Crystal structures ofLactobacillus buchneriisoleucine 2-epimerase, a novel branched-chain amino-acid racemase, were determined for the enzyme in the apo form, in complex with pyridoxal 5′-phosphate (PLP), in complex withN-(5′-phosphopyridoxyl)-L-isoleucine (PLP-L-Ile) and in complex withN-(5′-phosphopyridoxyl)-D-allo-isoleucine (PLP-D-allo-Ile) at resolutions of 2.77, 1.94, 2.65 and 2.12 Å, respectively. The enzyme assembled as a tetramer, with each subunit being composed of N-terminal, C-terminal and large PLP-binding domains. The active-site cavity in the apo structure was much more solvent-accessible than that in the PLP-bound structure. This indicates that a marked structural change occurs around the active site upon binding of PLP that provides a solvent-inaccessible environment for the enzymatic reaction. The main-chain coordinates of theL. buchneriisoleucine 2-epimerase monomer showed a notable similarity to those of α-amino-∊-caprolactam racemase fromAchromobactor obaeand γ-aminobutyrate aminotransferase fromEscherichia coli. However, the amino-acid residues involved in substrate binding in those two enzymes are only partially conserved inL. buchneriisoleucine 2-epimerase, which may account for the differences in substrate recognition by the three enzymes. The structures bound with reaction-intermediate analogues (PLP-L-Ile and PLP-D-allo-Ile) and site-directed mutagenesis suggest that L-isoleucine epimerization proceeds through abstraction of the α-hydrogen of the substrate by Lys280, while Asp222 serves as the catalytic residue adding an α-hydrogen to the quinonoid intermediate to form D-allo-isoleucine.