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Journal of Bacteriology, August 2002, p. 4134-4140, Vol. 184, No. 15
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.15.4134-4140.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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

Alexander D. Kim,1 Angela S. Baker,1,{dagger} Debra Dunaway-Mariano,1* W. W. Metcalf,2,{ddagger} B. L. Wanner,2 and Brian M. Martin3,§

Department of Chemistry, University of New Mexico, Albuquerque, New Mexico 87131,1 Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907,2 National Institute of Mental Health, Bethesda, Maryland 20892-44053

Received 15 February 2002/ Accepted 26 April 2002

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 Vm and log Vm/Km versus pH) revealed a pH optimum of 8.5. At pH 8.5, Keq is equal to 0.5 and the kcat values of the forward and reverse reactions are 7 and 9 s-1, respectively. The Km 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.

* Corresponding author. Mailing address: Department of Chemistry, University of New Mexico, Albuquerque, NM 87131. Phone: (505) 277-3383. Fax: (505) 277-6202. E-mail: dd39{at}unm.edu.

{dagger} Present address: Paragon Bioservices, Johns Hopkins Bayview Research Campus, Baltimore, MD 21224.

{ddagger} Present address: Department of Microbiology, University of Illinois, Urbana, IL 61801.

§ Present address: USAMRIID, MCMR-UIT-C, Fort Detrick, MD 21702-5011.

Journal of Bacteriology, August 2002, p. 4134-4140, Vol. 184, No. 15
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.15.4134-4140.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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