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Site-directed mutagenesis alters DnaK-dependent folding process.

Kazuaki Yoshimune·Nobuyoshi Esaki·Mitsuaki Moriguchi
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Abstract

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.

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AlcaligenesAmidohydrolasesAmino Acid SequenceAmino Acid SubstitutionBinding SitesConserved SequenceEnzyme ActivationEscherichia coliEscherichia coli ProteinsHSP70 Heat-Shock ProteinsKineticsMolecular Sequence DataMutagenesis, Site-DirectedProtein BindingProtein FoldingSequence Analysis, ProteinSequence Homology, Amino AcidStructure-Activity RelationshipSubstrate SpecificityJournal ArticleResearch Support, Non-U.S. Gov't

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Kazuaki Yoshimune, Nobuyoshi Esaki, Mitsuaki Moriguchi. 2005-01-07. Site-directed mutagenesis alters DnaK-dependent folding process.. https://doi.org/10.1016/j.bbrc.2004.11.007

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