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 NH(2)-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 D. 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 Mn(2+), Co(2+) and Ni(2+). 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.
Maeda
Takayuki
T
Division of Applied Bioscience, Research Faculty of Agriculture, Hokkaido University, Kita-9, Nishi-9, Kita-ku, Sapporo, Japan.
Takeda
Yuki
Y
Murakami
Tomoko
T
Yokota
Atsushi
A
Wada
Masaru
M
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D001483:Base Sequence; D020134:Catalytic Domain; D003001:Cloning, Molecular; D042742:Delftia / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli; D006836:Hydro-Lyases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D007700:Kinetics; D008239:Lysine / Q000378:metabolism*; D019216:Metals, Heavy / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011732:Pyridoxal Phosphate / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D016415:Sequence Alignment; D013379:Substrate Specificity; D013696:Temperature
D001426:Bacterial Proteins; D019216:Metals, Heavy; D011994:Recombinant Proteins; D011732:Pyridoxal Phosphate; D006836:Hydro-Lyases; D008239:Lysine