Abstract
The gene encoding low specificity D-threonine aldolase, catalyzing the interconversion of D-threonine/D-allo-threonine and glycine plus acetaldehyde, was cloned from the chromosomal DNA of Arthrobacter sp. strain DK-38. The gene contains an open reading frame consisting of 1,140 nucleotides corresponding to 379 amino acid residues. The enzyme was overproduced in recombinant Escherichia coli cells and purified to homogeneity by ammonium sulfate fractionation and three-column chromatography steps. The recombinant aldolase was identified as a pyridoxal enzyme with the capacity of binding 1 mol of pyridoxal 5'-phosphate per mol of subunit, and Lys59 of the enzyme was determined to be the cofactor binding site by chemical modification with NaBH4. In addition, Mn2+ ion was demonstrated to be an activator of the enzyme, although the purified enzyme contained no detectable metal ions. Equilibrium dialysis and atomic absorption studies revealed that the recombinant enzyme could bind 1 mol of Mn2+ ion per mol of subunit. Remarkably, the predicted amino acid sequence of the enzyme showed no significant similarity to those of the currently known pyridoxal 5'-phosphate-dependent enzymes, indicating that low specificity D-threonine aldolase is a new pyridoxal enzyme with a unique primary structure. Taken together, low specificity D-threonine aldolase from Arthrobacter sp. strain DK-38, with a unique primary structure, is a novel metal-activated pyridoxal enzyme.
Highlights
The gene encoding low specificity D-threonine aldolase, catalyzing the interconversion of D-threonine/Dallo-threonine and glycine plus acetaldehyde, was cloned from the chromosomal DNA of Arthrobacter sp. strain DK-38
Cloning of the dtaAS Gene—The primers used for cloning of the dtaAS gene by PCR were based on the NH2-terminal amino acid sequence of the purified low specificity D-Threonine aldolase (TA) from Arthrobacter sp. strain DK-38
We have previously reported the occurrence, isolation, and catalytic properties of a novel enzyme, D-threonine aldolase, that catalyzes the interconversion of D-threonine/D-allo-threonine and glycine plus acetaldehyde from Arthrobacter sp. strain DK-38 (16)
Summary
Ji-Quan Liu‡, Tohru Dairi‡, Nobuya Itoh‡, Michihiko Kataoka§, Sakayu Shimizu§, and Hideaki Yamada‡¶. The gene encoding low specificity D-threonine aldolase, catalyzing the interconversion of D-threonine/Dallo-threonine and glycine plus acetaldehyde, was cloned from the chromosomal DNA of Arthrobacter sp. The predicted amino acid sequence of the enzyme showed no significant similarity to those of the currently known pyridoxal 5-phosphatedependent enzymes, indicating that low specificity Dthreonine aldolase is a new pyridoxal enzyme with a unique primary structure. Strain DK-38, with a unique primary structure, is a novel metal-activated pyridoxal enzyme. The present paper describes cloning and sequencing of the dtaAS gene encoding the low specificity D-TA from Arthrobacter sp. Strain DK-38, the expression of the gene in Escherichia coli cells, and further characterization of the recombinant enzyme. Evidence is presented that low specificity D-TA has a unique primary structure, probably representing a new family of pyridoxal enzyme
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