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

化学信号分子A因子在极低浓度下控制灰色链霉菌的次级代谢和形态发育。建立了一种快速纯化A因子受体蛋白的方法，用于受体的结晶。受体和含重金属受体的晶体呈现出多种形态，这阻碍了衍射数据的收集。我们目前正在收集春8在播磨serenomethione取代晶体的数据。超过20个DNA片段结合的转录因子AdpA，A因子受体的目标之一。克隆覆盖分离片段的较大DNA片段，并测定其核苷酸序列。通过S1核酸酶作图证实了克隆片段中基因的A因子依赖性转录。其中一个A因子依赖基因被鉴定为RNA聚合酶的胞质外功能σ因子。该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。（1）通过蛋白质间的相互作用，与一个蛋白质丝氨酸/苏氨酸激酶AfsK结合蛋白KbpA相互作用，调节AfsK的自磷酸化。（2）克隆了A因子调控下的黄色素产生基因簇，并测定了其核苷酸序列。该基因簇由一种特异的σ^B样因子转录。iii）克隆了黑色素产生基因rppA和P-450。揭示了RppA的酶促功能，它将5分子丙二酰辅酶A缩合并环化形成四羟基萘。进一步研究阐明结合和解离的A-因子/受体的三维结构的基础上的分子机制，并揭示在A-因子调节级联反应的每一个步骤将揭示在链霉菌激素控制的全貌。

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.