The 2-aminoethylphosphonate-specific transaminase of the 2-aminoethylphosphonate degradation pathway.
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.
Kim
Alexander D
AD
Department of Chemistry, University of New Mexico, Albuquerque, New Mexico 87131, USA.
Baker
Angela S
AS
Dunaway-Mariano
Debra
D
Metcalf
W W
WW
Wanner
B L
BL
Martin
Brian M
BM
D000615:Aminoethylphosphonic Acid / Q000378:metabolism*; D001120:Arginine / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D006863:Hydrogen-Ion Concentration; D008239:Lysine / Q000235:genetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000096:biosynthesis; D012486:Salmonella typhimurium / Q000201:enzymology*; D013379:Substrate Specificity; D000637:Transaminases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics
D011994:Recombinant Proteins; D001224:Aspartic Acid; D001120:Arginine; D000615:Aminoethylphosphonic Acid; D000637:Transaminases; C038231:2-aminoethylphosphonate-pyruvate aminotransferase; D008239:Lysine