Detection and function of the intramolecular disulfide bond in arginine racemase: an enzyme with broad substrate specificity.
We found that a 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 phosphate (PLP)-dependent amino acid racemase that contains a disulfide bond. The amino acid racemase activity was still detected, when the disulfide bond of ArgR was disrupted by site-directed mutagenesis or reduced with dithiothreitol (DTT). 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 did not change when the disulfide bond of ArgR was reduced with DTT after the protein was matured. However, these properties changed when the disulfide bond of ArgR was disrupted by site-directed mutagenesis before protein maturation. 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, we can conclude that the disulfide bond of ArgR is essential for ArgR to fold and mature as an amino acid racemase with broad substrate specificity.
Matsui
Daisuke
D
Department of Life Science and Biotechnology, Faculty of Chemistry, Materials, and Bioengineering, Kansai University, 3-3-35 Yamate-Cho, Suita, Osaka-Fu 564-8680, Japan.
Oikawa
Tadao
T
D000591:Amino Acid Isomerases; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism*; D004229:Dithiothreitol / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011549:Pseudomonas / Q000201:enzymology; D019998:Racemases and Epimerases / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D013696:Temperature
D001426:Bacterial Proteins; D004220:Disulfides; D019998:Racemases and Epimerases; D000591:Amino Acid Isomerases; D004229:Dithiothreitol