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.

在极低浓度下的化学信号分子A因子控制晶状体链霉菌中的二级代谢和形态发展。建立了一种快速纯化A因子受体蛋白来结晶的方法。受体和含重金属受体的晶体显示多种形式，这阻碍了扩散数据的收集。我们目前正在Harima的春季8号收集有关Serenomethione取代的晶体的数据。多于20个DNA片段，由转录因子ADPA绑定，这是A因子受体的靶标之一。克隆覆盖隔离片段的较大的DNA片段，并确定其核苷酸序列。 S1核酸酶映射证实了克隆片段中基因的A因子依赖性转录。 A因子依赖性基因之一被鉴定为RNA聚合酶的外质函数σ因子。该σ因子仅与形态发展有关，而不涉及继发代谢功能。其他结果总结如下。 i）AFSK的自磷酸化是一种蛋白丝氨酸/苏氨酸激酶，通过与AFSK结合蛋白kbpa.ii的蛋白质蛋白质相互作用来调节。基因簇由特定的σ^b样因子转录。 iii）克隆了一种黑色素生产基因RPPA和P-450。揭示了RPPA的酶促功能；它凝结了5个丙二酰辅酶A分子，并循环形成四羟基苯二甲酸。进一步研究了A因子/受体根据其3D结构的结合和解离的分子机制的进一步研究，并揭示了A因子调节性级联反应中的每个步骤，将揭示链霉菌中马匹控制的整体情况。