Gene cloning, nucleotide sequencing, and purification and characterization of the low-specificity L-threonine aldolase from Pseudomonas sp. strain NCIMB 10558

Abstract

A low-specificity L-threonine aldolase (L-TA) gene from Pseudomonas sp. strain NCIMB 10558 was cloned and sequenced. The gene contains an open reading frame consisting of 1,041 nucleotides corresponding to 346 amino acid residues. The gene was overexpressed in Escherichia coli cells, and the recombinant enzyme was purified and characterized. The enzyme, requiring pyridoxal 5'-phosphate as a coenzyme, is strictly L specific at the alpha position, whereas it cannot distinguish between threo and erythro forms at the beta position. In addition to threonine, the enzyme also acts on various other L-beta-hydroxy-alpha-amino acids, including L-beta-3,4-dihydroxyphenylserine, L-beta-3,4-methylenedioxyphenylserine, and L-beta-phenylserine. The predicted amino acid sequence displayed less than 20% identity with those of low-specificity L-TA from Saccharomyces cerevisiae, L-allo-threonine aldolase from Aeromonas jandaei, and four relevant hypothetical proteins from other microorganisms. However, lysine 207 of low-specificity L-TA from Pseudomonas sp. strain NCIMB 10558 was found to be completely conserved in these proteins. Site-directed mutagenesis experiments showed that substitution of Lys207 with Ala or Arg resulted in a significant loss of enzyme activity, with the corresponding disappearance of the absorption maximum at 420 nm. Thus, Lys207 of the L-TA probably functions as an essential catalytic residue, forming an internal Schiff base with the pyridoxal 5'-phosphate of the enzyme to catalyze the reversible aldol reaction.

FIG. 1

Purification of the recombinant low-specificity l-TA from Pseudomonas sp. strain NCIMB 10558. Samples from each of the purification steps were loaded on an SDS–10% polyacrylamide gel and stained with Coomassie blue after electrophoresis. Lanes: 1, cell extract (10 μg); 2, Butyl-Toyopearl pool (10 μg); 3, DEAE-Toyopearl pool (20 μg); 4, molecular mass standards. The numbers to the left are the molecular masses of the standards.

FIG. 2

Absorption spectra of the wild-type (A) and K207R mutant (B) enzymes (the absorption spectrum of the K207A mutant enzyme is superimposed upon that of the K207R mutant enzyme, for which data are not shown). The absorption spectra were measured with 20 mM potassium phosphate buffer (pH 7.0) at a protein concentration of 1.5 mg/ml.

FIG. 3

Segmental sequence alignment of the low-specificity l-TA of Pseudomonas sp. strain NCIMB 10558 with other proteins. From top to bottom in each set, the proteins were the low-specificity l-TA from Pseudomonas sp. strain NCIMB 10558, l-allo-TA from A. jandaei, hypothetical protein from E. coli, low-specificity l-TA from S. cerevisiae, and hypothetical proteins from S. pombe, C. albicans, and T. inflatum. Identical residues are boxed in black. The numbers on the left are the residue numbers for each amino acid sequence, and those on the top are the residue numbers for the Pseudomonas aldolase sequence.