Characterization of Structure of Arginine Racemase, the Essential Enzyme for Microorganism to Utilize D-Lysine as a Carbon Source

Abstract

We found that the single intramolecular disulfide bond between the cysteines C47 and C73 exists in the primary structure of arginine racemase (ArgR) from Pseudomonas taetrolens NBRC 3460, and this is the first example of a pyridoxal 5ʼ-phosphate (PLP)-dependent amino acid racemase that contains a disulfide bond. The thermal and pH profiles and the quaternary structure of ArgR did not change when the disulfide bond of ArgR was disrupted by site-directed mutagenesis. The substrate specificity and the overall structure changed when the disulfide bond of ArgR was disrupted by site-directed mutagenesis. The total activity of ArgR decreased when the disulfide bond of ArgR was disrupted by site-directed mutagenesis before the protein was matured or when ArgR was expressed in the cytoplasm. Based on these results, the disulfide bond of ArgR is probably essential for ArgR to fold and mature as an amino acid racemase with a broad substrate specificity.