L-ALLO-THREONINE ALDOLASE FROM AEROMONAS-JANDAEI DK-39 - GENE CLONING, NUCLEOTIDE SEQUENCING, AND IDENTIFICATION OF THE PYRIDOXAL 5'-PHOSPHATE-BINDING LYSINE RESIDUE BY SITE-DIRECTED MUTAGENESIS

we have isolated the gene encoding L-all-threonine aldolase (L-allo-TA ) from Aeromonas jandaei DK-39, a pyridoxal 5'-phosphate (PLP)-depende nt enzyme that stereospecifically catalyzes the interconversion of L-a llo-threonine and glycine. The gene contains an open reading frame con sisting of 1,014 nucleotides corresponding to 338 amino acid residues. The protein molecular weight was estimated to be 36,294, which is in good agreement with the subunit molecular weight of the enzyme determi ned by polyacrylamide gel electrophoresis. The enzyme was overexpresse d in recombinant Escherichia coil cells and purified to homogeneity by one hydrophobic column chromatography step. The predicted amino acid sequence showed no significant similarity to those of the currently kn own PLP-dependent enzymes but displayed 40 and 41% identity with those of the hypothetical GLY1 protein of Saccharomyces cerevisiae and the GLY1-like protein of Caenorhabditis elegans, respectively. Accordingly , L-allo-TA might represent a new type of PLP-dependent enzyme. To det ermine the PLP-binding site of the enzyme, all of the three conserved lysine residues of L-allo-TA were replaced by alanine by site-directed mutagenesis. The purified mutant enzymes, K51A and K224A, showed prop erties similar to those of the wild type, while the mutant enzyme K199 A was catalytically inactive, with corresponding disappearance of the absorption maximum at 420 nm. Thus, Lys199 of L-allo-TA probably funct ions as an essential catalytic residue forming an internal Schiff base with PLP of the enzyme to catalyze the reversible aldol reaction.