Substrate analogues were synthesized and examined as inhibitors of diaminopimelic acid (DAP) dehydrogenase from Bacillus sphaericus and of DAP epimerase from Escherichia coli. These enzymes produce meso-DAP (3) (a precursor for L-lysine and for peptidoglycan) from L-tetrahydrodipicolinic acid (1) and LL-DAP (2), respectively. The epimerase was purified by an improved procedure and confirmed to require both carboxyl and both amino groups for substrate recognition using deuterium-exchange experiments with DAP isomers, L-lysine, D-lysine, L-alpha-aminopimelate, and racemic alpha-aminopimelate. An imidazole analogue of DAP, (2S)-2-amino-3-(4-carboxyimidazol-1-yl)propanoic acid (4), was synthesized by condensation of benzyl imidazole-4-carboxylate (8) with N-benzyloxycarbonyl(Cbz)-L-serine beta-lactone (9) (product structure confirmed by X-ray analysis) followed by hydrogenolytic deprotection. Two other analogues, (2S,5’R)-2-amino-3-(3-carboxy-2-isoxazolin-5-yl)propanoic acid (5) and its 5’S diastereomer 6, were prepared by condensation of methyl N-Cbz-L-allylglycinate (13) with methyl chlorooximidoacetate (14) followed by separation of isomers and deprotection with NaOH and Me(3)SiCl/NaI. Similar condensation of ethyl chlorooximidoacetate with ethylene and of 14 with ethyl acrylate generated isoxazolines, which were saponified to 2-isoxazoline-3-carboxylic acid (25) and 2-isoxazoline-3,5-dicarboxylic acid (26), respectively. None of the compounds show significant inhibition of DAP epimerase or DAP dehydrogenase with the exception of 6, which is a potent and specific inhibitor of DAP dehydrogenase. At pH 7.5 or 7.8, compound 6 shows competitive inhibition (K-i = 4.2 mu M) with tetrahydrodipicolinic acid (1) for the forward reaction and noncompetitive inhibition (K-i = 23 mu M) with meso-DAP (3) for the reverse process. Preliminary tests for antimicrobial activity demonstrate that 6 inhibits the growth of B. sphaericus, which relies exclusively on DAP dehydrogenase to produce 3.