Yoshimune Kazuaki, Esaki Nobuyoshi, Moriguchi Mitsuaki
Department of Applied Chemistry, Faculty of Engineering, Oita University, Dannoharu 700, Oita 870-1192, Japan.
Biochem Biophys Res Commun. 2005 Jan 7;326(1):74-8. doi: 10.1016/j.bbrc.2004.11.007.
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.
木糖氧化产碱杆菌木糖氧化亚种A-6（产碱杆菌A-6）的d-氨基酰化酶（A6-d-ANase）过量生产伴随着过量生产蛋白的聚集，并且DnaK-DnaJ-GrpE（DnaKJE）的共表达促进了其可溶性表达。当A6-d-ANase基因在大肠杆菌dnaK突变体dnaK756中表达时，在可溶部分几乎观察不到活性，并且通过DnaKJE的共表达或用赖氨酸替代A6-d-ANase的R354残基可使其恢复。这些结果表明，在没有DnaK的情况下，A6-d-ANase的R354残基的胍基会干扰其正确折叠，而在有DnaK的情况下，DnaK与R354残基的结合会消除这种干扰。这是第一份关于通过定点诱变改变该酶的DnaK依赖性折叠过程的报告。
