cysteine biosynthesis by a hyperthermophilic archaeon, Aeropyrum pernix K1, at 2.0 A resolution.

  Oda Y., Mino K., Ishikawa K., Ataka M.

  O-Phosphoserine sulfhydrylase is a new enzyme found in a hyperthermophilic archaeon, Aeropyrum pernix K1. This enzyme catalyzes a novel cysteine synthetic reaction from O-phospho-l-serine and sulfide. The crystal structure of the enzyme was determined at 2.0A resolution using the method of multi-w ... >> More

  O-Phosphoserine sulfhydrylase is a new enzyme found in a hyperthermophilic archaeon, Aeropyrum pernix K1. This enzyme catalyzes a novel cysteine synthetic reaction from O-phospho-l-serine and sulfide. The crystal structure of the enzyme was determined at 2.0A resolution using the method of multi-wavelength anomalous dispersion. A monomer consists of three domains, including an N-terminal domain with a new alpha/beta fold. The topology folds of the middle and C-terminal domains were similar to those of the O-acetylserine sulfhydrylase-A from Salmonella typhimurium and the cystathionine beta-synthase from human. The cofactor, pyridoxal 5'-phosphate, is bound in a cleft between the middle and C-terminal domains through a covalent linkage to Lys127. Based on the structure determined, O-phospho-l-serine could be rationally modeled into the active site of the enzyme. An enzyme-substrate complex model and a mutation experiment revealed that Arg297, unique to hyperthermophilic archaea, is one of the most crucial residues for O-phosphoserine sulfhydrylation activity. There are more hydrophobic areas and less electric charges at the dimer interface, compared to the S.typhimurium O-acetylserine sulfhydrylase. << Less

  J. Mol. Biol. 351:334-344(2005) [PubMed] [EuropePMC]

• CysK2 from Mycobacterium tuberculosis is an O-phospho-L-serine-dependent S-sulfocysteine synthase.

  Steiner E.M., Boeth D., Loessl P., Vilaplana F., Schnell R., Schneider G.

  Mycobacterium tuberculosis is dependent on cysteine biosynthesis, and reduced sulfur compounds such as mycothiol synthesized from cysteine serve in first-line defense mechanisms against oxidative stress imposed by macrophages. Two biosynthetic routes to l-cysteine, each with its own specific cyste ... >> More

  Mycobacterium tuberculosis is dependent on cysteine biosynthesis, and reduced sulfur compounds such as mycothiol synthesized from cysteine serve in first-line defense mechanisms against oxidative stress imposed by macrophages. Two biosynthetic routes to l-cysteine, each with its own specific cysteine synthase (CysK1 and CysM), have been described in M. tuberculosis, but the function of a third putative sulfhydrylase in this pathogen, CysK2, has remained elusive. We present biochemical and biophysical evidence that CysK2 is an S-sulfocysteine synthase, utilizing O-phosphoserine (OPS) and thiosulfate as substrates. The enzyme uses a mechanism via a central aminoacrylate intermediate that is similar to that of other members of this pyridoxal phosphate-dependent enzyme family. The apparent second-order rate of the first half-reaction with OPS was determined as kmax/Ks = (3.97 × 10(3)) ± 619 M(-1) s(-1), which compares well to the OPS-specific mycobacterial cysteine synthase CysM with a kmax/Ks of (1.34 × 10(3)) ± 48.2. Notably, CysK2 does not utilize thiocarboxylated CysO as a sulfur donor but accepts thiosulfate and sulfide as donor substrates. The specificity constant kcat/Km for thiosulfate is 40-fold higher than for sulfide, suggesting an annotation as S-sulfocysteine synthase. Mycobacterial CysK2 thus provides a third metabolic route to cysteine, either directly using sulfide as donor or indirectly via S-sulfocysteine. Hypothetically, S-sulfocysteine could also act as a signaling molecule triggering additional responses in redox defense in the pathogen upon exposure to reactive oxygen species during dormancy. << Less

  J. Bacteriol. 196:3410-3420(2014) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.