GENE CLONING, NUCLEOTIDE SEQUENCING, AND PURIFICATION AND CHARACTERIZATION OF THE LOW-SPECIFICITY L-THREONINE ALDOLASE FROM PSEUDOMONAS SP.STRAIN NCIMB-10558

A low-specificity L-threonine aldolase (L-TA) gene from Pseudomonas sp . strain NCIMB 10558 was cloned and sequenced. The gene contains an op en reading frame consisting of 1,041 nucleotides corresponding to 346 amino acid residues. The gene was overexpressed in Escherichia coli ce lls, and the recombinant enzyme was purified and characterized. The en zyme, requiring pyridoxal 5'-phosphate as a coenzyme, is strictly L sp ecific at the alpha position, whereas it cannot distinguish between th ree 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-dihhydroxy-phenylserine, L-beta-3,4-methylenedi oxyphenylserine, and L-beta-phenylserine. The predicted amino acid seq uence displayed less than 20% identity with those of low-specificity L -TA from Saccharomyces cerevisiae, L-allo-threonine aldolase from Aero monas jandaei, and four relevant hypothetical proteins from other micr oorganisms, However, lysine 207 of low-specificity L-TA from Pseudomon as sp. strain NCIMB 10558 was found to be completely conserved in thes e proteins. Site-directed mutagenesis experiments showed that substitu tion of Lys207 with Ala or Arg resulted in a significant loss of enzym e activity, with the corresponding disappearance of the absorption max imum at 120 nm, Thus, Lys207 of the L-TA probably functions as an esse ntial catalytic residue, forming an internal Schiff base with the pyri doxal 5'-phosphate of the enzyme to catalyze the reversible aldol reac tion.