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Studies on the structure and function of A-factor receptor based on its X-ray crystallographic structure

基于X射线晶体结构研究A因子受体的结构和功能

基本信息

批准号：
11306008
负责人：
HORINOUCHI Sueharu
金额：
$ 19.52万
依托单位：
THE UNIVERSITY OF TOKYO
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A).
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11306008/
关键词：
A-factor Streptomyces A-factor receptor X-ray chrystallography microbial hormone secondary metabolism morphogenesis

项目摘要

A chemical signaling molecule A-factor at an extremely low concentration controls secondary metabolism and morphological development in Streptomyces griseus. A rapid method for purification of the A-factor receptor protein was established for crystallization of the receptor. The crystals of the receptor and heavy metal-containing receptor show multiforms, which impeded collection of the difraction data. We are currently collecting data on serenomethione-substituted crystals on Spring 8 at Harima.More than 20 DNA fragments bound by a transcriptional factor AdpA, one of the targets of the A-factor recepto. Larger DNA fragments covering the isolated fragments were cloned, and their nucleotide sequences were determined. A-factor-dependent transcription of the genes in the cloned fragments was confirmed by S1 nuclease mapping. One of the A-factor-dependent genes was identified as an extracytoplasmic function σ factor of RNA polymerase. This σ factor concerned only with morphological development and not with secondary metabolic function. Additional results are summarized as follows. i) The autophosphorylation of AfsK, a protein serine/threonine kinase, was regulated by means of protein-protein interaction with an AfsK-binding protein KbpA.ii) The whole gene cluster for yellow pigment production, which is under the control of A-factor, was cloed and its nucleotide sequence aws determined. This gene cluster was transcribed by a specific σ^B-like factor. iii) A melanine production genes, rppA and P-450, were cloned. The enzymatic function of RppA was revealed ; it condensated 5 molecules of malonyl-CoA and circulized to form tetrahydroxynaphthalene. Further studied for elucidation of the molecular mechanism of binding and dissociation of A-factor/receptor on the basis of its 3D structure and for revealing each step in the A-factor regulatory cascade will reveal the overall picture of the hormonal control in Streptomyces.

一个极低浓度的化学信号分子A-因子控制着灰色链霉菌的次生代谢和形态发育。建立了一种快速纯化A因子受体蛋白的方法，用于A因子受体的结晶。受体和含重金属受体的晶体呈现多样性，这阻碍了数据的收集。我们目前正在收集哈里木春季8号的Serenomethione取代晶体的数据。20多个DNA片段与转录因子AdpA结合，这是A因子受体的目标之一。克隆了覆盖分离片段的较大DNA片段，并测定了它们的核苷酸序列。克隆片段中的基因经S1核酸酶定位证实为A因子依赖的转录。其中一个依赖于A因子的基因被鉴定为核糖核酸聚合酶的胞外功能σ因子。这种σ因子只与形态发育有关，与次生代谢功能无关。其他结果总结如下。I)通过与AfsK结合蛋白KbpA的蛋白-蛋白质相互作用来调节蛋白丝氨酸/苏氨酸激酶AfsK的自磷酸化。ii)关闭A因子控制的整个黄色素产生基因簇，并测定其核苷酸序列。该基因簇由一个特定的σ^B类因子转录而成。3)克隆了黑色素产生基因RPPA和P-450。揭示了RPPA的酶功能，它凝聚了5个丙二酰辅酶A分子，并循环形成四氢萘。根据A因子/受体的三维结构，进一步阐明A因子/受体结合和解离的分子机制，揭示A因子调控级联反应的每一步，将揭示链霉菌激素调控的全貌。