A dual cellular-heterogeneous catalyst strategy for the production of olefins from glucose.

Living systems provide a promising approach to chemical synthesis, having been optimized by evolution to convert renewable carbon sources, such as glucose, into an enormous range of small molecules. However, a large number of synthetic structures can still be difficult to obtain solely from cells, such as unsubstituted hydrocarbons. In this work, we demonstrate the use of a dual cellular-heterogeneous catalytic strategy to produce olefins from glucose using a selective hydrolase to generate an activated intermediate that is readily deoxygenated. Using a new family of iterative thiolase enzymes, we genetically engineered a microbial strain that produces 4.3 ± 0.4 g l-1 of fatty acid from glucose with 86% captured as 3-hydroxyoctanoic and 3-hydroxydecanoic acids. This 3-hydroxy substituent serves as a leaving group that enables heterogeneous tandem decarboxylation-dehydration routes to olefinic products on Lewis acidic catalysts without the additional redox input required for enzymatic or chemical deoxygenation of simple fatty acids.

Thioesterase superfamily member 1 undergoes stimulus-coupled conformational reorganization to regulate metabolism in mice.

In brown adipose tissue, thermogenesis is suppressed by thioesterase superfamily member 1 (Them1), a long chain fatty acyl-CoA thioesterase. Them1 is highly upregulated by cold ambient temperature, where it reduces fatty acid availability and limits thermogenesis. Here, we show that Them1 regulates metabolism by undergoing conformational changes in response to ß-adrenergic stimulation that alter Them1 intracellular distribution. Them1 forms metabolically active puncta near lipid droplets and mitochondria. Upon stimulation, Them1 is phosphorylated at the N-terminus, inhibiting puncta formation and activity and resulting in a diffuse intracellular localization. We show by correlative light and electron microscopy that Them1 puncta are biomolecular condensates that are inhibited by phosphorylation. Thus, Them1 forms intracellular biomolecular condensates that limit fatty acid oxidation and suppress thermogenesis. During a period of energy demand, the condensates are disrupted by phosphorylation to allow for maximal thermogenesis. The stimulus-coupled reorganization of Them1 provides fine-tuning of thermogenesis and energy expenditure.

Ligand-induced transition in conformations of vicinal cysteine disulfides in proteins.

Vicinal cysteine disulfides are thought to be associated with specific conformations of cysteine disulfides due to the restricted rotation of single bonds in an eight-membered cyclic disulfide loop. Conformations of vicinal cysteine disulfides are analyzed using χ1 , χ2 , χ3 , χ2 ', χ1 ' torsion angles in the crystal structures of proteins retrieved from Protein Data Bank (PDB). 85% of vicinal disulfides have (+, -)LHStaple conformation with trans configuration of the peptide bond and 9% have (-, -)RHStaple conformation with cis configured peptide bond. Conformational analysis of dipeptide Cys-Cys vicinal disulfide by density functional theory (DFT) further supported (+, -)LHStaple, (-, -)RHStaple, and (+, +)RHStaple as the preferred conformations of vicinal disulfides. Interestingly, the rare conformations of vicinal disulfides are observed in the ligand-bound forms of proteins and have higher disulfide strain energy. Conformations of vicinal disulfides in palmitoyl protein thioesterase 1, AChBP, and α7 nicotinic receptor are changed from preferred (+, -)LHStaple to rare (+, -)AntiLHHook/(+, -)AntiRHHook/(+, +)RHStaple conformation due to binding of ligands. Surprisingly, ligands are proximal to the vicinal disulfides in protein complexes that exhibited rare conformations of vicinal disulfides. The report has identified (+, -) LHStaple/(-, -) RHStaple as the hallmark conformations of vicinal disulfides and unraveled ligand-induced transition in conformations of vicinal cysteine disulfides in proteins.

Allosteric regulation of thioesterase superfamily member 1 by lipid sensor domain binding fatty acids and lysophosphatidylcholine.

Nonshivering thermogenesis occurs in brown adipose tissue to generate heat in response to cold ambient temperatures. Thioesterase superfamily member 1 (Them1) is transcriptionally up-regulated in brown adipose tissue upon exposure to the cold and suppresses thermogenesis in order to conserve energy reserves. It hydrolyzes long-chain fatty acyl-CoAs that are derived from lipid droplets, preventing their use as fuel for thermogenesis. In addition to its enzymatic domains, Them1 contains a C-terminal StAR-related lipid transfer (START) domain with unknown ligand or function. By complementary biophysical approaches, we show that the START domain binds to long-chain fatty acids, products of Them1's enzymatic reaction, as well as lysophosphatidylcholine (LPC), lipids shown to activate thermogenesis in brown adipocytes. Certain fatty acids stabilize the START domain and allosterically enhance Them1 catalysis of acyl-CoA, whereas 18:1 LPC destabilizes and inhibits activity, which we verify in cell culture. Additionally, we demonstrate that the START domain functions to localize Them1 near lipid droplets. These findings define the role of the START domain as a lipid sensor that allosterically regulates Them1 activity and spatially localizes it in proximity to the lipid droplet.

Fatty Acyl Synthetases and Thioesterases in Plant Lipid Metabolism: Diverse Functions and Biotechnological Applications.

Plants use fatty acids to synthesize acyl lipids for many different cellular, physiological, and defensive roles. These roles include the synthesis of essential membrane, storage, or surface lipids, as well as the production of various fatty acid-derived metabolites used for signaling or defense. Fatty acids are activated for metabolic processing via a thioester linkage to either coenzyme A or acyl carrier protein. Acyl synthetases metabolically activate fatty acids to their thioester forms, and acyl thioesterases deactivate fatty acyl thioesters to free fatty acids by hydrolysis. These two enzyme classes therefore play critical roles in lipid metabolism. This review highlights the surprisingly complex and varying roles of fatty acyl synthetases in plant lipid metabolism, including roles in the intracellular trafficking of fatty acids. This review also surveys the many specialized fatty acyl thioesterases characterized to date in plants, which produce a great diversity of fatty acid products in a tissue-specific manner. While some acyl thioesterases produce fatty acids that clearly play roles in plant-insect or plant-microbial interactions, most plant acyl thioesterases have yet to be fully characterized both in terms of their substrate specificities and their functions. The biotechnological applications of plant acyl thioesterases and synthetases are also discussed, as there is significant interest in these enzymes as catalysts for the sustainable production of fatty acids and their derivatives for industrial uses.

Elucidating the substrate specificities of acyl-lipid thioesterases from diverse plant taxa.

Acyl-ACP thioesterase enzymes, which cleave fatty acyl thioester bonds to release free fatty acids, contribute to much of the fatty acid diversity in plants. In Arabidopsis thaliana, a family of four single hot-dog fold domain, plastid-localized acyl-lipid thioesterases (AtALT1-4) generate medium-chain (C6-C14) fatty and ß-keto fatty acids as secondary metabolites. These volatile products may serve to attract insect pollinators or deter predatory insects. Homologs of AtALT1-4 are present in all plant taxa, but are nearly all uncharacterized. Despite high sequence identity, AtALT1-4 generate different lipid products, suggesting that ALT homologs in other plants also have highly varied activities. We investigated the catalytic diversity of ALT-like thioesterases by screening the substrate specificities of 15 ALT homologs from monocots, eudicots, a lycophyte, a green microalga, and the ancient gymnosperm Gingko biloba, via expression in Escherichia coli. Overall, these enzymes had highly varied substrate preferences compared to one another and to AtALT1-4, and could be classified into four catalytic groups comprising members from diverse taxa. Group 1 ALTs primarily generated 14:1 ß-keto fatty acids, Group 2 ALTs produced 6-10 carbon fatty/ß-keto fatty acids, Group 3 ALTs predominantly produced 12-14 carbon fatty acids, and Group 4 ALTs mainly generated 16 carbon fatty acids. Enzymes in each group differed significantly in the quantities of lipids and types of minor products they generated in E. coli. Medium-chain fatty acids are used to manufacture insecticides, pharmaceuticals, and biofuels, and ALT-like proteins are ideal candidates for metabolic engineering to produce specific fatty acids in significant quantities.

Mycobacteria Encode Active and Inactive Classes of TesB Fatty-Acyl CoA Thioesterases Revealed through Structural and Functional Analysis.

Mycobacteria contain a large number of highly divergent species and exhibit unusual lipid metabolism profiles, believed to play important roles in immune invasion. Thioesterases modulate lipid metabolism through the hydrolysis of activated fatty-acyl CoAs; multiple copies are present in mycobacteria, yet many remain uncharacterized. Here, we undertake a comprehensive structural and functional analysis of a TesB thioesterase from Mycobacterium avium (MaTesB). Structural superposition with other TesB thioesterases reveals that the Asp active site residue, highly conserved across a wide range of TesB thioesterases, is mutated to Ala. Consistent with these structural data, the wild-type enzyme failed to hydrolyze an extensive range of acyl-CoA substrates. Mutation of this residue to an active Asp residue restored activity against a range of medium-chain length fatty-acyl CoA substrates. Interestingly, this Ala mutation is highly conserved across a wide range of Mycobacterium species but not found in any other bacteria or organism. Our structural homology analysis revealed that at least one other TesB acyl-CoA thioesterase also contains an Ala residue at the active site, while two other Mycobacterium TesB thioesterases harbor an Asp residue at the active site. The inactive TesBs display a common quaternary structure that is distinct from that of the active TesB thioesterases. Investigation of the effect of expression of either the catalytically active or inactive MaTesB in Mycobacterium smegmatis exposed, to the best of our knowledge, the first genotype-phenotype association implicating a mycobacterial tesB gene. This is the first report that mycobacteria encode active and inactive forms of thioesterases, the latter of which appear to be unique to mycobacteria.

A Peptidyl-Transesterifying Type†I Thioesterase in Salinamide Biosynthesis.

Salinamide A belongs to a rare class of bicyclic depsipeptide antibiotics in which the installation of a (4-methylhexa-2,4-dienoyl)glycine handle across a hexadepsipeptide core contributes to its chemical complexity and biological properties. Herein, we report the genetic and biochemical basis for salinamide construction in the marine bacterium Streptomyces sp. CNB-091, which involves a novel intermolecular transesterification reaction catalyzed by a type I thioesterase. Heterologous expression studies revealed the central role of the nonribosomal peptide synthetase Sln9 in constructing and installing the distinctive acylglycine "basket handle" of salinamide. Biochemical characterization of the Sln9 thioesterase domain established that transesterification of the serine residue of desmethylsalinamide E with acylated glycyl thioesters yields desmethylsalinamide C.

Crystal structure and potential physiological role of zebra fish thioesterase superfamily member 2 (fTHEM2).

Thioesterase superfamily member 2 (THEM2) is an essential protein for mammalian cell proliferation. It belongs to the hotdog-fold thioesterase superfamily and catalyzes hydrolysis of thioester bonds of acyl-CoA in vitro, while its in vivo function remains unrevealed. In this study, Zebra fish was selected as a model organism to facilitate the investigations on THEM2. First, we solved the crystal structure of recombinant fTHEM2 at the resolution of 1.80 Å, which displayed a similar scaffolding as hTHEM2. Second, functional studies demonstrated that fTHEM2 is capable of hydrolyzing palmitoyl-CoA in vitro. In addition, injection of morpholino against fTHEM2 at one-cell stage resulted in distorted early embryo development, including delayed cell division, retarded development and increased death rate. The above findings validated our hypothesis that fTHEM2 could serve as an ideal surrogate for studying the physiological functions of THEM2.

Toward production of jet fuel functionality in oilseeds: identification of FatB acyl-acyl carrier protein thioesterases and evaluation of combinatorial expression strategies in Camelina seeds.

Seeds of members of the genus Cuphea accumulate medium-chain fatty acids (MCFAs; 8:0-14:0). MCFA- and palmitic acid- (16:0) rich vegetable oils have received attention for jet fuel production, given their similarity in chain length to Jet A fuel hydrocarbons. Studies were conducted to test genes, including those from Cuphea, for their ability to confer jet fuel-type fatty acid accumulation in seed oil of the emerging biofuel crop Camelina sativa. Transcriptomes from Cuphea viscosissima and Cuphea pulcherrima developing seeds that accumulate >90% of C8 and C10 fatty acids revealed three FatB cDNAs (CpuFatB3, CvFatB1, and CpuFatB4) expressed predominantly in seeds and structurally divergent from typical FatB thioesterases that release 16:0 from acyl carrier protein (ACP). Expression of CpuFatB3 and CvFatB1 resulted in Camelina oil with capric acid (10:0), and CpuFatB4 expression conferred myristic acid (14:0) production and increased 16:0. Co-expression of combinations of previously characterized Cuphea and California bay FatBs produced Camelina oils with mixtures of C8-C16 fatty acids, but amounts of each fatty acid were less than obtained by expression of individual FatB cDNAs. Increases in lauric acid (12:0) and 14:0, but not 10:0, in Camelina oil and at the sn-2 position of triacylglycerols resulted from inclusion of a coconut lysophosphatidic acid acyltransferase specialized for MCFAs. RNA interference (RNAi) suppression of Camelina ß-ketoacyl-ACP synthase II, however, reduced 12:0 in seeds expressing a 12:0-ACP-specific FatB. Camelina lines presented here provide platforms for additional metabolic engineering targeting fatty acid synthase and specialized acyltransferases for achieving oils with high levels of jet fuel-type fatty acids.

A study on the chiral inversion of mandelic acid in humans.

Mandelic acid is a chiral metabolite of the industrial pollutant styrene and is used in chemical skin peels, as a urinary antiseptic and as a component of other medicines. In humans, S-mandelic acid undergoes rapid chiral inversion to R-mandelic acid by an undefined pathway but it has been proposed to proceed via the acyl-CoA esters, S- and R-2-hydroxy-2-phenylacetyl-CoA, in an analogous pathway to that for Ibuprofen. This study investigates chiral inversion of mandelic acid using purified human recombinant enzymes known to be involved in the Ibuprofen chiral inversion pathway. Both S- and R-2-hydroxy-2-phenylacetyl-CoA were hydrolysed to mandelic acid by human acyl-CoA thioesterase-1 and -2 (ACOT1 and ACOT2), consistent with a possible role in the chiral inversion pathway. However, human α-methylacyl-CoA racemase (AMACR; P504S) was not able to catalyse exchange of the α-proton of S- and R-2-hydroxy-2-phenylacetyl-CoA, a requirement for chiral inversion. Both S- and R-2-phenylpropanoyl-CoA were epimerised by AMACR, showing that it is the presence of the hydroxy group that prevents epimerisation of R- and S-2-hydroxy-2-phenylacetyl-CoAs. The results show that it is unlikely that 2-hydroxy-2-phenylacetyl-CoA is an intermediate in the chiral inversion of mandelic acid, and that the chiral inversion of mandelic acid is via a different pathway to that of Ibuprofen and related drugs.

Hydrolysis of ibuprofenoyl-CoA and other 2-APA-CoA esters by human acyl-CoA thioesterases-1 and -2 and their possible role in the chiral inversion of profens.

Ibuprofen and related 2-arylpropanoic acid (2-APA) drugs are often given as a racemic mixture and the R-enantiomers undergo activation in vivo by metabolic chiral inversion. The chiral inversion pathway consists of conversion of the drug to the coenzyme A ester (by an acyl-CoA synthetase) followed by chiral inversion by α-methylacyl-CoA racemase (AMACR; P504S). The enzymes responsible for hydrolysis of the product S-2-APA-CoA ester to the active S-2-APA drug have not been identified. In this study, conversion of a variety of 2-APA-CoA esters by human acyl-CoA thioesterase-1 and -2 (ACOT-1 and -2) was investigated. Human recombinant ACOT-1 and -2 (ACOT-1 and -2) were both able to efficiently hydrolyse a variety of 2-APA-CoA substrates. Studies with the model substrates R- and S-2-methylmyristoyl-CoA showed that both enzymes were able to efficiently hydrolyse both of the epimeric substrates with (2R)- and (2S)- methyl groups. ACOT-1 is located in the cytosol and is able to hydrolyse 2-APA-CoA esters exported from the mitochondria and peroxisomes for inhibition of cyclo-oxygenase-1 and -2 in the endoplasmic reticulum. It is a prime candidate to be the enzyme responsible for the pharmacological action of chiral inverted drugs. ACOT-2 activity may be important in 2-APA toxicity effects and for the regulation of mitochondrial free coenzyme A levels. These results support the idea that 2-APA drugs undergo chiral inversion via a common pathway.

Molecular cloning, expression, purification and crystallographic analysis of zebrafish THEM2.

Thioesterase superfamily member 2 (THEM2) is essential for cell proliferation of mammalian cells. It belongs to the hotdog-fold thioesterase superfamily and catalyzes the hydrolysis of the thioester bonds of acyl-CoA in vitro. In this study, THEM2 protein from zebrafish (fTHEM2) was expressed in Escherichia coli and purified by Ni-affinity and gel-filtration chromatography. fTHEM2 crystals were obtained using the sitting-drop vapour-diffusion method with PEG 10 000 as precipitant. X-ray diffraction data were collected to 1.80 Šresolution using a synchrotron-radiation source. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a=77.1, b=74.4, c=96.6 Å, ß=93.7°.

Acyl coenzyme A thioesterase Them5/Acot15 is involved in cardiolipin remodeling and fatty liver development.

Acyl coenzyme A (acyl-CoA) thioesterases hydrolyze thioester bonds in acyl-CoA metabolites. The majority of mammalian thioesterases are α/ß-hydrolases and have been studied extensively. A second class of Hotdog-fold enzymes has been less well described. Here, we present a structural and functional analysis of a new mammalian mitochondrial thioesterase, Them5. Them5 and its paralog, Them4, adopt the classical Hotdog-fold structure and form homodimers in crystals. In vitro, Them5 shows strong thioesterase activity with long-chain acyl-CoAs. Loss of Them5 specifically alters the remodeling process of the mitochondrial phospholipid cardiolipin. Them5(-/-) mice show deregulation of lipid metabolism and the development of fatty liver, exacerbated by a high-fat diet. Consequently, mitochondrial morphology is affected, and functions such as respiration and ß-oxidation are impaired. The novel mitochondrial acyl-CoA thioesterase Them5 has a critical and specific role in the cardiolipin remodeling process, connecting it to the development of fatty liver and related conditions.

The emerging role of acyl-CoA thioesterases and acyltransferases in regulating peroxisomal lipid metabolism.

The importance of peroxisomes in lipid metabolism is now well established and peroxisomes contain approximately 60 enzymes involved in these lipid metabolic pathways. Several acyl-CoA thioesterase enzymes (ACOTs) have been identified in peroxisomes that catalyze the hydrolysis of acyl-CoAs (short-, medium-, long- and very long-chain), bile acid-CoAs, and methyl branched-CoAs, to the free fatty acid and coenzyme A. A number of acyltransferase enzymes, which are structurally and functionally related to ACOTs, have also been identified in peroxisomes, which conjugate (or amidate) bile acid-CoAs and acyl-CoAs to amino acids, resulting in the production of amidated bile acids and fatty acids. The function of ACOTs is to act as auxiliary enzymes in the α- and ß-oxidation of various lipids in peroxisomes. Human peroxisomes contain at least two ACOTs (ACOT4 and ACOT8) whereas mouse peroxisomes contain six ACOTs (ACOT3, 4, 5, 6, 8 and 12). Similarly, human peroxisomes contain one bile acid-CoA:amino acid N-acyltransferase (BAAT), whereas mouse peroxisomes contain three acyltransferases (BAAT and acyl-CoA:amino acid N-acyltransferases 1 and 2: ACNAT1 and ACNAT2). This review will focus on the human and mouse peroxisomal ACOT and acyltransferase enzymes identified to date and discuss their cellular localizations, emerging structural information and functions as auxiliary enzymes in peroxisomal metabolic pathways.

Insights into the substrate specificity of a thioesterase Rv0098 of mycobacterium tuberculosis through X-ray crystallographic and molecular dynamics studies.

The crystal structure of Rv0098, a long-chain fatty acyl-CoA thioesterase from Mycobacterium tuberculosis with bound dodecanoic acid at the active site provided insights into the mode of substrate binding but did not reveal the structural basis of substrate specificities of varying chain length. Molecular dynamics studies demonstrated that certain residues of the substrate binding tunnel are flexible and thus modulate the length of the tunnel. The flexibility of the loop at the base of the tunnel was also found to be important for determining the length of the tunnel for accommodating appropriate substrates. A combination of crystallographic and molecular dynamics studies thus explained the structural basis of accommodating long chain substrates by Rv0098 of M. tuberculosis.

Ligand-induced conformational changes within a hexameric Acyl-CoA thioesterase.

Acyl-coenzyme A (acyl-CoA) thioesterases play a crucial role in the metabolism of activated fatty acids, coenzyme A, and other metabolic precursor molecules including arachidonic acid and palmitic acid. These enzymes hydrolyze coenzyme A from acyl-CoA esters to mediate a range of cellular functions including ß-oxidation, lipid biosynthesis, and signal transduction. Here, we present the crystal structure of a hexameric hot-dog domain-containing acyl-CoA thioesterase from Bacillus halodurans in the apo-form and provide structural and comparative analyses to the coenzyme A-bound form to identify key conformational changes induced upon ligand binding. We observed dramatic ligand-induced changes at both the hot-dog dimer and the trimer-of-dimer interfaces; the dimer interfaces in the apo-structure differ by over 20% and decrease to about half the size in the ligand-bound state. We also assessed the specificity of the enzyme against a range of fatty acyl-CoA substrates and have identified a preference for short-chain fatty acyl-CoAs. Coenzyme A was shown both to negatively regulate enzyme activity, representing a direct inhibitory feedback, and consistent with the structural data, to destabilize the quaternary structure of the enzyme. Coenzyme A-induced conformational changes in the C-terminal helices of enzyme were assessed through mutational analysis and shown to play a role in regulating enzyme activity. The conformational changes are likely to be conserved from bacteria through to humans and provide a greater understanding, particularly at a structural level, of thioesterase function and regulation.

Comparative structural analysis of lipid binding START domains.

BACKGROUND: Steroidogenic acute regulatory (StAR) protein related lipid transfer (START) domains are small globular modules that form a cavity where lipids and lipid hormones bind. These domains can transport ligands to facilitate lipid exchange between biological membranes, and they have been postulated to modulate the activity of other domains of the protein in response to ligand binding. More than a dozen human genes encode START domains, and several of them are implicated in a disease. PRINCIPAL FINDINGS: We report crystal structures of the human STARD1, STARD5, STARD13 and STARD14 lipid transfer domains. These represent four of the six functional classes of START domains. SIGNIFICANCE: Sequence alignments based on these and previously reported crystal structures define the structural determinants of human START domains, both those related to structural framework and those involved in ligand specificity. ENHANCED VERSION: This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1.

Characterization of a novel thioesterase (PtTE) from Phaeodactylum tricornutum.

The Phaeodactylum tricornutum (P. tricornutum) thioesterase PtTE was encoded by a 648 bp open reading frame. The deduced 216 amino acids showed no similarity with plant acyl-acyl carrier protein (ACP) thioesterases and bacterial thioesterases. Southern blot analysis revealed that one copy of PtTE was present in the P. tricornutum genome, and Real-time quantitative PCR showed that PtTE was up-regulated upon nitrogen deprivation. Thioesterase activity of PtTE was established by heterologous expression of PtTE cDNA in Escherichia coli (E. coli) XL1-Blue and K27fadD88, a mutant strain of fatty acid ß-oxidation pathway. The substrate specificity of PtTE was determined by fatty acid profile analyses of the culture supernatant and membrane lipid of recombinant strains. Recombinant PtTE in E.coli enhanced total fatty acid content of XL1-Blue by 21%, and also changed the fatty acid compositions of membrane lipid and culture supernatant. These changes were directed predominantly towards C18:0 and C18:1 fatty acids. Overexpression of PtTE alone in P. tricornutum did not alter the fatty acid composition of P. tricornutum, but enhanced total fatty acid content by 72%. This novel thioesterase gene shows its potential in metabolic engineering for enhancing lipid yield of microalgae. This is so far the first report of thioesterase from eukaryotic microalgae.

Terminal alkene formation by the thioesterase of curacin A biosynthesis: structure of a decarboxylating thioesterase.

Curacin A is a polyketide synthase (PKS)-non-ribosomal peptide synthetase-derived natural product with potent anticancer properties generated by the marine cyanobacterium Lyngbya majuscula. Type I modular PKS assembly lines typically employ a thioesterase (TE) domain to off-load carboxylic acid or macrolactone products from an adjacent acyl carrier protein (ACP) domain. In a striking departure from this scheme the curacin A PKS employs tandem sulfotransferase and TE domains to form a terminal alkene moiety. Sulfotransferase sulfonation of ß-hydroxy-acyl-ACP is followed by TE hydrolysis, decarboxylation, and sulfate elimination (Gu, L., Wang, B., Kulkarni, A., Gehret, J. J., Lloyd, K. R., Gerwick, L., Gerwick, W. H., Wipf, P., Håkansson, K., Smith, J. L., and Sherman, D. H. (2009) J. Am. Chem. Soc. 131, 16033-16035). With low sequence identity to other PKS TEs (<15%), the curacin TE represents a new thioesterase subfamily. The 1.7-Å curacin TE crystal structure reveals how the familiar α/ß-hydrolase architecture is adapted to specificity for ß-sulfated substrates. A Ser-His-Glu catalytic triad is centered in an open active site cleft between the core domain and a lid subdomain. Unlike TEs from other PKSs, the lid is fixed in an open conformation on one side by dimer contacts of a protruding helix and on the other side by an arginine anchor from the lid into the core. Adjacent to the catalytic triad, another arginine residue is positioned to recognize the substrate ß-sulfate group. The essential features of the curacin TE are conserved in sequences of five other putative bacterial ACP-ST-TE tridomains. Formation of a sulfate leaving group as a biosynthetic strategy to facilitate acyl chain decarboxylation is of potential value as a route to hydrocarbon biofuels.