Cloning and expression of the pyridoxal 5'-phosphate-dependent aspartate racemase gene from the bivalve mollusk Scapharca broughtonii and characterization of the recombinant enzyme.
D-aspartate is present at high concentrations in the tissues of Scapharca broughtonii, and its production depends on aspartate racemase. This enzyme is the first aspartate racemase purified from animal tissues and unique in its pyridoxal 5'-phosphate (PLP)-dependence in contrast to microbial aspartate racemases thus far characterized. The enzyme activity is markedly increased in the presence of AMP and decreased in the presence of ATP. To analyze the structure-function relationship of the enzyme further, we cloned the cDNA of aspartate racemase, and then purified and characterized the recombinant enzyme expressed in Escherichia coli. The cDNA included an open reading frame of 1,017 bp encoding a protein of 338 amino acids, and the deduced amino acid sequence contained a PLP-binding motif. The sequence exhibits the highest identity (43-44%) to mammalian serine racemase, followed mainly by threonine dehydratase. These relationships are fully supported by phylogenetic analyses of the enzymes. The active recombinant aspartate racemase found in the Escherichia coli extract represented about 10% of total bacterial protein and was purified to display essentially identical physicochemical and catalytic properties with those of the native enzyme. In addition, the enzyme showed a dehydratase activity toward L-threo-3-hydroxyaspartate, similar to the mammalian serine racemase that produces pyruvate from D- and L-serine.
Abe
Katsumasa
K
Department of Environmental Systems Engineering, Nagaoka University of Technology, Nagaoka, Niigata 940-2188, Japan.
Takahashi
Shouji
S
Muroki
Yoshinori
Y
Kera
Yoshio
Y
Yamada
Ryo-hei
RH
D000591:Amino Acid Isomerases / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D008974:Mollusca / Q000201:enzymology*; D011732:Pyridoxal Phosphate / Q000737:chemistry*; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D017385:Sequence Homology; D013379:Substrate Specificity
D011994:Recombinant Proteins; D011732:Pyridoxal Phosphate; D000591:Amino Acid Isomerases; C019749:aspartate racemase