A NOVEL METAL-ACTIVATED PYRIDOXAL ENZYME WITH A UNIQUE PRIMARY STRUCTURE, LOW SPECIFICITY D-THREONINE ALDOLASE FROM ARTHROBACTER SP. STRAINDK-38 - MOLECULAR-CLONING AND COFACTOR CHARACTERIZATION

The gene encoding low specificity D-threonine aldolase, catalyzing the interconversion of D-threonine/D-allo-threonine and glycine plus acet aldehyde, was cloned from the chromosomal DNA of Arthrobacter sp, stra in DK-38, The gene contains an open reading frame consisting of 1,140 nucleotides corresponding to 379 amino acid residues. The enzyme was o verproduced in recombinant Escherichia coli cells and purified to homo geneity by ammonium sulfate fractionation and three-column chromatogra phy steps. The recombinant aldolase was identified as a pyridoxal enzy me with the capacity of binding 1 mol of pyridoxal 5'-phosphate per mo l of subunit, and Lys(59) Of the enzyme was determined to be the cofac tor 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 dialys is and atomic absorption studies revealed that the recombinant enzyme could bind 1 mol of Mn2+ ion per mol of subunit, Remarkably, the predi cted amino acid sequence of the enzyme showed no significant similarit y to those of the currently known pyridoxal 5'-phosphate-dependent enz ymes, indicating that low specificity D-threonine aldolase is a new py ridoxal enzyme with a unique primary structure. Taken together, low sp ecificity D-threonine aldolase from Arthrobacter sp. strain DK-38, wit h a unique primary structure, is a novel metal-activated pyridoxal enz yme.