RESUMO
Heparan sulfate (HS) is a polysaccharide fundamentally important for biologically activities. T/Tn antigens are universal carbohydrate cancer markers. Here, we report the specific imaging of these carbohydrates using a mesenchymal stem cell line and human umbilical vein endothelial cells (HUVEC). The staining specificities were demonstrated by comparing imaging of different glycans and validated by either removal of target glycans, which results in loss of signal, or installation of target glycans, which results in gain of signal. As controls, representative key glycans including O-GlcNAc, lactosaminyl glycans and hyaluronan were also imaged. HS staining revealed novel architectural features of the extracellular matrix (ECM) of HUVEC cells. Results from T/Tn antigen staining suggest that O-GalNAcylation is a rate-limiting step for O-glycan synthesis. Overall, these highly specific approaches for HS and T/Tn antigen imaging should greatly facilitate the detection and functional characterization of these biologically important glycans.
Assuntos
Glicosiltransferases/metabolismo , Heparitina Sulfato/metabolismo , Animais , Antígenos/metabolismo , Linhagem Celular , Química Click , Matriz Extracelular/metabolismo , Heparitina Sulfato/química , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Ácido Hialurônico/metabolismo , Células-Tronco Mesenquimais/metabolismo , Camundongos , Processamento de Proteína Pós-TraducionalRESUMO
Natural product chemical diversity is fuelled by the emergence and ongoing evolution of biosynthetic pathways in secondary metabolism. However, co-evolution of enzymes for metabolic diversification is not well understood, especially at the biochemical level. Here, two parallel assemblies with an extraordinarily high sequence identity from Lyngbya majuscula form a beta-branched cyclopropane in the curacin A pathway (Cur), and a vinyl chloride group in the jamaicamide pathway (Jam). The components include a halogenase, a 3-hydroxy-3-methylglutaryl enzyme cassette for polyketide beta-branching, and an enoyl reductase domain. The halogenase from CurA, and the dehydratases (ECH(1)s), decarboxylases (ECH(2)s) and enoyl reductase domains from both Cur and Jam, were assessed biochemically to determine the mechanisms of cyclopropane and vinyl chloride formation. Unexpectedly, the polyketide beta-branching pathway was modified by introduction of a gamma-chlorination step on (S)-3-hydroxy-3-methylglutaryl mediated by Cur halogenase, a non-haem Fe(ii), alpha-ketoglutarate-dependent enzyme. In a divergent scheme, Cur ECH(2) was found to catalyse formation of the alpha,beta enoyl thioester, whereas Jam ECH(2) formed a vinyl chloride moiety by selectively generating the corresponding beta,gamma enoyl thioester of the 3-methyl-4-chloroglutaconyl decarboxylation product. Finally, the enoyl reductase domain of CurF specifically catalysed an unprecedented cyclopropanation on the chlorinated product of Cur ECH(2) instead of the canonical alpha,beta C = C saturation reaction. Thus, the combination of chlorination and polyketide beta-branching, coupled with mechanistic diversification of ECH(2) and enoyl reductase, leads to the formation of cyclopropane and vinyl chloride moieties. These results reveal a parallel interplay of evolutionary events in multienzyme systems leading to functional group diversity in secondary metabolites.
Assuntos
Cianobactérias/enzimologia , Ciclopropanos/metabolismo , Enzimas/biossíntese , Enzimas/química , Enzimas/metabolismo , Evolução Molecular , Halogenação , Tiazóis/metabolismo , Cloreto de Vinil/metabolismoRESUMO
The secreted anthrax toxin consists of three components: the protective antigen (PA), edema factor (EF) and lethal factor (LF). LF, a zinc metalloproteinase, compromises the host immune system primarily by targeting mitogen-activated protein kinase kinases in macrophages. Peptide substrates and small-molecule inhibitors bind LF in the space between domains 3 and 4 of the hydrolase. Domain 3 is attached on a hinge to domain 2 via residues Ile300 and Pro385, and can move through an angular arc of greater than 35° in response to the binding of different ligands. Here, multiple LF structures including five new complexes with co-crystallized inhibitors are compared and three frequently populated LF conformational states termed `bioactive', `open' and `tight' are identified. The bioactive position is observed with large substrate peptides and leaves all peptide-recognition subsites open and accessible. The tight state is seen in unliganded and small-molecule complex structures. In this state, domain 3 is clamped over certain substrate subsites, blocking access. The open position appears to be an intermediate state between these extremes and is observed owing to steric constraints imposed by specific bound ligands. The tight conformation may be the lowest-energy conformation among the reported structures, as it is the position observed with no bound ligand, while the open and bioactive conformations are likely to be ligand-induced.
Assuntos
Antígenos de Bactérias/química , Bacillus anthracis/química , Toxinas Bacterianas/química , Metaloendopeptidases/química , Antraz/microbiologia , Antígenos de Bactérias/metabolismo , Bacillus anthracis/metabolismo , Toxinas Bacterianas/antagonistas & inibidores , Toxinas Bacterianas/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Desenho de Fármacos , Humanos , Ligantes , Metaloendopeptidases/metabolismo , Modelos Moleculares , Peptídeos , Conformação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína/efeitos dos fármacos , Sulfonamidas/química , Sulfonamidas/farmacologiaRESUMO
7,8-Diaminopelargonic acid synthase (BioA) of Mycobacterium tuberculosis is a recently validated target for therapeutic intervention in the treatment of tuberculosis (TB). Using biophysical fragment screening and structural characterization of compounds, we have identified a potent aryl hydrazine inhibitor of BioA that reversibly modifies the pyridoxal-5'-phosphate (PLP) cofactor, forming a stable quinonoid. Analogous hydrazides also form covalent adducts that can be observed crystallographically but are incapable of inactivating the enzyme. In the X-ray crystal structures, small molecules induce unexpected conformational remodeling in the substrate binding site. We compared these conformational changes to those induced upon binding of the substrate (7-keto-8-aminopelargonic acid), and characterized the inhibition kinetics and the X-ray crystal structures of BioA with the hydrazine compound and analogues to unveil the mechanism of this reversible covalent modification.
Assuntos
Proteínas de Bactérias/metabolismo , Hidrazinas/metabolismo , Mycobacterium tuberculosis/enzimologia , Transaminases/metabolismo , Aminoácidos/química , Aminoácidos/metabolismo , Proteínas de Bactérias/antagonistas & inibidores , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Hidrazinas/química , Cinética , Simulação de Acoplamento Molecular , Transaminases/antagonistas & inibidoresRESUMO
Germicidin synthase (Gcs) from Streptomyces coelicolor is a type III polyketide synthase (PKS) with broad substrate flexibility for acyl groups linked through a thioester bond to either coenzyme A (CoA) or acyl carrier protein (ACP). Germicidin synthesis was reconstituted in vitro by coupling Gcs with fatty acid biosynthesis. Since Gcs has broad substrate flexibility, we directly compared the kinetic properties of Gcs with both acyl-ACP and acyl-CoA. The catalytic efficiency of Gcs for acyl-ACP was 10-fold higher than for acyl-CoA, suggesting a strong preference toward carrier protein starter unit transfer. The 2.9 Å germicidin synthase crystal structure revealed canonical type III PKS architecture along with an unusual helical bundle of unknown function that appears to extend the dimerization interface. A pair of arginine residues adjacent to the active site affect catalytic activity but not ACP binding. This investigation provides new and surprising information about the interactions between type III PKSs and ACPs that will facilitate the construction of engineered systems for production of novel polyketides.
Assuntos
Proteína de Transporte de Acila/metabolismo , Proteínas de Bactérias/metabolismo , Policetídeo Sintases/metabolismo , Pironas/metabolismo , Streptomyces coelicolor/enzimologia , Acil Coenzima A/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Cristalografia por Raios X , Modelos Moleculares , Mutação , Policetídeo Sintases/química , Policetídeo Sintases/genética , Ligação Proteica , Transdução de Sinais , Streptomyces coelicolor/química , Streptomyces coelicolor/genética , Especificidade por SubstratoRESUMO
Differential scanning fluorimetry (DSF) is a practical and accessible technique that allows the assessment of multiphasic unfolding behavior resulting from subsaturating binding of ligands. Multiphasic unfolding is indicative of a heterogenous protein solution, which frequently interferes with crystallization and complicates functional characterization of proteins of interest. Along with UV-Vis spectroscopy, DSF was used to guide purification and crystallization improvements for the pyridoxal 5'-phosphate (PLP) dependent transaminase BioA from Mycobacterium tuberculosis. The incompatibility of the primary amine-containing buffer 2-amino-2-(hydroxymethyl)-1,3-propanediol (Tris) and PLP was identified as a significant contributor to heterogeneity. It is likely that the utility of DSF for ligand-binding assessment is not limited to the cofactor PLP but will be applicable to a variety of ligand-dependent enzymes.
Assuntos
Proteínas de Bactérias/isolamento & purificação , Fluorometria/métodos , Fosfato de Piridoxal/metabolismo , Transaminases/isolamento & purificação , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Cristalização , Mycobacterium tuberculosis/enzimologia , Fosfato de Piridoxal/química , Transaminases/química , Transaminases/metabolismoRESUMO
BioA catalyzes the second step of biotin biosynthesis, and this enzyme represents a potential target to develop new antitubercular agents. Herein we report the design, synthesis, and biochemical characterization of a mechanism-based inhibitor (1) featuring a 3,6-dihydropyrid-2-one heterocycle that covalently modifies the pyridoxal 5'-phosphate (PLP) cofactor of BioA through aromatization. The structure of the PLP adduct was confirmed by MS/MS and X-ray crystallography at 1.94 Å resolution. Inactivation of BioA by 1 was time- and concentration-dependent and protected by substrate. We used a conditional knock-down mutant of M. tuberculosis to demonstrate the antitubercular activity of 1 correlated with BioA expression, and these results provide support for the designed mechanism of action.
Assuntos
Proteínas de Bactérias/metabolismo , Biotina/biossíntese , Mycobacterium tuberculosis/enzimologia , Transaminases/metabolismo , Antituberculosos/síntese química , Antituberculosos/química , Antituberculosos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Biocatálise , Testes de Sensibilidade Microbiana , Modelos Moleculares , Estrutura Molecular , Mycobacterium tuberculosis/efeitos dos fármacos , Piridonas/síntese química , Piridonas/química , Piridonas/farmacologia , Estereoisomerismo , Relação Estrutura-Atividade , Transaminases/antagonistas & inibidores , Transaminases/químicaRESUMO
The aggregation behavior of two model proteins- i) bovine serum albumin (BSA) and ii) ß-galactosidase (ß-gal), was investigated by micro-flow imaging (MFI) during freeze-thaw cycling in phosphate buffered solutions. The pH shift was measured upon cooling the solutions from 20 to -25 °C. When the buffer concentration was 100 mM, cooling caused a pH decrease of 3.1 and 2.7 units (for BSA and ß-gal, respectively) attributed to selective crystallization of disodium hydrogen phosphate as a dodecahydrate. The crystallizing solute phase was characterized by low temperature powder X-ray diffractometry. The pH shift resulted in protein aggregation, evident from the pronounced increase in particle count (by MFI). The addition of cellobiose attenuated the pH shift on cooling (pH decrease of ~1.0 unit), and no evidence of either buffer salt crystallization or protein aggregation was observed. Decreasing the buffer concentration to 10 mM, also prevented protein aggregation. The protein, by inhibiting buffer crystallization, prevented the pH shift and then the buffer, by maintaining the pH, enhanced protein stability.
Assuntos
Agregados Proteicos , Soluções Tampão , Cristalização , Congelamento , Concentração de Íons de HidrogênioRESUMO
The plus-strand RNA genome of Sindbis virus (SINV) encodes four nonstructural proteins (nsP1 to nsP4) that are involved in the replication of the viral RNA. The approximately 800-amino-acid nsP2 consists of an N-terminal domain with nucleoside triphosphatase and helicase activities and a C-terminal protease domain. Recently, the structure determined for Venezuelan equine encephalitis virus nsP2 indicated the presence of a previously unrecognized methyltransferase (MTase)-like domain within the C-terminal approximately 200 residues and raised a question about its functional importance. To assess the role of this MTase-like region in viral replication, highly conserved arginine and lysine residues were mutated to alanine. The plaque phenotypes of these mutants ranged from large/wild-type to small plaques with selected mutations demonstrating temperature sensitive lethality. The proteolytic polyprotein processing activity of nsP2 was unaffected in most of the mutants. Some of the temperature-sensitive mutants showed reduction in the minus-strand RNA synthesis, a function that has not yet been ascribed to nsP2. Mutation of SINV residue R615 rendered the virus noncytopathic and incapable of inhibiting the host cell translation but with no effects on the transcriptional inhibition. This property differentiated the mutation at R615 from previously described noncytopathic mutations. These results implicate nsP2 in regulation of minus-strand synthesis and suggest that different regions of the nsP2 MTase-like domain differentially modulate host defense mechanisms, independent of its role as the viral protease.
Assuntos
Cisteína Endopeptidases/metabolismo , Metiltransferases/metabolismo , RNA Viral/biossíntese , Sindbis virus/enzimologia , Sindbis virus/fisiologia , Replicação Viral , Substituição de Aminoácidos/genética , Cisteína Endopeptidases/genética , Efeito Citopatogênico Viral , Genes Essenciais , Temperatura Alta , Metiltransferases/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Poliproteínas/metabolismo , Sindbis virus/genética , Ensaio de Placa Viral , Proteínas Virais/metabolismoRESUMO
Anthrax is an infectious disease caused by Bacillus anthracis, a Gram-positive, rod-shaped, anaerobic bacterium. The lethal factor (LF) enzyme is secreted by B. anthracis as part of a tripartite exotoxin and is chiefly responsible for anthrax-related cytotoxicity. As LF can remain in the system long after antibiotics have eradicated B. anthracis from the body, the preferred therapeutic modality would be the administration of antibiotics together with an effective LF inhibitor. Although LF has garnered a great deal of attention as an attractive target for rational drug design, relatively few published inhibitors have demonstrated activity in cell-based assays and, to date, no LF inhibitor is available as a therapeutic or preventive agent. Here we present a novel in silico high-throughput virtual screening protocol that successfully identified 5 non-hydroxamic acid small molecules as new, preliminary LF inhibitor scaffolds with low micromolar inhibition against that target, resulting in a 12.8% experimental hit rate. This protocol screened approximately 35 million nonredundant compounds for potential activity against LF and comprised topomeric searching, docking and scoring, and drug-like filtering. Among these 5 hit compounds, none of which has previously been identified as a LF inhibitor, three exhibited experimental IC(50) values less than 100 microM. These three preliminary hits may potentially serve as scaffolds for lead optimization as well as templates for probe compounds to be used in mechanistic studies. Notably, our docking simulations predicted that these novel hits are likely to engage in critical ligand-receptor interactions with nearby residues in at least two of the three (S1', S1-S2, and S2') subsites in the LF substrate binding area. Further experimental characterization of these compounds is in process. We found that micromolar-level LF inhibition can be attained by compounds with non-hydroxamate zinc-binding groups that exhibit monodentate zinc chelation as long as key hydrophobic interactions with at least two LF subsites are retained.
Assuntos
Antitoxinas/química , Antitoxinas/farmacologia , Toxinas Bacterianas/antagonistas & inibidores , Avaliação Pré-Clínica de Medicamentos/métodos , Modelos Moleculares , Antígenos de Bactérias/química , Antígenos de Bactérias/metabolismo , Antitoxinas/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Ácidos Hidroxâmicos/química , Inibidores de Metaloproteinases de Matriz , Metaloproteinases da Matriz/química , Metaloproteinases da Matriz/metabolismo , Estrutura Terciária de Proteína , Reprodutibilidade dos Testes , Interface Usuário-ComputadorRESUMO
The PLP-dependent transaminase (BioA) of Mycobacterium tuberculosis and other pathogens that catalyzes the second step of biotin biosynthesis is a now well-validated target for antibacterial development. Fragment screening by differential scanning fluorimetry has been performed to discover new chemical scaffolds and promote optimization of existing inhibitors. Calorimetry confirms binding of six molecules with high ligand efficiency. Thermodynamic data identifies which molecules bind with the enthalpy driven stabilization preferred in compounds that represent attractive starting points for future optimization. Crystallographic characterization of complexes with these molecules reveals the dynamic nature of the BioA active site. Different side chain conformational states are stabilized in response to binding by different molecules. A detailed analysis of conformational diversity in available BioA structures is presented, resulting in the identification of two states that might be targeted with molecular scaffolds incorporating well-defined conformational attributes. This new structural data can be used as part of a scaffold hopping strategy to further optimize existing inhibitors or create new small molecules with improved therapeutic potential.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Transaminases/química , Transaminases/metabolismo , Tuberculose/tratamento farmacológico , Sítios de Ligação , Varredura Diferencial de Calorimetria , Domínio Catalítico , Cristalografia por Raios X , Modelos Moleculares , Conformação Molecular , Mycobacterium tuberculosis/efeitos dos fármacos , TermodinâmicaRESUMO
DmmA is a haloalkane dehalogenase (HLD) identified and characterized from the metagenomic DNA of a marine microbial consortium. Dehalogenase activity was detected with 1,3-dibromopropane as substrate, with steady-state kinetic parameters typical of HLDs (K(m) = 0.24 ± 0.05 mM, k(cat) = 2.4 ± 0.1 s(-1) ). The 2.2-Å crystal structure of DmmA revealed a fold and active site similar to other HLDs, but with a substantially larger active site binding pocket, suggestive of an ability to act on bulky substrates. This enhanced cavity was shown to accept a range of linear and cyclic substrates, suggesting that DmmA will contribute to the expanding industrial applications of HLDs.
Assuntos
Organismos Aquáticos/enzimologia , Hidrolases/química , Hidrolases/metabolismo , Animais , Organismos Aquáticos/química , Sítios de Ligação , Cristalografia por Raios X , Cianobactérias/química , Cianobactérias/enzimologia , Cianobactérias/metabolismo , Ativação Enzimática , Cinética , Modelos Biológicos , Modelos Moleculares , Multimerização Proteica/fisiologia , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Especificidade por SubstratoRESUMO
The mycobacterial biotin protein ligase (MtBPL) globally regulates lipid metabolism in Mtb through the posttranslational biotinylation of acyl coenzyme A carboxylases involved in lipid biosynthesis that catalyze the first step in fatty acid biosynthesis and pyruvate coenzyme A carboxylase, a gluconeogenic enzyme vital for lipid catabolism. Here we describe the design, development, and evaluation of a rationally designed bisubstrate inhibitor of MtBPL. This inhibitor displays potent subnanomolar enzyme inhibition and antitubercular activity against multidrug resistant and extensively drug resistant Mtb strains. We show that the inhibitor decreases in vivo protein biotinylation of key enzymes involved in fatty acid biosynthesis and that the antibacterial activity is MtBPL dependent. Additionally, the gene encoding BPL was found to be essential in M. smegmatis. Finally, the X-ray cocrystal structure of inhibitor bound MtBPL was solved providing detailed insight for further structure-activity analysis. Collectively, these data suggest that MtBPL is a promising target for further antitubercular therapeutic development.
Assuntos
Antituberculosos/química , Proteínas de Bactérias/antagonistas & inibidores , Carbono-Nitrogênio Ligases/antagonistas & inibidores , Inibidores Enzimáticos/química , Mycobacterium tuberculosis/enzimologia , Antituberculosos/síntese química , Antituberculosos/farmacologia , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Carbono-Nitrogênio Ligases/metabolismo , Cristalografia por Raios X , Desenho de Fármacos , Farmacorresistência Bacteriana , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Cinética , Testes de Sensibilidade Microbiana , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Especificidade por Substrato , TermodinâmicaRESUMO
Bacterial type I polyketide synthases (PKSs) assemble structurally diverse natural products of significant clinical value from simple metabolic building blocks. The synthesis of these compounds occurs in a processive fashion along a large multiprotein complex. Transfer of the acyl intermediate across interpolypeptide junctions is mediated, at least in large part, by N- and C-terminal docking domains. We report here a comprehensive analysis of the binding affinity and selectivity for the complete set of discrete docking domain pairs in the pikromycin and erythromycin PKS systems. Despite disconnection from their parent module, each cognate pair of docking domains retained exquisite binding selectivity. Further insights were obtained by X-ray crystallographic analysis of the PikAIII/PikAIV docking domain interface. This new information revealed a series of key interacting residues that enabled development of a structural model for the recently proposed H2-T2 class of polypeptides involved in PKS intermodular molecular recognition.
Assuntos
Bactérias/enzimologia , Eritromicina/metabolismo , Macrolídeos/metabolismo , Policetídeo Sintases/química , Policetídeo Sintases/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Eritromicina/química , Escherichia coli/genética , Polarização de Fluorescência , Macrolídeos/química , Modelos Moleculares , Dados de Sequência Molecular , Domínios e Motivos de Interação entre Proteínas , Especificidade por Substrato , Ressonância de Plasmônio de SuperfícieRESUMO
An unexpected biochemical strategy for chain initiation is described for the loading module of the polyketide synthase of curacin A, an anticancer lead derived from the marine cyanobacterium Lyngbya majuscula. A central GCN5-related N-acetyltransferase (GNAT) domain bears bifunctional decarboxylase/S-acetyltransferase activity, both unprecedented for the GNAT superfamily. A CurA loading tridomain, consisting of an adaptor domain, the GNAT domain, and an acyl carrier protein, was assessed biochemically, revealing that a domain showing homology to GNAT (GNAT(L)) catalyzes (i) decarboxylation of malonyl-coenzyme A (malonyl-CoA) to acetyl-CoA and (ii) direct S-acetyl transfer from acetyl-CoA to load an adjacent acyl carrier protein domain (ACP(L)). Moreover, the N-terminal adapter domain was shown to facilitate acetyl-group transfer. Crystal structures of GNAT(L) were solved at 1.95 angstroms (ligand-free form) and 2.75 angstroms (acyl-CoA complex), showing distinct substrate tunnels for acyl-CoA and holo-ACP(L) binding. Modeling and site-directed mutagenesis experiments demonstrated that histidine-389 and threonine-355, at the convergence of the CoA and ACP tunnels, participate in malonyl-CoA decarboxylation but not in acetyl-group transfer. Decarboxylation precedes acetyl-group transfer, leading to acetyl-ACP(L) as the key curacin A starter unit.
Assuntos
Acetiltransferases/química , Acetiltransferases/metabolismo , Cianobactérias/enzimologia , Ciclopropanos/metabolismo , Policetídeo Sintases/química , Policetídeo Sintases/metabolismo , Tiazóis/metabolismo , Acetilcoenzima A/metabolismo , Proteína de Transporte de Acila/química , Proteína de Transporte de Acila/metabolismo , Sequência de Aminoácidos , Carboxiliases/química , Carboxiliases/metabolismo , Cristalografia por Raios X , Cianobactérias/genética , Descarboxilação , Malonil Coenzima A/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Policetídeo Sintases/genética , Conformação Proteica , Estrutura Terciária de ProteínaRESUMO
Curacin A is a mixed polyketide/nonribosomal peptide possessing anti-mitotic and anti-proliferative activity. In the biosynthesis of curacin A, the N-terminal domain of the CurF multifunctional protein catalyzes decarboxylation of 3-methylglutaconyl-acyl carrier protein (ACP) to 3-methylcrotonyl-ACP, the postulated precursor of the cyclopropane ring of curacin A. This decarboxylase is encoded within an "HCS cassette" that is used by several other polyketide biosynthetic systems to generate chemical diversity by introduction of a beta-branch functional group to the natural product. The crystal structure of the CurF N-terminal ECH(2) domain establishes that the protein is a crotonase superfamily member. Ala(78) and Gly(118) form an oxyanion hole in the active site that includes only three polar side chains as potential catalytic residues. Site-directed mutagenesis and a biochemical assay established critical functions for His(240) and Lys(86), whereas Tyr(82) was nonessential. A decarboxylation mechanism is proposed in which His(240) serves to stabilize the substrate carboxylate and Lys(86) donates a proton to C-4 of the acyl-ACP enolate intermediate to form the Delta(2) unsaturated isopentenoyl-ACP product. The CurF ECH(2) domain showed a 20-fold selectivity for ACP-over CoA-linked substrates. Specificity for ACP-linked substrates has not been reported for any other crotonase superfamily decarboxylase. Tyr(73) may select against CoA-linked substrates by blocking a contact of Arg(38) with the CoA adenosine 5'-phosphate.