Crystal structure of the novel amino‐acid racemase isoleucine 2‐epimerase from Lactobacillus buchneri

Crystal structures of Lactobacillus buchneri isoleucine 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 with N ‐(5′‐phosphopyridoxyl)‐ l ‐isoleucine (PLP‐ l ‐Ile) and in complex with N ‐(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 the L. buchneri isoleucine 2‐epimerase monomer showed a notable similarity to those of α‐amino‐ϵ‐caprolactam racemase from Achromobactor obae and γ‐aminobutyrate aminotransferase from Escherichia coli . However, the amino‐acid residues involved in substrate binding in those two enzymes are only partially conserved in L. buchneri isoleucine 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.