Abstract
RppA is a type III polyketide synthase (PKS) that catalyzes condensation of five molecules of malonyl-CoA to form 1,3,6,8-tetrahydroxynaphthalene (THN). In Streptomyces antibioticus IFO13271 and several other Streptomyces species, an open reading frame, named momA, is present as a neighbor of rppA. MomA belonged to the "cupin" superfamily because it contained a set of two motifs that is responsible for binding one equivalent of metal ions. MomA catalyzed monooxygenation of the THN produced from malonyl-CoA by the action of RppA to form flaviolin. In addition, it used several polyketides as substrates and formed the corresponding quinones. MomA required redox-active transition metal ions (Ni(2+), Cu(2+), Fe(3+), Fe(2+), Mn(2+), and Co(2+)) for its activity, whereas it was inhibited by a redox-inert transition metal ion (Zn(2+)). MomA neither possessed any flavin prosthetic group nor required nicotinamide cofactors for monooxygenation, which shows that MomA as a member of the cupin superfamily is a novel monooxygenase. Consistent with the catalytic property of MomA, WhiE-ORFII showing similarity in amino acid sequence to MomA and containing a cupin domain also catalyzed monooxygenation of THN. whiE-ORFII is located immediately upstream of the "minimal PKS" gene within the whiE type II PKS gene cluster for biosynthesis of a gray spore pigment in Streptomyces coelicolor A3(2), and a number of whiE-ORFII homologues are present in the biosynthetic gene cluster for polyketides of type II in various Streptomyces species. These findings show that a novel class of quinone-forming monooxygenases is involved in modification of aromatic polyketides synthesized by PKSs of types II and III.
Highlights
Aromatic polyketides are widely distributed in bacteria, fungi, and plants, and their structural diversity reflects the variety of pharmacological and veterinary properties (1)
Identification of the rppA-momA Operon in S. antibioticus—We reported previously (10) that rppA, a type III polyketide synthase catalyzing the synthesis of THN from malonyl-CoA, is responsible for melanin biosynthesis in S. griseus
Our preliminary data showed that a gene, named P-450mel, located just upstream of rppA was responsible for the biosynthesis of a dark brown, THN-derived melanin in this strain (Fig. 1A)
Summary
Materials—Emodin, naphthalene, 2-hydroxy-1,4-naphthoquinone, and 1,3-dihydroxynaphthalene (DHN) were purchased from Aldrich. 1-Naphthol was purchased from Sigma. 2-Naphthol, resorcinol, phloroglucinol, 8-quinolinol, tiron (1,2-dihydroxy-3,5-benzene-disulfonic acid), and 1,10-phenanthroline were purchased from Wako. 5-Methylresorcinol was purchased from Tokyo Kasei. Construction of pET16b-MomA— The NdeI-BamHI fragment excised from pUC19-MF3 was cloned between the NdeI and BamHI sites of pET16b (Novagen), resulting in pET16b-MomA. Prosthetic Group Investigation of MomA and WhiE-ORFII—For determination of the prosthetic group and metal ion, the standard reaction mixture contained THN (1 M for MomA and 100 M for WhiEORFII), 500 M each of organic cofactors or 100 M each of metal ions in 100 mM sodium phosphate (pH 7.5), and 0.59 g of MomA or 56 g of WhiE-ORFII in a total volume of 400 l. Reverse phase HPLC conditions were as follows: ODS-80Ts (C18) column (4.6 ϫ 150 mm; Tosoh), maintained at 40 °C, eluted with 25% CH3CN in H2O (each contained 2% acetic acid) with detection at 260 nm; flow rate, 1.0 ml/min.
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