Alanine racemases are ubiquitous prokaryotic enzymes providing the essential peptidoglycan precursor D-alanine. We present evidence that the enzymes from Pseudomonas aeruginosa and Escherichia coli function exclusively as homodimers. Moreover, we demonstrate that expression of a K35A Y235A double mutation of dadX in E. coli suppresses bacterial growth in a dominant negative fashion.In most bacteria the D-alanine-D-alanyl dipeptide is essential for cross-linking within the peptidoglycan cell wall. It is produced from the natural amino acid L-alanine by the subsequent action of two enzymes, alanine racemases, to create D-alanine and D-alanine-D-alanyl ligase. While some organisms contain only one alanine racemase, others have two isozymes, encoded by the alr and the dadX genes. The latter group includes Escherichia coli (4) and Pseudomonas aeruginosa (12). Little is known about the advantages or disadvantages of having two enzymes or about the interaction between the enzymes. alr expression is constitutive and seems to provide the D-alanine necessary to maintain cell growth, while dadX gene expression is induced by L-alanine to a level much greater than that of alr and thus is most active when L-alanine is used as a carbon and energy source (4).Alanine racemase is an attractive target for the development of new antibiotics; the enzyme is ubiquitous among bacteria and generally absent in higher eukaryotes, although it has recently been found to function as a means of osmoregulation in some lower marine eukaryotes (7, 10).There are still open questions regarding the phylogenetic history of the two alanine racemase genes. For instance, the sequence identity between Alr and DadX enzymes from the same organism is not significantly greater than between enzymes from different bacteria (12). Likewise, the structural differences between alanine racemases deserve further attention. While the alanine racemases from Salmonella enterica serovar Typhimurium (1, 15), Thermus thermophilus (8), and Shigella sp. (19) have been described as monomers, the crystal structure of the alanine racemase from Bacillus stearothermophilus reveals a dimer with two identical polypeptide chains (9). The dimer has two active sites, each of which contains residues from both monomers. It is noteworthy that, especially within these active center domains, alanine racemase protein sequences from different bacteria share a high level of conservation. Pyridoxal-5Ј-phosphate (PLP), the essential cofactor of alanine racemases, is in aldimine linkage to an N-terminal lysine residue as a protonated Schiff's base (6). This residue (K39), together with a tyrosine residue near the C terminus (Y265) from the other subunit in the dimer, has been identified as being critical for the racemization of alanine (16,17). The contribution of these two residues to enzyme activity was further confirmed through cocrystallization of the B. stearothermophilus alanine racemase with one of its inhibitors, 1-aminoethylphosphonate (11). While the lysine residue is clearly the cat...