Inhibition of the fungal fatty acid synthase type I multienzyme complex

Abstract

Fatty acids are among the major building blocks of living cells, making lipid biosynthesis a potent target for compounds with antibiotic or antineoplastic properties. We present the crystal structure of the 2.6-MDa Saccharomyces cerevisiae fatty acid synthase (FAS) multienzyme in complex with the antibiotic cerulenin, representing, to our knowledge, the first structure of an inhibited fatty acid megasynthase. Cerulenin attacks the FAS ketoacyl synthase (KS) domain, forming a covalent bond to the active site cysteine C1305. The inhibitor binding causes two significant conformational changes of the enzyme. First, phenylalanine F1646, shielding the active site, flips and allows access to the nucleophilic cysteine. Second, methionine M1251, placed in the center of the acyl-binding tunnel, rotates and unlocks the inner part of the fatty acid binding cavity. The importance of the rotational movement of the gatekeeping M1251 side chain is reflected by the cerulenin resistance and the changed product spectrum reported for S. cerevisiae strains mutated in the adjacent glycine G1250. Platensimycin and thiolactomycin are two other potent inhibitors of KSs. However, in contrast to cerulenin, they show selectivity toward the prokaryotic FAS system. Because the flipped F1646 characterizes the catalytic state accessible for platensimycin and thiolactomycin binding, we superimposed structures of inhibited bacterial enzymes onto the S. cerevisiae FAS model. Although almost all side chains involved in inhibitor binding are conserved in the FAS multienzyme, a different conformation of the loop K1413-K1423 of the KS domain might explain the observed low antifungal properties of platensimycin and thiolactomycin.cerulenin ͉ platensimycin ͉ thiolactomycin ͉ fatty acid synthesis ͉ yeast T hree different schemes for de novo synthesis of fatty acids are found in nature. Eukaryotes and advanced prokaryotes generally use the type I fatty acid synthase system (FAS I), composed of complexes of large multifunctional enzymes. Bacteria, in contrast, use the dissociated FAS II system that consists of a set of separate enzymes, each catalyzing one of the reactions of the fatty acid synthase cycle (1). A third system exists in some parasites that use membrane-bound fatty acid elongases for the synthesis of aliphatic chains (2). Despite this considerable variation, the individual reaction steps of fatty acid biosynthesis are essentially conserved in all kingdoms of life. Four basic reactions constitute a single round of elongation. In the first step, an acceptor CoA or acyl carrier protein (ACP) associated acetyl unit is condensed with malonyl-ACP to form ␤-ketobutyryl-ACP, which is subsequently reduced by an NADPH-dependent ketoacyl-ACP reductase. The resulting ␤-hydroxyacyl-ACP is dehydrated to produce enoyl-ACP and finally reduced by an enoyl reductase (ER) to form the saturated acyl-ACP, which can be further elongated in a new cycle [see supporting information (SI) Fig. S1].The importance of the fatty acid biosynthesis pathway makes the FAS sys...

Structure and function of eukaryotic fatty acid synthases

“…Refinement using the 3Á1 Å resolution structure by Leibundgut et al as a starting model resulted in the visualization of the additionally soaked classic FAS inhibitor cerulenin, which covalently binds to the active site of the KS. The remaining electron density for the PPT domain was located at the same position as observed by Lomakin et al (Johansson et al 2008 ;Leibundgut et al 2007 ;Lomakin et al 2007). Very recently, Johansson et al also reported the high-resolution structure of the isolated PPT domain from yeast FAS (Johansson et al 2009), which finally completes the atomic picture of the entire fungal FAS complex, apart from two flexible linkers and few solvent-exposed loops.…”

“…The Steitz lab obtained two crystal forms, a particularly radiation-sensitive monoclinic form (P2 1 , 217r347r263 Å 3 ) and bipyramidal tetragonal crystals in space group P4 3 2 1 2 with unit cell dimensions of a=b=231 Å and c=754 Å , which both diffracted weakly to 4 Å resolution (Lomakin et al 2007). The latter crystal form was also obtained in the Oesterhelt group with identical diffraction limits ( Johansson et al 2008). Leibundgut et al however, obtained tetragonal crystals with related unit cell dimensions of a=b=231 Å and c=784 Å , but now in space group P4 1 2 1 2, with a solvent content of 69 % and half a FAS particle per ASU, which diffracted to about 3 Å resolution.…”

“…The purification of fungal FAS has been carried out through a combination of sucrose cushion and sucrose gradient ultracentrifugation followed by ion exchange chromatography Leibundgut et al 2007 ;Lomakin et al 2007), sometimes combined with fractionated ammonium sulfate precipitation Leibundgut et al 2007). Johansson et al chose a different strategy and used homologous recombination to produce a yeast strain expressing a C-terminally His 8 -tagged FAS b-chain and purified the intact FAS particle through metal chelate affinity chromatography followed by ultracentrifugation ( Johansson et al 2008). Commercial crystallization screens targeted at crystallizing standard-sized proteins commonly use precipitants at concentrations too high for large macromolecular complexes.…”

“…Leibundgut et al however, obtained tetragonal crystals with related unit cell dimensions of a=b=231 Å and c=784 Å , but now in space group P4 1 2 1 2, with a solvent content of 69 % and half a FAS particle per ASU, which diffracted to about 3 Å resolution. To avoid a spatial overlap of reflections, the long c-axis had to be aligned to the spindle axis during data collection from yeast FAS crystals, which was achieved either using bent loops (Dauter, 1999 ;Johansson et al 2008 ;Leibundgut et al 2007) or a kappa goniostat ( Johansson et al 2008).…”

(±) cis-bisamido epoxides: A novel series of potent FXIII-A inhibitors

“…Thus intracellular glutathione is present at millimolar concentrations, but even if it were desirable to selectively inhibit plasma FXIII-A with membrane impermeant reagents, glutathione is present at micromolar concentrations in plasma. However, cerulenin 2 which contains an epoxide moiety as its Cerulenin has previously been shown to inhibit the fatty acid synthase enzyme (FAS) complex, which involves covalent bond formation via attack of an active site cysteine thiol on the epoxide moiety within the natural product with subsequent ring opening [16]. In light of the interesting inhibitory properties displayed by cerulenin in the presence of FXIII-A, we were keen to explore the possible binding mode of this natural product with a view to designing variants that would be amenable to synthetic manipulation in order to enable SAR studies.…”