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"Streptomyces avermitilis" mutants defective in methylation of avermectins

M D Schulman, D Valentino, S Streicher

    Antimicrobial Agents and Chemotherapy
    |May 1, 1987
    PubMed

    Insights

    Mutants of Streptomyces avermitilis were studied for avermectin methylation defects. Two distinct enzymes are responsible for methylating the macrolide and oleandrose moieties, with oleandrose methylation occurring first.

    Area of Science:

    • Microbial biochemistry
    • Natural product biosynthesis
    • Enzymology

    Background:

    • Avermectins are potent antiparasitic compounds produced by Streptomyces avermitilis.
    • Post-biosynthetic modifications, such as methylation, are crucial for avermectin activity.
    • Understanding the enzymatic pathways involved in avermectin modification is essential for metabolic engineering and drug discovery.

    Purpose of the Study:

    • To identify and characterize Streptomyces avermitilis mutants with defects in avermectin methylation.
    • To elucidate the specific enzymatic steps and the order of methylation reactions in avermectin biosynthesis.
    • To differentiate the enzymes responsible for methylating the macrolide and oleandrose moieties.

    Main Methods:

    • Isolation and characterization of spontaneous mutants of Streptomyces avermitilis.
    • Analysis of avermectin components produced by mutant strains using chromatographic techniques.
    • Enzyme assays to determine avermectin B2 O-methyltransferase (B2OMT) activity in cell extracts and purified enzymes.
    • In vitro methylation reactions using demethylated avermectin precursors and S-adenosylmethionine.

    Main Results:

    • Four mutants (CR-1 to CR-4) lacked avermectin B2 O-methyltransferase (B2OMT) activity, indicating impaired methylation at the C5 oxygen of the macrolide moiety, producing only avermectin B components.
    • Two mutants (CR-5 and CR-6) showed defects in methylating the oleandrose moiety at C3' and C3'', producing demethylavermectin components.
    • In vitro studies confirmed that distinct enzymes methylate the macrolide (C5 oxygen) and the oleandrose (C3 oxygen), with oleandrose methylation preceding macrolide attachment.

    Conclusions:

    • Two separate enzymatic activities are responsible for the methylation of the macrolide and oleandrose sugar moieties of avermectins.
    • The methylation of the oleandrose moiety occurs prior to its attachment to the macrolide ring structure.
    • These findings provide critical insights into the avermectin biosynthetic pathway, enabling targeted manipulation for novel avermectin derivatives.

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