MlrA Located in the Inner Membrane Is Essential for Initial Degradation of Microcystin in Sphingopyxis sp. C-1
MlrA Located in the Inner Membrane Is Essential for Initial Degradation of Microcystin in Sphingopyxis sp. C-1
复制标题
位于内膜的 MlrA 对于鞘颈藻中微囊藻毒素的初始降解至关重要。
DOI：
10.2521/jswtb.48.99
复制
发表时间：
2012
期刊：
影响因子：
--
通讯作者：
Michihiko Kobayashi
中科院分区：
文献类型：
--
作者：
H. Maseda;K. Shimizu;Y. Doi;Y. Inamori;M. Utsumi;N. Sugiura;Michihiko Kobayashi
利用E.杆菌然而，目前还不清楚这种蛋白质是否仅参与微囊藻毒素降解细菌的初始降解步骤，以及相应的酶位于何处。因此，我们构建了一个基因特异性破坏系统，敲除Sphingopyxis sp. C - 1菌株中的染色体mlrA，我们以前报道的微囊藻毒素降解细菌在其染色体上具有mlr基因簇。破坏mlrA完全阻止了环状微囊藻毒素的降解。这是第一次报告，MlrA是唯一的酶负责降解的初始切割的环状微囊藻毒素在C - 1菌株。此外，我们在MlrA的C -末端连接了一个六聚组氨酸标签，即，MlrA his 6的表达，以确定MlrA在细胞中的定位及其功能。尽管我们制备并研究了C - 1菌株的膜和胞质溶胶组分，但在用抗penta-His抗体进行的Western印迹中，我们仅在膜中检测到MlrA his 6条带。这表明MlrA是一种膜相关蛋白，它降解周质中的微囊藻毒素。因此，我们认为，MlrB和MlrC降解线性微囊藻毒素及其四肽在周质或胞质后，MlrA降解环微囊藻毒素在周质。
MlrA encoded in the mlr gene cluster, mlrA-mlrB-mlrC-mlrD , appears to be responsible for the initial hydrolytic cleavage of the cyclic microcystin structure, based on the results of gene cluster analysis using E. coli . However, it is unknown whether this protein is solely involved in the initial degrading step in microcystin-degrading bacteria and where the corresponding enzyme is located. Thus, we constructed a gene-specific disruption system to knock out chromosomal mlrA in Sphingopyxis sp. C － 1 strain, which we previously reported as a microcystin-degrading bacterium possessing the mlr gene cluster in its chromosome. Disruption of mlrA completely prevented the degradation of cyclic microcystin. This is the first report that MlrA is the sole enzyme responsible for degrading the initial cleavage of cyclic microcystin in the C － 1 strain. Moreover, we attached a hexahistidine tag to the C － terminal end of MlrA, i.e., MlrA his6 , to determine the location of MlrA and its functions in the cell. We detected MlrA his6 band only in the membrane in Western blot with anti-penta-His antibody although we prepared and investigated the membrane and cytosol fractions of the C － 1 strain. This suggests that MlrA is a membrane-associated protein and that it degrades microcystin in the periplasm. Thus, we suggest that MlrB and MlrC degrade linear microcystin and its tetrapeptide in the periplasm or cytosol after MlrA degrades cyclic microcystin in the periplasm.
DOI：
10.1099/ijs.0.63789-0
发表时间：
2006-01-01
影响因子：
2.8
作者：
Maruyama, T;Park, HD;Kato, K
通讯作者：
Kato, K