P-66 Mechanistic and Structural Analysis of Bacterial Type III PKSs

DOI

Bibliographic Information

Other Title
  • P-66 バクテリア由来Type III PKSの構造及び機能解析(ポスター発表の部)

Abstract

In bacteria, a structurally simple type III polyketide synthase (PKS) known as 1,3,6,8-tetrahydroxynaphthalene synthase (THNS) catalyzes the iterative condensation of five CoA-linked malonyl units to form a pentaketide intermediate. THNS subsequently catalyzes dual intramolecular Claisen and aldol condensations of this linear intermediate to produce the fused-ring THN skeleton. The type III PKS-catalyzed polyketide extension mechanism, utilizing a conserved Cys-His-Asn catalytic triad in an internal active site cavity, is fairly well understood. However, the mechanistic basis for THNS's unusual production and dual cyclization of its malonyl-primed pentaketide is obscure. Here we present the first bacterial type III PKS crystal structure, that of Streptomyces coelicolor THNS, and identify by mutagenesis, structural modeling, and chemical analysis the unexpected catalytic participation of an additional THNS-conserved cysteine residue in polyketide extension beyond the tirketide stage. Our crystal structure also reveals an unanticipated novel cavity extending into the 'floor' of the traditional active site cavity, providing the first plausible structural and mechanistic explanation for yet another unusual THNS catalytic activity: its previously inexplicable extra polyketide extension step when primed with a long acyl starter. In our observation, this tunnel structure contributes nothing to form THN or other products from several acyl starters. However this tunnel allows for selective expansion of available active site cavity volume by sequestration of aliphatic starter-derived polyketide tails, and further suggests another distinct protection mechanism involving maintenance of a linear polyketide conformation. Cytochrome P450 158A2 (CYP158A2) is encoded within a three-gene operon in the prototypic soil bacterium S. coelicolor A3(2). This operon is widely conserved among streptomycetes. CYP158A2 has been suggested to produce polymers of flaviolin, a pigment that many protect microbes from UV radiation, in combination with THNS. Following cloning, expression, and purification of this cytochrome P450, we have shown that it can produce dimer and trimer products from the substrate flaviolin and that the structures of two of the dimeric products were established using mass spectrometry and multiple NMR methods. A comparison of the x-ray structure of ligand-free (1.75A) and flaviolin-bound (1.62A) forms of CYP158A2 demonstrates a major conformational change upon ligand binding that closes the entry into the active site, partly due to repositioning of the F and G helices. Particularly interesting is the presence of two molecules of flaviolin in the closed active site. The flaviolin molecules form a quasi-planar three-molecule stack including the heme of CYP158A2, suggesting that oxidative C-C coupling of these phenolic molecules leads to the production of flaviolin dimers.

Journal

Details 詳細情報について

  • CRID
    1390282681054237824
  • NII Article ID
    110006682619
  • DOI
    10.24496/tennenyuki.47.0_635
  • ISSN
    24331856
  • Text Lang
    ja
  • Data Source
    • JaLC
    • CiNii Articles
  • Abstract License Flag
    Disallowed

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