We have isolated the gene encoding L-allo-threonine aldolase (L-allo-TA) from Aeromonas jandaei DK-39, a pyridoxal 5-phosphate (PLP)-dependent enzyme that stereospecifically catalyzes the interconversion of L-allothreonine and glycine. The gene contains an open reading frame consisting 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 determined by polyacrylamide gel electrophoresis. The enzyme was overexpressed in recombinant Escherichia coli 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 known 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 determine the PLPbinding 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 properties similar to those of the wild type, while the mutant enzyme K199A was catalytically inactive, with corresponding disappearance of the absorption maximum at 420 nm. Thus, Lys199 of L-allo-TA probably functions as an essential catalytic residue forming an internal Schiff base with PLP of the enzyme to catalyze the reversible aldol reaction.The bioorganic synthesis of -hydroxy-␣-amino acids is attracting a great deal of attention because of their potential application as chiral building blocks for the synthesis of biologically active molecules (6,7,24,32,33). A variety of -hydroxy-␣-amino acids are present in complex natural compounds with interesting biological properties. 4-Hydroxy-Lthreonine, for instance, is a precursor of rizobitoxine, a potent inhibitor of pyridoxal 5Ј-phosphate (PLP)-dependent enzymes (32). 3,4,5-Trihydroxy-L-aminopentanoic acid is a key component of polyoxins (32). -Hydroxy-␣-amino acids are also constituents of a range of antibiotics, for example, cyclosporin A, lysobactin, and vancomycin (30) and bouvardin and deoxybouvardin (9). Threonine aldolase (EC 4.1.2.5), which catalyzes the retro-aldol cleavage of certain -hydroxy-␣-amino acids, has also been shown to catalyze synthesis of the substituted amino acids from aldehyde and glycine (24,32,33).Thus far, two types of threonine aldolases have been purified and characterized. Low-specific-activity L-threonine aldolase (L-TA), previously named L-threonine aldolase, catalyzes the cleavage of both L-threonine and L-allo-threonine to glycine and acetaldehyde, as well as the reverse aldol condensation. The enzymes have been purified from Candida humicola (13,35) and Pseudomonas sp. strain NCIMB 11097 (5). L-TA activity has also been shown to exist in mammals (10,20,23) and a variety of other microbial ...
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