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

Abstract

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 ...

Cloning and characterization of the aru genes encoding enzymes of the catabolic arginine succinyltransferase pathway in Pseudomonas aeruginosa

“…The amino acid sequence of the deduced protein of the last ORF, ORF1 (334 residues; nt 8328 to 9214) (Fig. 4), showed 57 and 38% identities with L-allo-threonine aldolases of Aeromonas jandaei (18) and Saccharomyces cerevisiae (19, An ORF capable of encoding a polypeptide with 329 residues (M r , 36,854) was found 514 bp upstream of aruC. This protein had significant homology with the AraC-XylS family of regulatory proteins (7).…”

A Novel Metal-activated Pyridoxal Enzyme with a Unique Primary Structure, Low Specificity d-Threonine Aldolase fromArthrobacter sp. Strain DK-38

“…The ␤ and ␥ enzymes catalyze ␤-replacement or ␤-elimination and ␥-replacement or ␥-elimination reactions, respectively. We have recently cloned and determined the primary structures of several L-type threonine aldolases, L-allo-TA from A. jandaei DK-39 (15) and three low specificity L-TAs from S. cerevisiae S288C (13), Pseudomonas sp. strain NCIMB 10558 (14), and E. coli GS245.…”

“…The genes encoding for L-allo-TA of A. jandaei and low specificity L-TAs of S. cerevisiae and Pseudomonas sp. strain have been cloned and sequenced (13)(14)(15). Likewise, Dtype TA, acting on D-threonine and/or D-allo-threonine, might include D-allo-TA, D-TA, and low specificity D-TA.…”

Synthesis of 4-Hydroxyisoleucine by the Aldolase–Transaminase Coupling Reaction and Basic Characterization of the Aldolase fromArthrobacter simplexAKU 626

“…There have been some reports on PLPdependent aldolases. [13][14][15][16] It is not clear that the Arthrobacter aldolase requires PLP as a coenzyme, because the transaminase in the reaction mixture also requires PLP as a cofactor. Further characterization, cloning, and expression of the aldolase are in progress.…”