Crystal structure and molecular mechanism underlying broad specificity of racemase RacX from Bacillus subtilis.

Abstract

Bacterial D-amino acids play key roles in as constituents of cell-wall peptidoglycan and peptide antibiotics, and in cell-shape maintenance, stress tolerance, and host-immune interactions. They are mainly produced from L-amino acids by amino acid racemases that invert the α‑carbon of L- or D-amino acids and are classified into well-studied pyridoxal 5'-phosphate (PLP)-dependent and less characterized PLP-independent racemases. Among the latter, glutamate and aspartate racemases are best documented. RacX from Bacillus subtilis is a PLP-independent racemase with broad specificity that preferentially converts basic amino acids (e.g. arginine and lysine) to racemize 15 different amino acids. The molecular basis underlying the broad specificity of RacX is little known. Here, crystal structures of RacX were determined: In the Type -I crystal, two electron-density clusters corresponding to oxamate and imidazole (components of the crystallization solution) were observed in the active site. Structural comparisons of RacX with aspartate racemases (RacDs) indicate that the side chain of Asp33* (belonging to the other subunit of the dimer) facilitates binding of positively charged amino-acid side chains while hindering binding of negatively charged ones. Moreover, unlike RacDs, no basic residues recognizing negatively-charged amino acids were present in the active site of RacX. The broader substrate specificity of RacX relative to RacD was attributed to differences in the flexible lid structure located between β2 and α3, covering the active site. Specifically, while the lid of RacD adopts a helical conformation, the corresponding region in RacX forms a flexible loop, thereby allowing accommodation of a wider range of substrates.

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