Cerulenin, an antifungal antibiotic produced by
Cephalosporium caerulens, is a potent inhibitor of fatty acid synthase in various organisms, including
Saccharomyces cerevisiae. The antibiotic inhibits the enzyme by binding covalently to the active center cysteine of the condensing enzyme domain. We isolated 12 cerulenin-resistant mutants of
S. cerevisiae following treatment with ethyl methanesulfonate. The mechanism of cerulenin resistance in one of the mutants, KNCR-1, was studied. Growth of the mutant was over 20 times more resistant to cerulenin than that of the wild-type strain. Tetrad analysis suggested that all mutants mapped at the same locus,
FAS2, the gene encoding the subunit of the fatty acid synthase. The isolated fatty acid synthase, purified from the mutant KNCR-1, was highly resistant to cerulenin. The cerulenin concentration causing 50% inhibition (IC
50) of the enzyme activity was measured to be 400 M, whereas the IC
50 value was 15 M for the enzyme isolated from the wild-type strain, indicating a 30-fold increase in resistance to cerulenin. The
FAS2 gene was cloned from the mutant. Sequence replacement experiments suggested that an 0.8 kb
EcoRV-
HindIII fragment closely correlated with cerulenin resistance. Sequence analysis of this region revealed that the GGT codon encoding Gly-1257 of the
FAS2 gene was altered to AGT in the mutant, resulting in the codon for Ser. Furthermore, a recombinant
FAS2 gene, in which the 0.8 Kb
EcoRV-
HindIII fragment of the wild-type
FAS2 gene was replaced with the same region from the mutant, when introduced into
FAS2-defective
S. cerevisiae complemented the
FAS2 pheno-type and showed cerulenin resistance. These data indicate that one amino acid substitution (Gly Ser) in the subunit of fatty acid synthase is responsible for the cerulenin resistance of the mutant KNCR-1.
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