Cerulemn Resistance in a Cerulenin-Producing Fungus. II. Characterization of Fatty Acid Synthetase from Cephalosporium caerulens1

Abstract

Cerulenin, an antifungal antibiotic isolated from a culture filtrate of Cephalosporium caerulens, is a potent inhibitor of fatty acid synthetase systems of various microorganisms and animal tissues. This antibiotic specifically blocks the activity of β-ketoacyl thioester synthetase (condensing enzyme) by binding to the functional cysteine-SH in the active center of the condensing enzyme domain (the peripheral SH-group). However, fatty acid synthetase from C. caerulens is much less sensitive to cerulenin than fatty acid synthetases from other sources. The properties of C. caerutens synthetase were investigated and compared to those of Saccharomyces cerevisiae synthetase, which is sensitive to the antibiotic. The molecular weight of the enzymically active form of C. caerulens synthetase was 2.53 × 10⁶. The enzyme consisted of two multifunctional proteins, α and β, which are arranged in a complex, α₆β₆. The synthetase was inactivated by iodoacetamide. At 0°C and pH 7.15, the second-order rate constant of k = 15.6 M⁻¹.S⁻¹ was obtained for the inactivation by iodoacetamide. This value was about 15 times greater than that for S. cerevisiae synthetase. Treatment of C. caerulens synthetase with iodoacetamide, while impairing the synthetase activity, induced malonyl-CoA decarboxylase activity. When S. cerevisiae synthetase was preincubated with cerulenin, malonylCoA decarboxylase activity could not be detected even after treatment of the enzyme with iodoacetamide (Kawaguchi, A., Tomoda, H., Nozoe, S., Ömura, S., & Okuda, S. (1982) J. Biochem.92, 7–12). In the case of C. caerulens synthetase, on the other hand, malonyl-CoA decarboxylase activity was induced by lodoacetamide even after the preincubation of the enzyme with cerulenin. The amount of [²H]- cerulenin bound to C. caerulens synthetase was not enough to occupy all the functional cysteine-SH of the condensing enzyme domain. The results reported in this paper suggest that the low reactivity of the functional cysteine-SH with cerulenin plays an important role in the cerulenin resistance of this fungus.