In vivo inactivation of the mycobacterial integral membrane stearoyl coenzyme a desaturase DesA3 by a C-terminus-specific degradation process
In vivo inactivation of the mycobacterial integral membrane stearoyl coenzyme a desaturase DesA3 by a C-terminus-specific degradation process
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DOI：
10.1128/jb.00585-08
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发表时间：
2008-10-01
影响因子：
3.2
通讯作者：
Fox, Brian G.
中科院分区：
文献类型：
--
作者：
Chang, Yong;Wesenberg, Gary E.;Fox, Brian G.
来自结核分枝杆菌的 DesA3 (Rv3229c) 是一种膜结合硬脂酰辅酶 A Delta(9) 去饱和酶，可与氧化还原酶 Rv3230c 反应产生油酸。这项工作为分枝杆菌调节这种必需酶活性的机制提供了证据。表达为与 C 端 His(6) 或 c-myc 标签融合的 DesA3 始终比具有 LAA 天然 C 端序列的天然 DesA3 具有更高的活性和稳定性，LAA 显然是针对 DesA3 的分枝杆菌蛋白酶/降解系统的结合决定簇。仅天然 DesA3 的最后 12 个残基与绿色荧光蛋白 (GFP) C 末端的融合就足以使 GFP 不稳定。此外，来自耻垢分枝杆菌 DesA3 同源物 Msmeg_1886 的类似 C 端序列也赋予 GFP 融合稳定性。系统检查表明，最后两个位置带有带电侧链、大的非极性侧链或无侧链的残基对于稳定构建体最重要，而在倒数第三个位置观察到的影响较小。使用这些规则，DesA3 最后三个残基的组合替换表明，DKD 或 LEA 为耻垢分枝杆菌中修饰的 GFP 提供了最佳的稳定性增强。此外，将天然 DesA3 中 C 末端的 LAA 诱变为这些三肽中的任一种后，修饰酶具有增强的催化活性和稳定性。由于许多蛋白酶在细菌家族中是保守的，因此结核分枝杆菌使用类似的 C 末端降解系统来翻译后调节 DesA3 和其他蛋白质的活性是合理的。将这些规则应用于结核分枝杆菌基因组表明，大约 10% 的必需基因编码的蛋白质可能对 C 末端蛋白水解敏感。其中，已知注释的不到一半，这凸显了人们普遍缺乏对仅在细胞中暂时存在的蛋白质的理解。
DesA3 (Rv3229c) from Mycobacterium tuberculosis is a membrane-bound stearoyl coenzyme A Delta(9) 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 His(6) 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 similar to 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.