Checa S K, Viale A M
Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Argentina.
Eur J Biochem. 1997 Sep 15;248(3):848-55. doi: 10.1111/j.1432-1033.1997.00848.x.
We have studied the in vivo requirements of the DnaK chaperone system for the folding of recombinant ribulose-bisphosphate carboxylase/oxygenase in Escherichia coli. Expression of functional dimeric or hexadecameric ribulose-bisphosphate carboxylase from different bacterial sources (including purple bacteria and cyanobacteria) was severely impaired in E. coli dnaK, dnaJ, or grpE mutants. These enzymes were synthesized mostly in soluble, fully enzymatically active forms in wild-type E. coli cells cultured in the temperature range 20-42 degrees C, but aggregated extensively in dnaK null mutants. Co-expression of dnaK, but not groESL, markedly reduced the aggregation of ribulose-bisphosphate carboxylase subunits in dnaK null mutants and restored the enzyme activity to levels found in isogenic wild-type strains. Ribulose-bisphosphate carboxylase expression in wild-type E. coli cells growing at 30 degrees C promoted an enhanced synthesis of stress proteins, apparently by sequestering DnaK from its negative regulatory role in this response. The overall results indicate that the DnaK chaperone system assists in vivo the folding pathway of ribulose-bisphosphate carboxylase large subunits, most probably at its very early stages.
我们研究了大肠杆菌中DnaK伴侣系统对重组核酮糖-1,5-二磷酸羧化酶/加氧酶折叠的体内需求。来自不同细菌来源（包括紫色细菌和蓝细菌）的功能性二聚体或十六聚体核酮糖-1,5-二磷酸羧化酶在大肠杆菌dnaK、dnaJ或grpE突变体中的表达严重受损。这些酶在20-42摄氏度温度范围内培养的野生型大肠杆菌细胞中大多以可溶性、具有完全酶活性的形式合成，但在dnaK缺失突变体中大量聚集。共表达dnaK而非groESL可显著减少dnaK缺失突变体中核酮糖-1,5-二磷酸羧化酶亚基的聚集，并将酶活性恢复到同基因野生型菌株中的水平。在30摄氏度生长的野生型大肠杆菌细胞中核酮糖-1,5-二磷酸羧化酶的表达促进了应激蛋白的增强合成，显然是通过将DnaK从其在该反应中的负调控作用中分离出来实现的。总体结果表明，DnaK伴侣系统在体内协助核酮糖-1,5-二磷酸羧化酶大亚基的折叠途径极有可能在其非常早期的阶段。
