In recent years, dihydrodipicolinate synthase (DHDPS, E.C. 220.127.116.11) has received considerable attention from a mechanistic and structural viewpoint. DHDPS catalyzes the reaction of (S)-aspartate-β-semialdehyde with pyruvate, which is bound via a Schiff base to a conserved active-site lysine (Lys161 in the enzyme from Escherichia coli). To probe the mechanism of DHDPS, we have studied the inhibition of E. coli DHDPS by the substrate analog, β-hydroxypyruvate. The Ki was determined to be 0.21 (±0.02) mM, similar to that of the allosteric inhibitor, (S)-lysine, and β-hydroxypyruvate was observed to cause time-dependent inhibition. The inhibitory reaction with β-hydroxypyruvate could be qualitatively followed by mass spectrometry, which showed initial noncovalent adduct formation, followed by the slow formation of the covalent adduct. It is unclear whether β-hydroxypyruvate plays a role in regulating the biosynthesis of meso-diaminopimelate and (S)-lysine in E. coli, although we note that it is present in vivo. The crystal structure of DHDPS complexed with β-hydroxypyruvate was solved. The active site clearly showed the presence of the inhibitor covalently bound to the Lys161. Interestingly, the hydroxyl group of β-hydroxypyruvate was hydrogen-bonded to the main-chain carbonyl of Ile203. This provides insight into the possible catalytic role played by this peptide unit, which has a highly strained torsion angle (ω ˜201°). A survey of the known DHDPS structures from other organisms shows this distortion to be a highly conserved feature of the DHDPS active site, and we propose that this peptide unit plays a critical role in catalysis.