PilO of Pseudomonas aeruginosa 1244: subcellular location and domain assignment.
PilO of Pseudomonas aeruginosa 1244: subcellular location and domain assignment.
复制标题
铜绿假单胞菌 1244 的 Pil0：亚细胞定位和结构域分配。
DOI：
10.1111/j.1365-2958.2007.06001.x
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发表时间：
2007
影响因子：
3.6
通讯作者：
Castric,Peter
中科院分区：
文献类型：
--
作者：
Qutyan,Mohammed;Paliotti,Michael;Castric,Peter
铜绿假单胞菌1244的PilO催化O抗原重复单元与菌毛蛋白C末端残基（丝氨酸）的β碳的连接。本研究旨在定位这种酶在催化中的重要区域，并建立菌毛蛋白糖基化反应的细胞定位。虽然PilO在铜绿假单胞菌或大肠杆菌的提取物中检测不到，但即使在过表达的条件下，也发现大量产生了完整的MalE-PilO融合蛋白。 该融合体与含有api 10缺失的aP.aerosa1244突变体互补，并靶向大肠杆菌的胞质膜。 Wzy和WaaL，也利用O抗原重复单元作为底物的酶，被发现与PilO共享序列模式，即使这些蛋白质具有很少的总体序列相似性。其中该共同序列的部分被缺失或通过定点诱变改变的PilO构建体缺乏菌毛蛋白糖基化活性。共同区域下游片段的缺失也阻止了酶的活性。拓扑学研究表明，与酶活性相关的两个PilO区域位于周质中。这些结果建立了这种酶的催化和目前的证据表明，菌毛蛋白糖基化发生在这种生物体的周质空间的重要区域。
PilO ofPseudomonas aeruginosa1244 catalyses the attachment of an O‐antigen repeating unit to the β‐carbon of the pilin C‐terminal residue, a serine. The present study was conducted to locate the regions of this enzyme important in catalysis and to establish the cellular location of the pilin glycosylation reaction. While PilO was not detectable in extracts ofP. aeruginosaorEscherichia coli, even under conditions of overexpression, it was found that an intact MalE–PilO fusion protein was produced in significant amounts. This fusion complemented aP. aeruginosa1244 mutant containing apilOdeletion and targeted to the cytoplasmic membrane ofE. coli. Wzy and WaaL, enzymes that also utilize the O‐antigen repeating unit as substrate, were found to share a sequence pattern with PilO even though these proteins have little overall sequence similarity. PilO constructs in which portions of this common sequence were deleted or altered by site‐directed mutagenesis lacked pilin glycosylating activity. Deletions of segments downstream from the common region also prevented enzyme activity. Topology studies showed that the two PilO regions associated with enzyme activity were located in the periplasm. These results establish regions of this enzyme important for catalysis and present evidence that pilin glycosylation occurs in the periplasmic space of this organism.