Gene cloning and overproduction of low-specificity D-threonine aldolase from Alcaligenes xylosoxidans and its application for production of a key intermediate for parkinsonism drug

The dtaAX gene encoding a pyridoxal 5'-phosphate (pyridoxal-P)-dependent lo w-specificity D-threonine aldolase was cloned from the chromosomal DNA of A lcaligenes xylosoxidans IFO 12669. It contains an open reading frame consis ting of 1,134 nucleotides corresponding to 377 amino acid residues. The pre dicted amino acid sequence displayed 54% identity with that of D-threonine aldolase from gram-positive bacteria Ar-throbacter sp. DK-38, but showed no significant similarity with those of other known pyridoxal-P enzymes. This gram-negative bacterial enzyme was highly overproduced in recombinant Esch erickia coli cells, and the specific activity of the enzyme in the cell ext ract was as high as 18 U/mg (purified enzyme 38.6 U/mg), which was 6,000 ti mes higher than that from the wild-type Alcaligenes cell extract. The recom binant enzyme was thus feasibly purified to homogeneity by ammonium sulfate fractionation and DEAE-Toyopearl chromatography steps. The recombinant low -specificity D-threonine aldolase was shown to be an efficient biocatalyst for resolution of L-beta-3,4-methylenedioxyphenylserine, an intermediate fo r production of a therapeutic drug for Parkinson's disease.