Molecular Cloning and Heterologous Expression of the Gene Encoding Dihydrogeodin Oxidase, a Multicopper Blue Enzyme from Aspergillus terreus

Huang, Kai; Fujii, Isao; Ebizuka, Yutaka; Gomi, Katsuya; Sankawa, Ushio

doi:10.1074/jbc.270.37.21495

Cited by 69 publications

(77 citation statements)

References 41 publications

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“…In this paper we mainly report the isolation and structure determination of the new diphenyl ether (1), and its preliminary in vitro cytotoxicity against the K562 cell line. These kinds of compounds are formed biosynthetically from the anthraquinone emodin, via sulochrin, and grisandienes such as geodin was previously reported [3,4]. …”

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A New Diphenyl Ether from Marine-derived Fungus Aspergillus sp. B-F-2

Li¹,

Zhu

Zhang

et al. 2006

J Antibiot

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A new diphenyl ether dimethyl 2,3Ј-dimethylosoate (1) together with three known compounds monomethylsulochrin (2), emodin (3), and questin (4) were isolated through bioassay-guided fractionations from the culture of a marine-derived fungus Aspergillus sp. B-F-2. The structures of these compounds were determined by spectroscopic methods. Cytotoxicities of compounds 1 and 2 against K562 cell line were preliminarily evaluated by the MTT method and flow cytometry.Keywords Aspergillus sp., diphenyl ether, asterric acid, anthraquinone, cytotoxicity IntroductionIn our research for new anticancer microbial metabolites, a culture broth of a marine-derived fungus Aspergillus sp. B-F-2, exerted significant cytotoxic activity against K562 cell line. Bioassay-guided isolation led to one new asterric acid derivative dimethyl 2,3Ј-dimethoxyosoate (1), and three known fungal metabolites monomethylsulochrin (2), emodin (3), and questin (4) (Fig. 1).Asterric acid, possessing a diphenyl ether structure, was the first non-peptide endothelin (ET) binding inhibitor discovered [1,2]. In this paper we mainly report the isolation and structure determination of the new diphenyl ether (1), and its preliminary in vitro cytotoxicity against the K562 cell line. These kinds of compounds are formed biosynthetically from the anthraquinone emodin, via sulochrin, and grisandienes such as geodin was previously reported [3,4]. Materials and Methods Fungal MaterialThe fungus Aspergillus sp. B-F-2 was isolated from the marine sediments collected in Behai Bay, China. It was preserved in the China Center for Type Culture Collection (patent depositary number: B-F-2 CCTCC M 205148). Working stocks were prepared on Potato Dextrose agar slants stored at 4°C. Fermentation and ExtractionThe fungus was grown under static conditions at 24°C for 25 days in 1000 mlϫ150 conical flasks containing the liquid medium (300 ml/flask) composed of glucose (20 g/liter), peptone (5.0 g/liter), malt extract (3.0 g/liter) and yeast extract (3.0 g/liter) and sea-water after adjusting its pH to 7.0. The fermented whole broth (45 liter

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confidence: 98%

A New Diphenyl Ether from Marine-derived Fungus Aspergillus sp. B-F-2

Li¹,

Zhu

Zhang

et al. 2006

J Antibiot

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“…Other well-known MCOs include ascorbate oxidase (EC 1.10.3.3), cytochrome c oxidase (EC 1.9.3.1), and ceruloplasmin (sometimes referred to as ferroxidase; EC 1.16.3.1). However, in recent years, the MCO family has grown rapidly through the addition of new enzymes, such as phenoxazinone synthase (22), bilirubin oxidase (42), and dihydrogeodin oxidase (27). Most of these new MCOs are of fungal origin, and in these organisms they often catalyze oxidative steps in the biosynthesis of secondary metabolites, including antibiotics of commercial interest.…”

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Oxidation of Phenolate Siderophores by the Multicopper Oxidase Encoded by the Escherichia coli yacK Gene

et al. 2001

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A gene (yacK) encoding a putative multicopper oxidase (MCO) was cloned from Escherichia coli, and the expressed enzyme was demonstrated to exhibit phenoloxidase and ferroxidase activities. The purified protein contained six copper atoms per polypeptide chain and displayed optical and electron paramagnetic resonance (EPR) spectra consistent with the presence of type 1, type 2, and type 3 copper centers. The strong optical A 610 (⌭ 610 ‫؍‬ 10,890 M ؊1 cm ؊1) and copper stoichiometry were taken as evidence that, similar to ceruloplasmin, the enzyme likely contains multiple type 1 copper centers. The addition of copper led to immediate and reversible changes in the optical and EPR spectra of the protein, as well as decreased thermal stability of the enzyme. Copper addition also stimulated both the phenoloxidase and ferroxidase activities of the enzyme, but the other metals tested had no effect. In the presence of added copper, the enzyme displayed significant activity against two of the phenolate siderophores utilized by E. coli for iron uptake, 2,3-dihydroxybenzoate and enterobactin, as well as 3-hydroxyanthranilate, an iron siderophore utilized by Saccharomyces cerevisiae. Oxidation of enterobactin produced a colored precipitate suggestive of the polymerization reactions that characterize microbial melanization processes. As oxidation should render the phenolate siderophores incapable of binding iron, yacK MCO activity could influence levels of free iron in the periplasm in response to copper concentration. This mechanism may explain, in part, how yacK MCO moderates the sensitivity of E. coli to copper.

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“…However, whereas secreted homologous protein yields can reach several grams per liter, production of proteins of mammalian or avian origin usually remains in the range of milligrams per liter (18,34). Fungal metalloproteins, such as laccases, heme peroxidases, and other oxidases, although of fungal origin, are also normally produced in limited amounts (1,5,22,24,36,38,40).…”

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Calnexin Overexpression Increases Manganese Peroxidase Production in Aspergillus niger

Conesa

Jeenes

Archer

et al. 2002

Appl Environ Microbiol

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Heme-containing peroxidases from white rot basidiomycetes, in contrast to most proteins of fungal origin, are poorly produced in industrial filamentous fungal strains. Factors limiting peroxidase production are believed to operate at the posttranslational level. In particular, insufficient availability of the prosthetic group which is required for peroxidase biosynthesis has been proposed to be an important bottleneck. In this work, we analyzed the role of two components of the secretion pathway, the chaperones calnexin and binding protein (BiP), in the production of a fungal peroxidase. Expression of the Phanerochaete chrysosporium manganese peroxidase (MnP) in Aspergillus niger resulted in an increase in the expression level of the clxA and bipA genes. In a heme-supplemented medium, where MnP was shown to be overproduced to higher levels, induction of clxA and bipA was also higher. Overexpression of these two chaperones in an MnP-producing strain was analyzed for its effect on MnP production. Whereas bipA overexpression seriously reduced MnP production, overexpression of calnexin resulted in a four-to fivefold increase in the extracellular MnP levels. However, when additional heme was provided in the culture medium, calnexin overexpression had no synergistic effect on MnP production. The possible function of these two chaperones in MnP maturation and production is discussed.

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Molecular Cloning and Heterologous Expression of the Gene Encoding Dihydrogeodin Oxidase, a Multicopper Blue Enzyme from Aspergillus terreus

Cited by 69 publications

References 41 publications

A New Diphenyl Ether from Marine-derived Fungus Aspergillus sp. B-F-2

A New Diphenyl Ether from Marine-derived Fungus Aspergillus sp. B-F-2

Oxidation of Phenolate Siderophores by the Multicopper Oxidase Encoded by the Escherichia coli yacK Gene

Calnexin Overexpression Increases Manganese Peroxidase Production in Aspergillus niger

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