Site-directed mutagenesis alters DnaK-dependent folding process.
The overproduction of d-aminoacylase (A6-d-ANase) of Alcaligenes xylosoxydans subsp. xylosoxydans A-6 (Alcaligenes A-6) is accompanied by aggregation of the overproduced protein, and its soluble expression is facilitated by the coexpression of DnaK-DnaJ-GrpE (DnaKJE). When the A6-d-ANase gene was expressed in the Escherichia coli dnaK mutant dnaK756, little activity was observed in the soluble fraction, and it was restored by the coexpression of DnaKJE or the substitution of the R354 residue of A6-d-ANase for lysine. These results suggest that the guanidino group of the R354 residue of A6-d-ANase disturbs its proper folding in the absence of DnaK and the disturbance is eliminated by binding of DnaK to the R354 residue in the presence of DnaK. This is the first report that the DnaK-dependent folding process of the enzyme is altered by site-directed mutagenesis.
Yoshimune
Kazuaki
K
Department of Applied Chemistry, Faculty of Engineering, Oita University, Dannoharu 700, Oita 870-1192, Japan.
Esaki
Nobuyoshi
N
Moriguchi
Mitsuaki
M
D000421:Alcaligenes / Q000235:genetics / Q000378:metabolism*; D000581:Amidohydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D017124:Conserved Sequence; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017510:Protein Folding; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D000581:Amidohydrolases; C019672:aminoacylase I; C063842:dnaK protein, E coli