PMID:12107130
| Citation |
Kim, AD, Baker, AS, Dunaway-Mariano, D, Metcalf, WW, Wanner, BL and Martin, BM (2002) The 2-aminoethylphosphonate-specific transaminase of the 2-aminoethylphosphonate degradation pathway. J. Bacteriol. 184:4134-40 |
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| 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. |
| Links | |
| Keywords |
Aminoethylphosphonic Acid/metabolism; Arginine/genetics; Aspartic Acid/genetics; Escherichia coli/genetics; Genetic Vectors; Hydrogen-Ion Concentration; Lysine/genetics; Molecular Sequence Data; Molecular Weight; Mutagenesis, Site-Directed; Recombinant Proteins/biosynthesis; Salmonella typhimurium/enzymology; Substrate Specificity; Transaminases/biosynthesis; Transaminases/chemistry; Transaminases/genetics |
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