A pyridoxal-phosphate protein. The enzyme, purified from the bacterium Pseudomonas sp. T62, is highly specific, and does not accept any other stereoisomer of 3-hydroxyaspartate. Different from EC 4.3.1.20, erythro-3-hydroxy-L-aspartate ammonia-lyase and EC 4.3.1.27, threo-3-hydroxy-D-aspartate ammonia-lyase. Requires a divalent cation such as Mn2+, Mg2+, or Ca2+.
A pyridoxal-phosphate protein. The enzyme, purified from the bacterium Pseudomonas sp. T62, is highly specific, and does not accept any other stereoisomer of 3-hydroxyaspartate. Different from EC 4.3.1.20, erythro-3-hydroxy-L-aspartate ammonia-lyase and EC 4.3.1.27, threo-3-hydroxy-D-aspartate ammonia-lyase. Requires a divalent cation such as Mn2+, Mg2+, or Ca2+.
none of the other 3-hydroxy amino acids tested (DL-erythro- and D-threo-3-hydroxyaspartate, D-threonine, L-threonine, DL-allo-threonine, D-serine, L-serine, and DL-phenylserine) is a substrate for this enzyme, either at 5 mM or 50 mM, as in the case of the enzyme from the original strain
none of the other 3-hydroxy amino acids tested (DL-erythro- and D-threo-3-hydroxyaspartate, D-threonine, L-threonine, DL-allo-threonine, D-serine, L-serine, and DL-phenylserine) is a substrate for this enzyme, either at 5 mM or 50 mM, as in the case of the enzyme from the original strain
effect of divalent cations are measured using the enzyme as control dialyzed against the buffer minus MnCl2, showing a decrease of the specific activity of the enzyme to approximately 36% of the initial activity after overnight dialysis
none of the other 3-hydroxy amino acids tested (DL-erythro- and D-threo-3-hydroxyaspartate, D-threonine, L-threonine, DL-allo-threonine, D-serine, L-serine, and DL-phenylserine) is a substrate for this enzyme, either at 5 mM or 50 mM, as in the case of the enzyme from the original strain
DL-aspartate, L-serine, DL-threonine, DL-allo-threonine, DL-phenylserine, or malonic acid did not cause significant inhibition of the enzyme reaction at 5 mM
determined by using a MALDI-TOF mass spectrometer, measured value is lower than that determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (40 kDa), and that of the enzyme purified from the original strain (approximately 39 kDa by SDS-PAGE). The discrepancy may be due to the surface charge of the protein or some unknown factors such as the conformation of the enzyme
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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
crystals of D-THA DH belong to space group I4(1)22, with unit-cell parameters a = b = 157.3, c = 157.9 A. Single wavelength anomalous diffraction data are collected to a resolution of 2.0 A
after centrifugation of cultivated cell culture with the recombinant enzyme, resulting supernatant is applied to a Ni-sepharose column connected to a fast protein liquid chromatography system. The column is equilibrated with buffer supplemented with 20 mM imidazole. The enzyme is eluted with imidazole gradient.
the recombinant enzyme expressed in Escherichia coli is used to produce optically pure L-erythro-3-hydroxyaspartate and D-threo-3-hydroxyaspartate from the corresponding DL-racemic mixtures
Gene cloning and expression of pyridoxal 5-phosphate-dependent L-threo-3-hydroxyaspartate dehydratase from Pseudomonas sp. T62, and characterization of the recombinant enzyme
Nagano, H.; Shibano, K.; Matsumoto, Y.; Yokota, A.; Wada, M.
Isolation and amino acid sequence of a dehydratase acting on D-erythro-3-hydroxyaspartate from Pseudomonas sp. N99, and its application in the production of optically active 3-hydroxyaspartate