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首页> 外文期刊>Journal of bacteriology >In Vivo Inactivation of the Mycobacterial Integral Membrane Stearoyl Coenzyme A Desaturase DesA3 by a C-Terminus-Specific Degradation Process
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In Vivo Inactivation of the Mycobacterial Integral Membrane Stearoyl Coenzyme A Desaturase DesA3 by a C-Terminus-Specific Degradation Process

机译：通过C末端特定的降解过程的分枝杆菌整体膜硬脂酰辅酶A去饱和酶DesA3的体内失活。

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DesA3 (Rv3229c) from Mycobacterium tuberculosis is a membrane-bound stearoyl coenzyme A Δ9 desaturase that reacts with the oxidoreductase Rv3230c to produce oleic acid. This work provides evidence for a mechanism used by mycobacteria to regulate this essential enzyme activity. DesA3 expressed as a fusion with either a C-terminal His6 or c-myc tag had consistently higher activity and stability than native DesA3 having the native C-terminal sequence of LAA, which apparently serves as a binding determinant for a mycobacterial protease/degradation system directed at DesA3. Fusion of only the last 12 residues of native DesA3 to the C terminus of green fluorescent protein (GFP) was sufficient to make GFP unstable. Furthermore, the comparable C-terminal sequence from the Mycobacterium smegmatis DesA3 homolog Msmeg_1886 also conferred instability to the GFP fusion. Systematic examination revealed that residues with charged side chains, large nonpolar side chains, or no side chain at the last two positions were most important for stabilizing the construct, while lesser effects were observed at the third-from-last position. Using these rules, a combinational substitution of the last three residues of DesA3 showed that either DKD or LEA gave the best enhancement of stability for the modified GFP in M. smegmatis. Moreover, upon mutagenesis of LAA at the C terminus in native DesA3 to either of these tripeptides, the modified enzyme had enhanced catalytic activity and stability. Since many proteases are conserved within bacterial families, it is reasonable that M. tuberculosis will use a similar C-terminal degradation system to posttranslationally regulate the activity of DesA3 and other proteins. Application of these rules to the M. tuberculosis genome revealed that ～10% the proteins encoded by essential genes may be susceptible to C-terminal proteolysis. Among these, an annotation is known for less than half, underscoring a general lack of understanding of proteins that have only temporal existence in a cell.
机译：结核分枝杆菌的DesA3（Rv3229c）是膜结合的硬脂酰辅酶AΔ 9 去饱和酶，与氧化还原酶Rv3230c反应生成油酸。这项工作为分枝杆菌调节这种基本酶活性的机制提供了证据。表达为与C末端His 6 或c-myc标签融合的DesA3的活性和稳定性始终高于具有LAA天然C末端序列的天然DesA3，后者显然起着结合作用决定于DesA3的分枝杆菌蛋白酶/降解系统的决定因素。仅将天然DesA3的最后12个残基融合到绿色荧光蛋白（GFP）的C末端，足以使GFP不稳定。此外，来自耻垢分枝杆菌DesA3同源物Msmeg_1886的可比较的C末端序列也赋予GFP融合物不稳定。系统检查表明，在最后两个位置带有带电侧链，较大的非极性侧链或无侧链的残基对于稳定构建体最为重要，而在倒数第二个位置观察到的影响较小。使用这些规则，对DesA3的最后三个残基进行组合替换，表明DKD或LEA可使 M中修饰的GFP的稳定性得到最佳增强。包皮垢。此外，在天然DesA3的C末端将LAA诱变为这些三肽中的任何一个时，修饰的酶具有增强的催化活性和稳定性。由于细菌家族中许多蛋白酶是保守的，因此 M是合理的。结核病将使用类似的C末端降解系统来翻译后调节DesA3和其他蛋白质的活性。这些规则应用于 M。结核基因组显示，大约10％的必需基因编码的蛋白质可能对C端蛋白水解敏感。在这些注释中，已知的注释不到一半，这说明人们普遍缺乏对在细胞中仅存在时间的蛋白质的了解。

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