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

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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.