Binding Site of Cerulenin in Fatty Acid Synthetase1

Abstract

An antibiotic cerulenin, (2R, 3S)-2,3-epoxy-4-oxo-7,10-trans,trans- dodecadienamide, irreversibly inhibits fatty acid synthetase from Saccharomyces cerevisiae. Three moles of cerulenin were bound to 1 mol of the enzyme with concomitant loss of its activity. Pretreatment of the enzyme with iodoacetamide reduced the amount of cerulenin bound to the enzyme. Since iodoacetamide is known to specifically bind to the cysteine residue on the condensing reaction domain, cerulenin is considered to bind to the same domain. Tryptic digestion of the [3H] cerulenin-treated enzyme gave a radioactive peptide; its amino acid composition was Asx 1, Thr 1, Ser 1, Glx 2, Pro 1, Gly 1, Ala 1, Val 1, Ile 1, and Leu 2. This composition included all the amino acids of the condensing reaction site (Thr-Pro-Val-Gly-Ala-Cys) previously reported by Kresze et al. (Eur. J. Biochem., 79, 181 [1977] except for Cys. When the enzyme was treated with [3H]cerulenin and digested successively with trypsin and carboxypeptidase P, a [3H] cerulenin-cysteine adduct was isolated as the sole product. This was identified with the adduct chemically synthesized from non-labeled cerulenin and cysteine, and its structure was elucidated by 1H-, 13C-NMR, and fast atom bombardment mass spectrometry. These results indicate that cerulenin, forming a hydroxylactam ring, reacts at its epoxide carbon (C-2 position) with the SH-group of the cysteine residue in the condensing reaction domain of yeast fatty acid synthetase.

Effect of side‐chain structure on inhibition of yeast fatty‐acid synthase by cerulenin analogues

“…FAS was isolated from baker's yeast according to the method described previously [21]. The specific activity of FAS used in these experiments was 343 U/g protein.…”

“…FAS activity was assayed by the procedure described previously [21]. The assay was carried out at 25°C in a mixture containing 0.1 M potassium phosphate (PH 6.5), 2 mM EDTA, 10 mM cysteine, 0.2 mM NADPH, 0.05 mM acetylCoA, 0.08 mM malonyl-CoA, 0.3 mg bovine serum albumin, and 20.7 pg FAS in a total volume of 1 ml.…”

“…ester [20], and product 2a was isolated after enzymic digestion of the cerulenin-FAS adduct [21]. Cerulenin is known to equilibrate in favor of the acyclic structure l a ( Fig.…”

Key role of mitochondria in cerulenin-mediated apoptosis

“…1,2 Previous reports have shown that cerulenin is a cytotoxic agent for several tumor cells and apoptosis has been demonstrated to represent one mechanism of ceruleninmediated cytotoxicity. 3,4 As reported recently, we were able to confirm the apoptosis-inducing effect of cerulenin in various cell lines by Annexin V-binding and PARP cleavage.…”

Fatty acid synthase: a novel target for antiglioma therapy

“…The first identified 'specific' inactivator of FAS, cerulenin, (2R, 3S)-2,3-epoxy-4-oxo-7,10-trans,transdodecadienamide, is a natural antibiotic product of the fungus Cephalosporium ceruleans (Omura, 1976). Cerulenin irreversibly inhibits FAS by binding covalently to the active site cysteine thiol in the b-ketoacyl-synthase domain (Funabashi et al, 1989). Cerulenin is selectively cytotoxic to a number of established human cancer cell lines, including breast (Gabrielson et al, 2001), colon (Pizer et al, 1998a), and prostate (Furuya et al 1997;Pizer et al, 2001).…”