Visick J E, Cai H, Clarke S
Department of Chemistry and Biochemistry and the Molecular Biology Institute, University of California, Los Angeles, California 90095-1569, USA.
J Bacteriol. 1998 May;180(10):2623-9. doi: 10.1128/JB.180.10.2623-2629.1998.
Like its homologs throughout the biological world, the L-isoaspartyl protein repair methyltransferase of Escherichia coli, encoded by the pcm gene, can convert abnormal L-isoaspartyl residues in proteins (which form spontaneously from asparaginyl or aspartyl residues) to normal aspartyl residues. Mutations in pcm were reported to greatly reduce survival in stationary phase and when cells were subjected to heat or osmotic stresses (C. Li and S. Clarke, Proc. Natl. Acad. Sci. USA 89:9885-9889, 1992). However, we subsequently demonstrated that those strains had a secondary mutation in rpoS, which encodes a stationary-phase-specific sigma factor (J. E. Visick and S. Clarke, J. Bacteriol. 179:4158-4163, 1997). We now show that the rpoS mutation, resulting in a 90% decrease in HPII catalase activity, can account for the previously observed phenotypes. We further demonstrate that a new pcm mutant lacks these phenotypes. Interestingly, the newly constructed pcm mutant, when maintained in stationary phase for extended periods, is susceptible to environmental stresses, including exposure to methanol, oxygen radical generation by paraquat, high salt concentrations, and repeated heating to 42 degrees C. The pcm mutation also results in a competitive disadvantage in stationary-phase cells. All of these phenotypes can be complemented by a functional pcm gene integrated elsewhere in the chromosome. These data suggest that protein denaturation and isoaspartyl formation may act synergistically to the detriment of aging E. coli and that the repair methyltransferase can play a role in limiting the accumulation of the potentially disruptive isoaspartyl residues in vivo.
与生物界中的同源物一样，由pcm基因编码的大肠杆菌L-异天冬氨酰蛋白修复甲基转移酶可将蛋白质中异常的L-异天冬氨酰残基（由天冬酰胺酰或天冬氨酰残基自发形成）转化为正常的天冬氨酰残基。据报道，pcm突变会大大降低稳定期的存活率，以及细胞在受热或渗透胁迫时的存活率（C. Li和S. Clarke，《美国国家科学院院刊》89:9885 - 9889，1992）。然而，我们随后证明那些菌株在rpoS中有一个二次突变，rpoS编码一种稳定期特异性的σ因子（J. E. Visick和S. Clarke，《细菌学杂志》179:4158 - 4163，1997）。我们现在表明，导致HPII过氧化氢酶活性降低90%的rpoS突变可以解释先前观察到的表型。我们进一步证明一个新的pcm突变体没有这些表型。有趣的是，新构建的pcm突变体在稳定期维持较长时间后，对环境胁迫敏感，包括暴露于甲醇、百草枯产生的氧自由基、高盐浓度以及反复加热至42摄氏度。pcm突变在稳定期细胞中也导致竞争劣势。所有这些表型都可以通过整合在染色体其他位置的功能性pcm基因得到互补。这些数据表明，蛋白质变性和异天冬氨酰形成可能协同作用，对衰老的大肠杆菌产生不利影响，并且修复甲基转移酶可以在限制体内潜在破坏性异天冬氨酰残基的积累中发挥作用。
