title: Role of arginine residues of D-aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6.
abstract: To investigate the role of arginine in the folding of d-aminoacylase, seven arginine residues, R26, R152, R296, R302, R354, R377, and R391, among twelve arginine residues highly conserved in d-aminoacylase, N-acyl-d-aspartate amidohydrolase (d-AAase), and N-acyl-d-glutamate amidohydrolase (d-AGase) from Alcaligenes xylosoxydans subsp. xylosoxydans A-6 (Alcaligenes A-6) were substituted with lysine by site-directed mutagenesis. The mutants, R26K, R152K, R296K, and R302K were identified as mutations that increase partitioning of the enzyme into inclusion bodies. No mutants with substitutions within the carboxyterminal segment were found to increase partitioning into inclusion bodies (R354K, R377K, and R392K). These results suggest that arginine residues that position between the N-terminus and central region can play an important role in facilitating folding or stabilizing the structure of d-aminoacylase. By anaerobic cultivation, the production level of R302K in the soluble fraction was improved. Coexpression of the DnaK-DnaJ-GrpE chaperone assisted the folding of R302K, and reduced the effect of the aeration conditions on the solubility of R302K. We hypothesized that R302K requires a larger amount of chaperones for efficient folding than the wild type enzyme.
authors.affiliation: Department of Applied Chemistry, Faculty of Engineering, Oita University, Dannoharu, Oita 870-1192, Japan.
authors.forename: Kazuaki
authors.initials: K
authors.lastname: Yoshimune
authors.forename: Masanori
authors.initials: M
authors.lastname: Kanda
authors.forename: Mamoru
authors.initials: M
authors.lastname: Wakayama
authors.forename: Shun-Ichi
authors.initials: S
authors.lastname: Kanda
authors.forename: Akiko
authors.initials: A
authors.lastname: Sato
authors.forename: Kenji
authors.initials: K
authors.lastname: Sakai
authors.forename: Mitsuaki
authors.initials: M
authors.lastname: Moriguchi
mesh_terms: D000421:Alcaligenes / Q000201:enzymology*; D000581:Amidohydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001120:Arginine / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods; D008239:Lysine / Q000378:metabolism; D018832:Molecular Chaperones / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding; D016415:Sequence Alignment
chemical_list: D001426:Bacterial Proteins; D018832:Molecular Chaperones; D001120:Arginine; D000581:Amidohydrolases; C019672:aminoacylase I; D008239:Lysine