Structural insights into bacterial resistance to cerulenin Felipe Trajtenberg 1, *, Silvia Altabe 2, *, Nicole Larrieux 1 , Florencia Ficarra 2 , Diego de Mendoza 2 , Alejandro Buschiazzo 1,3 and Gustavo E. Schujman 2 1 Institut Pasteur de Montevideo, Unit of Protein Crystallography, Montevideo, Uruguay 2 Instituto de Biolog  ıa Molecular y Celular de Rosario (IBR) – CONICET, Facultad de Cs Bioqu  ımicas y Farmac euticas, Universidad Nacional de Rosario, Argentina 3 D epartement de Biologie Structurale et Chimie, Institut Pasteur, Paris, France Keywords antibiotic resistance; bacteria; enzyme inhibitors; fatty acids; thiolase superfamily Correspondence G. E. Schujman, Instituto de Biolog  ıa Molecular y Celular de Rosario (IBR) – CONICET, Facultad de Cs Bioqu  ımicas y Farmac euticas, Universidad Nacional de Rosario, Ocampo y Esmeralda, Predio CONICET Rosario, Rosario (S2000EZO), Argentina Fax: +54 341 4237070 ext 607 Tel: +54 341 4237070 E-mail: schujman@ibr-conicet.gov.ar *These authors contributed equally to this work. (Received 17 January 2014, revised 10 March 2014, accepted 13 March 2014) doi:10.1111/febs.12785 Cerulenin is a fungal toxin that inhibits both eukaryotic and prokaryotic ketoacyl-acyl carrier protein synthases or condensing enzymes. It has been used experimentally to treat cancer and obesity, and is a potent inhibitor of bacterial growth. Understanding the molecular mechanisms of resistance to cerulenin and similar compounds is thus highly relevant for human health. We have previously described a Bacillus subtilis cerulenin-resistant strain, expressing a point-mutated condensing enzyme FabF (FabF[I108F]) (i.e. FabF with isoleucine 108 substituted by phenylalanine). We now report the crystal structures of wild-type FabF from B. subtilis, both alone and in complex with cerulenin, as well as of the FabF[I108F] mutant pro- tein. The three-dimensional structure of FabF[I108F] constitutes the first atomic model of a condensing enzyme that remains active in the presence of the inhibitor. Soaking the mycotoxin into preformed wild-type FabF crystals allowed for noncovalent binding into its specific pocket within the FabF core. Interestingly, only co-crystallization experiments allowed us to trap the covalent complex. Our structure shows that the covalent bond between Cys163 and cerulenin, in contrast to that previously proposed, implicates carbon C3 of the inhibitor. The similarities between Escherichi- a coli and B. subtilis FabF structures did not explain the reported inability of ecFabF[I108F] (i.e. FabF from Escherichia coli with isoleucine 108 substituted by phenylalanine) to elongate medium and long-chain acyl- ACPs. We now demonstrate that the E. coli modified enzyme efficiently catalyzes the synthesis of medium and long-chain ketoacyl-ACPs. We also characterized another cerulenin-insensitive form of FabF, conferring a dif- ferent phenotype in B. subtilis. The structural, biochemical and physiologi- cal data presented, shed light on the mechanisms of FabF catalysis and resistance to cerulenin. Database Crystallographic data (including atomic coordinates and structure factors) have been deposited in the Protein Data Bank under accession codes 4LS5, 4LS6, 4LS7 and 4LS8. Abbreviations 3D, three-dimensional; AAS, acyl ACP synthase; ACP, acyl carrier protein; bsFabF, FabF from Bacillussubtilis; ecFabF, FabF from Escherichiacoli; Fab[I108M], FabF with isoleucine 108 substituted by methionine; FabF[I108F], FabF with isoleucine 108 substituted by phenylalanine; FASI, type I fatty-acid synthase; FASII, type II or dissociated fatty-acid synthase; IPTG, isopropyl thio-b-D-galactoside; MIC, minimum inhibitory concentration; PDB, Protein Data Bank; TLS, translation libration screw; UFA, unsaturated fatty acid; wtFabF, wild-type FabF. 2324 FEBS Journal 281 (2014) 2324–2338 ª 2014 FEBS