PHOSPHORYLATION OF THE AFSR PRODUCT, A GLOBAL REGULATORY PROTEIN FOR SECONDARY-METABOLITE FORMATION IN STREPTOMYCES-COELICOLOR A3(2)
PHOSPHORYLATION OF THE AFSR PRODUCT, A GLOBAL REGULATORY PROTEIN FOR SECONDARY-METABOLITE FORMATION IN STREPTOMYCES-COELICOLOR A3(2)
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DOI：
10.1128/jb.173.7.2311-2318.1991
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发表时间：
1991-04-01
影响因子：
3.2
通讯作者：
HORINOUCHI, S
中科院分区：
文献类型：
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作者：
HONG, SK;KITO, M;HORINOUCHI, S
AfsR蛋白是在野生型水平上合成a因子、放线菌素和绿链霉菌（Streptomyces colicolor A3）(2)和绿链霉菌（lividans）中必需的。由于afsR基因的过表达会对这些菌株产生一定的有害影响，因此将携带afsR全基因的多拷贝质粒引入灰色链霉菌中，从中制备粗细胞裂解液作为蛋白质来源。通过atp -琼脂糖亲和柱层析和抗AfsR抗体检测，从细胞质部分纯化出均匀的AfsR蛋白。经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和凝胶过滤测定，AfsR的分子量为105,300，与从AfsR的核苷酸序列推断的分子量吻合较好。在S. coelicolor A3(2)和S. lividans的细胞提取物存在下，纯化的AfsR蛋白通过ATP的γ -磷酸基团的转移被磷酸化。这种磷酸化过程非常迅速，没有观察到与CTP、GTP、UTP或环AMP的竞争。在金黄色葡萄球菌的细胞提取物中，没有检测到磷酸化AfsR蛋白的活性，这表明这种活性并不普遍存在于链霉菌中，而是特定物种的特异性活性。可以想象，AfsR蛋白的磷酸化程度调节了其调控活性，进而调节了一些参与s . coelicolor A3次生代谢物形成的靶基因的表达(2)。
The AfsR protein is essential for the biosynthesis at the wild-type level of A-factor, actinorhodin, and undecylprodigiosin in Streptomyces coelicolor A3(2) and Streptomyces lividans. Because overexpression of the afsR gene caused some deleterious effect on these strains, a multicopy plasmid carrying the whole afsR gene was introduced into Streptomyces griseus, from which a crude cell lysate was prepared as a protein source. The AfsR protein was purified to homogeneity from the cytoplasmic fraction through several steps of chromatography, including affinity column chromatography with ATP-agarose and use of anti-AfsR antibody for its detection. The molecular weight of AfsR was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by gel filtration to be 105,300, which is in good agreement with that deduced from the nucleotide sequence of afsR. The purified AfsR protein was found to be phosphorylated through the transfer of the gamma-phosphate group of ATP in the presence of the cell extracts of S. coelicolor A3(2) and S. lividans. This phosphorylation proceeded very rapidly, and no competition was observed with CTP, GTP, UTP, or cyclic AMP. In the cell extract of S. griseus, no activity phosphorylating the AfsR protein was detected, suggesting that this activity is not generally present in Streptomyces spp. but is specific to certain species. It is conceivable that the extent of phosphorylation of the AfsR protein modulates its regulatory activity which, in turn, regulates expression of some target gene(s) involved in the secondary-metabolite formation in S. coelicolor A3(2).