The 2-Aminoethylphosphonate-Specific Transaminase of the 2-Aminoethylphosphonate Degradation Pathway

Abstract

ABSTRACT The 2-aminoethylphosphonate transaminase (AEPT; the phnW gene product) of the Salmonella enterica serovar Typhimurium 2-aminoethylphosphonate (AEP) degradation pathway catalyzes the reversible reaction of AEP and pyruvate to form phosphonoacetaldehyde (P-Ald) and l -alanine ( l -Ala). Here, we describe the purification and characterization of recombinant AEPT. pH rate profiles (log V m and log V m / K m versus pH) revealed a pH optimum of 8.5. At pH 8.5, K eq is equal to 0.5 and the k cat values of the forward and reverse reactions are 7 and 9 s −1 , respectively. The K m for AEP is 1.11 ± 0.03 mM; for pyruvate it is 0.15 ± 0.02 mM, for P-Ald it is 0.09 ± 0.01 mM, and for l -Ala it is 1.4 ± 0.03 mM. Substrate specificity tests revealed a high degree of discrimination, indicating a singular physiological role for the transaminase in AEP degradation. The 40-kDa subunit of the homodimeric enzyme is homologous to other members of the pyridoxalphosphate-dependent amino acid transaminase superfamily. Catalytic residues conserved within well-characterized members are also conserved within the seven known AEPT sequences. Site-directed mutagenesis demonstrated the importance of three selected residues (Asp168, Lys194, and Arg340) in AEPT catalysis.