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

2-Aminoethylphosphonate (AEP) and its N-alkylated derivatives are the most abundant and ubiquitous of naturally occurring phosphonates (16). These are typically found as conjugates of glycans (7), lipids (3, 19, 31), and proteins (15), which in turn perform essential biochemical functions in specialized lower organisms. In pathogens, AEP conjugates are used for host infection and persistence. Thus, the enzymes responsible for AEP metabolism are prime targets for inhibitor development. AEP is synthesized by a variety of organisms according to the pathway shown in Fig. ​Fig.11 (6, 24, 36). Because of its natural abundance and resistance to acid-, base-, and phosphotransferase-catalyzed hydrolysis (16), soil-dwelling bacteria have acquired a unique pathway for the degradation of AEP to usable forms of carbon, nitrogen, and phosphorus (Fig. ​(Fig.1)1) (8, 18, 22, 28, 33). FIG. 1. Pathway of AEP synthesis. PEP, phosphoenolpyruvate. Cloning and sequencing of genes for the Salmonella enterica serovar Typhimurium AEP pathway operon revealed a cluster of seven genes (phnR to phnX) that are activated by the Pho regulon under conditions of phosphate deprivation (18, 23; W. W. Metcalf, W. Jiang, and B. L. Wanner, unpublished data). Based on sequence similarities at the protein level, PhnR is thought to act as a transcriptional regulator (S.-K. Kim, W. Jiang, K. A. Datsenko, K.-S. Lee, and B. L. Wanner, unpublished data), PhnS is thought to act as a periplasmic binding protein, PhnT is thought to act as an ABC family traffic ATPase, and PhnU and PhnV function as the integral membrane channel proteins. PhnW and PhnX are the AEP pathway enzymes AEP aminotransferase (AEPT) (EC 2.6.1.37) and a phosphonoacetaldehyde (P-Ald) hydrolase (trivial name, phosphonatase), respectively (35). Phosphonatase has been isolated from several bacterial sources, including Salmonella serovar Typhimurium, and both its three-dimensional structure (26) and mechanism of action are well characterized (5, 13, 22, 26, 27). AEPT was not as well understood. Previous studies of AEPT structure and catalysis have focused on the Pseudomonas aeruginosa enzyme (12, 13). In agreement with the mass predicted by its gene sequence (encoding a 147-residue protein; GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"U61982","term_id":"1470085","term_text":"U61982"}}U61982), the P. aeruginosa AEPT was reported to be a homotetramer of 16.5-kDa subunits (12). The enzyme requires the cofactor pyridoxalphosphate (PLP), has a pH optimum of 8.5 to 9.0, and is specific for pyruvate as the amino group acceptor and AEP as the amino group donor. During catalysis, the pro-S proton of AEP is abstracted (21). Based on the phnW sequence, Salmonella serovar Typhimurium AEPT is predicted to be 367 amino acids in length, about twice the reported size of the P. aeruginosa AEPT. Sequence analysis of the Bacillus cereus AEPT gene (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"AY077635","term_id":"19221219","term_text":"AY077635"}}AY077635) (4) identified a 355-amino-acid protein, similar in size to the Salmonella serovar Typhimurium AEPT. Sequence alignments revealed 40 to 52% sequence identity among different AEPT sequences. The P. aeruginosa AEPT sequence aligns with the C-terminal halves of the Salmonella serovar Typhimurium and B. cereus AEPT sequences, suggesting that they possess an N-terminal domain in addition to the C-terminal catalytic domain that they have in common. In this paper, we describe the isolation and kinetic properties of recombinant Salmonella serovar Typhimurium AEPT and provide evidence that it is a homodimer of 40-kDa subunits. Through sequence comparisons with amino acid transaminase homologues, we identified probable substrate and PLP binding residues. By using site-directed mutagenesis, we confirmed that these residues are important in catalytic functioning. Since two of the residues (D168 and K194) are located within its N-terminal half, the full-length protein is required for catalytic activity. Based on the P. aeruginosa genome sequence (30) (GenBank accession number NP 250001), the AEPT gene encodes a 371-residue protein homologous to the Salmonella serovar Typhimurium AEPT, thus providing a consistent picture of AEPT structure and catalysis. The initial sequence of the P. aeruginosa AEPT has an internal stop codon absent from the genome sequence. As in Salmonella serovar Typhimurium, the P. aeruginosa phnW and phnX genes are juxtaposed.