How the “South”Has Been Constructed? : Imaginary Africa of the Japanese Popular/Juvenile Literature and Comics

表象と文化(13

From "Tiryagyoni" through "Animal" to "Ferns" : A Critique of Western Religious Thinking with L. Tolstoy, V. Rozanov, and F. Kafka

表象と文化 (15

Gene Cloning and Expression of Pyridoxal 5'-Phosphate-Dependent L-threo-3-Hydroxyaspartate Dehydratase from Pseudomonas sp. T62, and Characterization of the Recombinant Enzyme

L-threo-3-Hydroxyaspartate dehydratase (L-THA DH, EC 4.3.1.16), which catalyses the cleavage of L-threo-3-hydroxyaspartate (L-THA) to oxalacetate and ammonia, has been purified from the soil bacterium Pseudomonas sp. T62. In this report, the gene encoding L-THA DH was cloned and expressed in Escherichia coli, and the gene product was purified and characterized in detail. A 957-bp nucleotide fragment was confirmed to be the gene encoding L-THA DH, based on the agreement of internal amino acid sequences. The deduced amino acid sequence, which belongs to the serine/threonine dehydratase family, shows similarity to YKL218c from Saccharomyces cerevisiae (64%), serine racemase from Schizosaccharomyces pombe (64%), and Mus musculus (36%), and biodegradative threonine dehydratase from E. coli (38%). Site-directed mutagenesis experiments revealed that lysine at position 53 is an important residue for enzymatic activity. This enzyme exhibited dehydratase activity specific only to L-THA (K_m = 0.54 mM, V_[max] = 39.0 μmol min^[-1] [mg protein]^[-1]), but not to other 3-hydroxyaspartate isomers, and exhibited no detectable serine/aspartate racemase activity. This is the first report of an amino acid sequence of the bacterial enzyme that acts on L-THA

Purification, characterization and amino acid sequence of a novel enzyme, D-threo-3-hydroxyaspartate dehydratase, from Delftia sp. HT23

D-threo-3-hydroxyaspartate dehydratase (D-THA DH) was purified from the cell-free extract of the soil-isolated bacterium Delftia sp. HT23. The enzyme exhibited dehydratase activity towards D-threo-3-hydroxyaspartate, L-threo-3-hydroxyaspartate, L-erythro-3-hydroxyaspartate and D-serine. Absorption of the purified enzyme at 412 nm suggests that it contains pyridoxal 5'-phosphate (PLP) as a cofactor. The NH2-terminal and internal amino acid sequences showed significant similarity to hypothetical alanine racemase of genome-sequenced Delftia acidovorans SPH-1; however, the purified enzyme showed no alanine racemase activity. Using the sequence information of Delftia acidovorans SPH-1, the gene encoding D-THA DH was cloned. The deduced amino acid sequence, which belongs to the alanine racemase family, shows significant (26-36%) similarity to D-serine dehydratase of both Saccharomyces cerevisiae and chicken. In order to obtain purified D-THA DH efficiently, the gene was expressed in Escherichia coli. The recombinant enzyme was highly activated by divalent cations, such as Mn2+, Co2+ and Ni2+. Site-directed mutagenesis experiment revealed that lysine 43 is an important residue involved in PLP binding and catalysis. This is the first reported enzyme that acts on D-THA. In addition, this enzyme is the first example of a prokaryotic dehydratase belonging to the fold-type III PLP-dependent enzyme family