1. 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
J Q Liu, T Dairi, M Kataoka, S Shimizu, H Yamada J Bacteriol. 1997 Jun;179(11):3555-60. doi: 10.1128/jb.179.11.3555-3560.1997.
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-allo-threonine 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 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 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.
2. Purification and characterization of L-allo-threonine aldolase from Aeromonas jandaei DK-39
M Kataoka, M Wada, K Nishi, H Yamada, S Shimizu FEMS Microbiol Lett. 1997 Jun 15;151(2):245-8. doi: 10.1111/j.1574-6968.1997.tb12577.x.
L-allo-Threonine aldolase (L-allo-threonine acetaldehyde-lyase), which exhibited specificity for L-allo-threonine but not for L-threonine, was purified from a cell-free extract of Aeromonas jandaei DK-39. The purified enzyme catalyzed the aldol cleavage reaction of L-allo-threonine (K(m) = 1.45 mM, Vmax = 45.2 mumol min-1 mg-1). The activity of the enzyme was inhibited by carbonyl reagents, which suggests that pyridoxal-5'-phosphate participates in the enzymatic reaction. The enzyme does not act on either L-serine or L-threonine, and thus it can be distinguished from serine hydroxy-methyltransferase (L-serine:tetrahydrofolate 5,10-hydroxy-methyltransferase, EC 2.1.2.1) or L-threonine aldolase (EC 4.1.2.5).
3. L-allo-threonine aldolase with an H128Y/S292R mutation from Aeromonas jandaei DK-39 reveals the structural basis of changes in substrate stereoselectivity
Hui-Min Qin, et al. Acta Crystallogr D Biol Crystallogr. 2014 Jun;70(Pt 6):1695-703. doi: 10.1107/S1399004714007664. Epub 2014 May 30.
L-allo-Threonine aldolase (LATA), a pyridoxal-5'-phosphate-dependent enzyme from Aeromonas jandaei DK-39, stereospecifically catalyzes the reversible interconversion of L-allo-threonine to glycine and acetaldehyde. Here, the crystal structures of LATA and its mutant LATA_H128Y/S292R were determined at 2.59 and 2.50 Å resolution, respectively. Their structures implied that conformational changes in the loop consisting of residues Ala123-Pro131, where His128 moved 4.2 Å outwards from the active site on mutation to a tyrosine residue, regulate the substrate specificity for L-allo-threonine versus L-threonine. Saturation mutagenesis of His128 led to diverse stereoselectivity towards L-allo-threonine and L-threonine. Moreover, the H128Y mutant showed the highest activity towards the two substrates, with an 8.4-fold increase towards L-threonine and a 2.0-fold increase towards L-allo-threonine compared with the wild-type enzyme. The crystal structures of LATA and its mutant LATA_H128Y/S292R reported here will provide further insights into the regulation of the stereoselectivity of threonine aldolases targeted for the catalysis of L-allo-threonine/L-threonine synthesis.