Katsumasa ABE

Molecular Cloning of cDNA Encoding Aspartate Racemese from the Bivalve Mollusk Scapharca broughtonii

Ryo-hei YAMADA, Yoshio KERA, Shouji TAKAHASHI

We have recently reported the purification of aspartate racemase from blood shell Scapharca broughtonii, and showed that it is strictly specific toward aspartate and is pyridoxal 5'-phosphate (PLP) - dependent. Further characterization of the enzyme demonstrates that the enzyme activity is markedly modulated by some nucleotides. No similar modulation of activity has been reported on any amino acid racemase to the best of our knowledge. In the present study, we have cloned the aspartate racemase gene to analyze the relationship between enzyme properties and structure. This is the first report on gene cloning of PLP - dependent aspartate racemase from eukaryotic organisms.
Aspartate racemase was purified from the foot muscle of the Scapharca broughtonii to homogeneity, and the amino acid sequences of internal peptides were determined. A partial cDNA fragment of the enzyme was isolated by PCR using degenerate primers based on the internal amino acid sequences of aspartate racemese, and the 5'- and 3'- cDNA ends were obtained by rapid amplification of cDNA ends PCR (RACE-PCR). The aspartate racemase specific activity in the cell-free extract of E. coli transformant cells expressing the aspartate racemase gene was 130-fold higher than that in homogenate of S. broughtonii foot muscle.