Thioester Hydrolysis Research Articles

Engineering thioesterase as a driving force for novel itaconate production via its degradation scheme

Incorporation of irreversible steps in pathway design enhances the overall thermodynamic favorability and often leads to better bioconversion yield given functional enzymes. Using this concept, here we constructed the first non-natural itaconate biosynthesis pathway driven by thioester hydrolysis. Itaconate is a commercially valuable platform chemical with wide applications in the synthetic polymer industry. Production of itaconate has long relied on the decarboxylation of TCA cycle intermediate cis-aconitate as the only biosynthetic route. Inspired by nature's design of itaconate detoxification, here we engineered a novel itaconate producing pathway orthogonal to native metabolism with no requirement of auxotrophic knock-out. The reversed degradation pathway initiates with pyruvate and acetyl-CoA condensation forming (S)-citramalyl-CoA, followed by its dehydration and isomerization into itaconyl-CoA then hydrolysis into itaconate. Phenylacetyl-CoA thioesterase (PaaI) from Escherichiacoli was identified via screening to deliver the highest itaconate formation efficiency when coupled to the reversible activity of citramalate lyase and itaconyl-CoA hydratase. The preference of PaaI towards itaconyl-CoA hydrolysis over acetyl-CoA and (S)-citramalyl-CoA also minimized the inevitable precursor loss due to enzyme promiscuity. With acetate recycling, acetyl-CoA conservation, and condition optimization, we achieved a final itaconate titer of 1 g/L using the thioesterase driven pathway, which is a significant improvement compared to the original degradation pathway based on CoA transferase. This study illustrates the significance of thermodynamic favorability as a design principle in pathway engineering.

Structural and mechanistic analysis of Ca2+-dependent regulation of transglutaminase 2 activity using a Ca2+-bound intermediate state

Mammalian transglutaminases, a family of Ca2+-dependent proteins, are implicated in a variety of diseases. For example, celiac disease (CeD) is an autoimmune disorder whose pathogenesis requires transglutaminase 2 (TG2) to deamidate select glutamine residues in diet-derived gluten peptides. Deamidation involves the formation of transient γ-glutamyl thioester intermediates. Recent studies have revealed that in addition to the deamidated gluten peptides themselves, their corresponding thioester intermediates are also pathogenically relevant. A mechanistic understanding of this relevance is hindered by the absence of any structure of Ca2+-bound TG2. We report the X-ray crystallographic structure of human TG2 bound to an inhibitory gluten peptidomimetic and two Ca2+ ions in sites previously designated as S1 and S3. Together with additional structure-guided experiments, this structure provides a mechanistic explanation for how S1 regulates formation of an inhibitory disulfide bond in TG2, while also establishing that S3 is essential for γ-glutamyl thioester formation. Furthermore, our crystallographic findings and associated analyses have revealed that i) two interacting residues, H305 and E363, play a critical role in resolving the thioester intermediate into an isopeptide bond (transamidation) but not in thioester hydrolysis (deamidation); and ii) residues N333 and K176 stabilize preferred TG2 substrates and inhibitors via hydrogen bonding to nonreactive backbone atoms. Overall, the intermediate-state conformer of TG2 reported here represents a superior model to previously characterized conformers for both transition states of the TG2-catalyzed reaction.

Role of Fast and Slow Inhibitors in Oscillatory Rhythm Design.

In biological or abiotic systems, rhythms occur, owing to the coupling between positive and negative feedback loops in a reaction network. Using the Semenov-Whitesides oscillatory network for thioester hydrolysis as a prototype, we experimentally and theoretically analyzed the role of fast and slow inhibitors in oscillatory reaction networks. In the presence of positive feedback, a single fast inhibitor generates a time delay, resulting in two saddle-node bifurcations and bistability in a continuously stirred tank reactor. A slow inhibitor produces a node-focus bifurcation, resulting in damped oscillations. With both fast and slow inhibitors present, the node-focus bifurcation repeatedly modulates the saddle-node bifurcations, producing stable periodic oscillations. These fast and slow inhibitions result in a pair of time delays between steeply ascending and descending dynamics, which originate from the positive and negative feedbacks, respectively. This pattern can be identified in many chemical relaxation oscillators and oscillatory models, e.g., the bromate-sulfite pH oscillatory system, the Belousov-Zhabotinsky reaction, the trypsin oscillatory system, and the Boissonade-De Kepper model. This study provides a novel understanding of chemical and biochemical rhythms and suggests an approach to designing such behavior.

Acyl-CoA thioesterase 9 promotes tumour growth and metastasis through reprogramming of fatty acid metabolism in hepatocellular carcinoma.

Acyl-CoA thioesterase 9 (ACOT9) is a critical regulator of cellular utilization of fatty acids by catalysing the hydrolysis of acyl-CoA thioesters to non-esterified fatty acid and coenzyme A (CoA). Recently, ACOT9 was reported to participate in the pathogenesis of non-alcoholic liver disease (NAFLD), which arises from aberrant lipid metabolism and serves as a risk factor for hepatocellular carcinoma (HCC). However, the functions of ACOT9 in carcinogenesis and aberrant lipid metabolism in HCC remain unexplored. Here, we found that ACOT9 expression is significantly elevated in HCC at least partially due to the down-regulation of miR-449c-3p. Upregulation of ACOT9 is closely associated with poor prognosis for patients with HCC. Knockdown of ACOT9 expression in HCC cells significantly decreased cell proliferation, colony formation, migration and invasion, mainly through suppression of G1-to-S cell cycle transition and epithelial-to-mesenchymal transition (EMT). By contrast, forced ACOT9 expression promoted HCC growth and metastasis. In addition, we found that ACOT9 reprogrammed lipid metabolism in HCC cells by increasing de novo lipogenesis. Furthermore, we demonstrated that increased lipogenesis was involved in ACOT9-promoted HCC growth and metastasis. Altogether, we demonstrate that ACOT9 plays a critical oncogenic role in the promotion of tumour growth and metastasis by reprogramming lipid metabolism in HCC, indicating ACOT9 as a potential therapeutic target in treatment of HCC.

Molecular characterization of substrate-induced ubiquitin transfer by UBR7-PHD finger, a newly identified histone H2BK120 ubiquitin ligase.

Monoubiquitination of histone H2B at lysine 120 plays a vital role in active transcription and DNA damage response pathways. Ubiquitin protein ligase E3 component N-recognin 7 (UBR7) has been recently identified as an H2BK120 monoubiquitin ligase. However, the molecular details of its ubiquitin transfer mechanism are not well understood. Here, we report that the plant homeodomain (PHD) finger of UBR7 is essential for its association with E2 UbcH6 and consequent ubiquitin transfer to its substrate histone H2B. We also identified the critical region of UbcH6 involved in this function and shown that the residues stretching from 114 to 125 of histone H2B C-terminal tail are sufficient for UBR7/UbcH6-mediated ubiquitin transfer. We also employed antibody-independent mass spectrometry to confirm UBR7-mediated ubiquitination of the H2B C-terminal tail. We demonstrated that the PHD finger of UBR7 forms a dimer and this dimerization is essential for ubiquitination of histone H2B. We mapped the critical residues involved in the dimerization and mutation of these residues that abrogate E3 ligase activity and are associated with cancer. Furthermore, we compared the mode of ubiquitin discharge from UbcH6 mediated by UBR7 and RING finger protein 20 (RNF20) through a thioester hydrolysis assay. Interestingly, binding of substrate H2B to UBR7 induces a conformational change in the PHD finger, which triggers ubiquitin transfer from UbcH6. However, the RNF20 RING finger alone is sufficient to promote the release of ubiquitin from UbcH6. Overall, the mechanism of ubiquitin transfer by the newly identified E3 ubiquitin ligase UBR7 is markedly different from that of RNF20.

Unveiling the CoA mediated salicylate catabolic mechanism in Rhizobium sp. X9.

Salicylate is a typical aromatic compound widely distributed in nature. Microbial degradation of salicylate has been well studied and salicylate hydroxylases play essential roles in linking the peripheral and ring-cleavage catabolic pathways. The direct hydroxylation of salicylate catalyzed by salicylate-1-hydroxylase or salicylate-5-hydroxylase has been well studied. However, the CoA mediated salicylate 5-hydroxylation pathway has not been characterized in detail. Here, we elucidate the molecular mechanism of the reaction in the conversion of salicylate to gentisate in the carbaryl-degrading strain Rhizobium sp. X9. Three enzymes (salicylyl-CoA ligase CehG, salicylyl-CoA hydroxylase CehH and gentisyl-CoA thioesterase CehI) catalyzed the conversion of salicylate to gentisate via a route, including CoA thioester formation, hydroxylation and thioester hydrolysis. Further analysis indicated that genes cehGHI are also distributed in other bacteria from terrestrial environment and marine sediments. These genomic evidences highlight the role of this salicylate degradation pathway in the carbon cycle of soil organic compounds and marine sediments. Our findings of this three-step strategy enhanced the current understanding of CoA mediated degradation of salicylate.

ACOT4 accumulation via AKT-mediated phosphorylation promotes pancreatic tumourigenesis.

The acyl-CoA thioesterase (ACOT) family catalyses the hydrolysis of acyl-CoA thioesters to their corresponding non-esterified fatty acid and coenzyme A (CoA). Increasing evidence suggests that cancer cells generally have altered lipid metabolism in different aspects. However, the roles of the ACOT family in cancer, especially in pancreatic ductal carcinoma (PDAC), are largely unknown. In the present study, we mined data to determine the clinical significance of all eleven ACOT genes among nine major solid tumour types from TCGA database and found that the expression of ACOT4 in PDAC was negatively correlated with patient survival, establishing ACOT4 as a potential biomarker of PDAC. Depletion of ACOT4 attenuated the proliferation and tumour formation of PDAC cells. Using mass spectrometry, HSPA1A was found to associate with ACOT4. Furthermore, we found that phosphorylation of ACOT4 at S392 by AKT decreased the binding of ACOT4 to HSPA1A, resulting in ACOT4 accumulation. The ACOT4 elevation promotes pancreatic tumourigenesis by producing excessive CoA to support tumour cell metabolism. Thus, our study expands the relationship between AKT signalling and lipid metabolism and establishes a functional role of ACOT4 in PDAC.

The Impact of Carboxylesterases in Drug Metabolism and Pharmacokinetics.

Background: Carboxylesterases (CES) play a critical role in catalyzing hydrolysis of esters, amides, carbamates and thioesters, as well as bioconverting prodrugs and soft drugs. The unique tissue distribution of CES enzymes provides great opportunities to design prodrugs or soft drugs for tissue targeting. Marked species differences in CES tissue distribution and catalytic activity are particularly challenging in human translation.Methods: Review and summarization of CES fundamentals and applications in drug discovery and development.Results: Human CES1 is one of the most highly expressed drug metabolizing enzymes in the liver, while human intestine only expresses CES2. CES enzymes have moderate to high inter-individual variability and exhibit low to no expression in the fetus, but increase substantially during the first few months of life. The CES genes are highly polymorphic and some CES ge-netic variants show significant influence on metabolism and clinical outcome of certain drugs. Monkeys appear to be more predictive of human pharmacokinetics for CES substrates than other species. Low risk of clinical drug-drug interaction is anticipated for CES, although they should not be overlooked, particularly interaction with alcohols. CES enzymes are moderately inducible through a number of transcription factors and can be repressed by inflammatory cy-tokines.Conclusion: Although significant advances have been made in our understanding of CESs, in vitro - in vivo extrapolation of clearance is still in its infancy and further exploration is needed. In vitro and in vivo tools are continuously being developed to characterize CES substrates and inhibitors

The molecular basis for the intramolecular migration (NIH shift) of the carboxyl group during para-hydroxybenzoate catabolism.

The NIH shift is a chemical rearrangement in which a substituent on an aromatic ring undergoes an intramolecular migration, primarily during an enzymatic hydroxylation reaction. The molecular mechanism for the NIH shift of a carboxyl group has remained a mystery for 40 years. Here, we elucidate the molecular mechanism of the reaction in the conversion of para-hydroxybenzoate (PHB) to gentisate (GA, 2, 5-dihydroxybenzoate). Three genes (phgABC) from the PHB utilizer Brevibacillus laterosporus PHB-7a encode enzymes (p-hydroxybenzoyl-CoA ligase, p-hydroxybenzoyl-CoA hydroxylase and gentisyl-CoA thioesterase, respectively) catalyzing the conversion of PHB to GA via a route involving CoA thioester formation, hydroxylation concomitant with a 1, 2-shift of the acetyl CoA moiety and thioester hydrolysis. The shift of the carboxyl group was established rigorously by stable isotopic experiments with heterologously expressed phgABC, converting 2, 3, 5, 6-tetradeutero-PHB and [carboxyl-13 C]-PHB to 3, 4, 6-trideutero-GA and [carboxyl-13 C]-GA respectively. This is distinct from the NIH shifts of hydrogen and aceto substituents, where a single oxygenase catalyzes the reaction without the involvement of a thioester. The discovery of this three-step strategy for carboxyl group migration reveals a novel role of the CoA thioester in biochemistry and also illustrates the diversity and complexity of microbial catabolism in the carbon cycle.

A novel synthetic pathway for glutarate production in recombinant Escherichia coli

Abstract Glutarate is an important C5 linear chain dicarboxylic acid having wide applications in chemical industry. In this work we report glutarate production by Escherichia coli with a newly constructed biosynthetic pathway containing part of known glutaconate biosynthetic pathway and a gap-filling module employing trans -enoyl-CoA reductase (Ter). Overall the artificial pathway comprises reduction of the central carbon metabolite α-ketoglutarate to 2-hydroxyglutarate, activation to 2-hydroxyglutaryl-CoA, dehydration to trans -glutaconyl-CoA, hydrogenation to glutaryl-CoA by Ter and thioester hydrolysis to finally yield glutarate. The pathway introduced into E. coli resulted in a recombinant strain that produced 3.8 mg/L of glutarate together with 27.7 mg/L of glutaconate in anaerobic culture mode. The glutarate production increased by approximately 50% through the mutation of Ter from Treponema denticola . The results demonstrated biosynthesis of glutarate via a non-natural synthetic pathway, which may enable its biobased production from renewable resources.

Structural analysis of the dual-function thioesterase SAV606 unravels the mechanism of Michael addition of glycine to an α,β-unsaturated thioester

Thioesterases catalyze hydrolysis of acyl thioesters to release carboxylic acid or macrocyclization to produce the corresponding macrocycle in the biosynthesis of fatty acids, polyketides, or nonribosomal peptides. Recently, we reported that the thioesterase CmiS1 from Streptomyces sp. MJ635-86F5 catalyzes the Michael addition of glycine to an α,β-unsaturated fatty acyl thioester followed by thioester hydrolysis in the biosynthesis of the macrolactam antibiotic cremimycin. However, the molecular mechanisms of CmiS1-catalyzed reactions are unclear. Here, we report on the functional and structural characterization of the CmiS1 homolog SAV606 from Streptomyces avermitilis MA-4680. In vitro analysis indicated that SAV606 catalyzes the Michael addition of glycine to crotonic acid thioester and subsequent hydrolysis yielding (R)-N-carboxymethyl-3-aminobutyric acid. We also determined the crystal structures of SAV606 both in ligand-free form at 2.4 Å resolution and in complex with (R)-N-carboxymethyl-3-aminobutyric acid at 2.0 Å resolution. We found that SAV606 adopts an α/β hotdog fold and has an active site at the dimeric interface. Examining the complexed structure, we noted that the substrate-binding loop comprising Tyr-53-Asn-61 recognizes the glycine moiety of (R)-N-carboxymethyl-3-aminobutyric acid. Moreover, we found that SAV606 does not contain an acidic residue at the active site, which is distinct from canonical hotdog thioesterases. Site-directed mutagenesis experiments revealed that His-59 plays a crucial role in both the Michael addition and hydrolysis via a water molecule. These results allow us to propose the reaction mechanism of the SAV606-catalyzed Michael addition and thioester hydrolysis and provide new insight into the multiple functions of a thioesterase family enzyme.

Engineering Escherichia coli for Microbial Production of Butanone.

To expand the chemical and molecular diversity of biotransformation using whole-cell biocatalysts, we genetically engineered a pathway in Escherichia coli for heterologous production of butanone, an important commodity ketone. First, a 1-propanol-producing E. coli host strain with its sleeping beauty mutase (Sbm) operon being activated was used to increase the pool of propionyl-coenzyme A (propionyl-CoA). Subsequently, molecular heterofusion of propionyl-CoA and acetyl-CoA was conducted to yield 3-ketovaleryl-CoA via a CoA-dependent elongation pathway. Lastly, 3-ketovaleryl-CoA was channeled into the clostridial acetone formation pathway for thioester hydrolysis and subsequent decarboxylation to form butanone. Biochemical, genetic, and metabolic factors affecting relative levels of ketogenesis, acidogenesis, and alcohol genesis under selected fermentative culture conditions were investigated. Using the engineered E. coli strain for batch cultivation with 30 g liter(-1)glycerol as the carbon source, we achieved coproduction of 1.3 g liter(-1)butanone and 2.9 g liter(-1)acetone. The results suggest that approximately 42% of spent glycerol was utilized for ketone biosynthesis, and thus they demonstrate potential industrial applicability of this microbial platform.

S- to N-Palmitoyl Transfer During Proteomic Sample Preparation.

N-palmitoylation has been reported in a number of proteins and suggested to play an important role in protein localization and functions. However, it remains unclear whether N-palmitoylation is a direct enzyme-catalyzed process, or results from intramolecular S- to N-palmitoyl transfer. Here, using the S-palmitoyl peptide standard, GCpalmLGNAK, as the model system, we observed palmitoyl migration from the cysteine residue to either the peptide N-terminus or the lysine side chain during incubation in both neutral and slightly basic buffers commonly used in proteomic sample preparation. Palmitoyl transfer can take place either intra- or inter-molecularly, with the peptide N-terminus being the preferred migration site, presumably because of its lower basicity. The extent of intramolecular palmitoyl migration was low in the system studied, as it required the formation of an entropically unfavored macrocycle intermediate. Intermolecular palmitoyl transfer, however, remained a tangible problem, and may lead to erroneous reporting of in vivo N-palmitoylation. It was found that addition of the MS-compatible detergent RapiGest could significantly inhibit intermolecular palmitoyl transfer, as well as thioester hydrolysis and DTT-induced thioester cleavage. Finally, palmitoyl transfer from the cysteine residue to the peptide N-terminus can also occur in the gas phase, during collision-induced dissociation, and result in false identification of N-palmitoylation. Therefore, one must be careful with both sample preparation and interpretation of tandem mass spectra in the study of N-palmitoylation. Graphical Abstract ᅟ.

Native chemical ligation between asparagine and valine: Application and limitations for the synthesis of tri-phosphorylated C-terminal tau

We present the successful native chemical ligation (NCL) at an Asn-Val site employing β-mercaptovaline and subsequent desulfurization in the synthesis of native phosphorylated C-terminal tau, relevant for Alzheimer’s disease related research. Despite recent progress in the field of NCL we illustrate limitations of this ligation site that stem from thioester hydrolysis and predominantly aspartimide formation. We systematically investigated the influence of pH, temperature, peptide concentration and thiol additives on the outcome of this ligation and identified conditions under which the ligation can be driven toward complete conversion, which required the deployment of a high surplus of thioester. Application of the optimized conditions allowed us to gain access to challenging tri-phosphorylated C-terminal tau peptide in practical yields.

Structural and functional analysis of the loading acyltransferase from avermectin modular polyketide synthase.

The loading acyltransferase (AT) domains of modular polyketide synthases (PKSs) control the choice of starter units incorporated into polyketides and are therefore attractive targets for the engineering of modular PKSs. Here, we report the structural and biochemical characterizations of the loading AT from avermectin modular PKS, which accepts more than 40 carboxylic acids as alternative starter units for the biosynthesis of a series of congeners. This first structural analysis of loading ATs from modular PKSs revealed the molecular basis for the relaxed substrate specificity. Residues important for substrate binding and discrimination were predicted by modeling a substrate into the active site. A mutant with altered specificity toward a panel of synthetic substrate mimics was generated by site-directed mutagenesis of the active site residues. The hydrolysis of the N-acetylcysteamine thioesters of racemic 2-methylbutyric acid confirmed the stereospecificity of the avermectin loading AT for an S configuration at the C-2 position of the substrate. Together, these results set the stage for region-specific modification of polyketides through active site engineering of loading AT domains of modular PKSs.

A mechanistic study of thioester hydrolysis with heavy atom kinetic isotope effects.

The carbonyl-C, carbonyl-O, and leaving-S kinetic isotope effects (KIEs) were determined for the hydrolysis of formylthiocholine. Under acidic conditions, (13)k(obs) = 1.0312, (18)k(obs) = 0.997, and (34)k(obs) = 0.995; for neutral conditions, (13)k(obs) = 1.022, (18)k(obs) = 1.010, and (34)k(obs) = 0.996; and for alkaline conditions, (13)k(obs) = 1.0263, (18)k(obs) = 0.992, and (34)k(obs) = 1.000. The observed KIEs provided helpful insights into a qualitative description of the bond orders in the transition state structure.

Phosphomimetic Mutation of the N-Terminal Lid of MDM2 Enhances the Polyubiquitination of p53 through Stimulation of E2-Ubiquitin Thioester Hydrolysis

Mouse double minute 2 (MDM2) has a phosphorylation site within a lid motif at Ser17 whose phosphomimetic mutation to Asp17 stimulates MDM2-mediated polyubiquitination of p53. MDM2 lid deletion, but not Asp17 mutation, induced a blue shift in the λmax of intrinsic fluorescence derived from residues in the central domain including Trp235, Trp303, Trp323, and Trp329. This indicates that the Asp17 mutation does not alter the conformation of MDM2 surrounding the tryptophan residues. In addition, Phe235 mutation enhanced MDM2 binding to p53 but did not stimulate its ubiquitination function, thus uncoupling increases in p53 binding from its E3 ubiquitin ligase function. However, the Asp17 mutation in MDM2 stimulated its discharge of the UBCH5a-ubiquitin thioester adduct (UBCH5a is a ubiquitin-conjugating enzyme E2D 1 UBC4/5 homolog yeast). This stimulation of ubiquitin discharge from E2 was independent of the p53 substrate. There are now four known effects of the Asp17 mutation on MDM2: (i) it alters the conformation of the isolated N-terminus as defined by NMR; (ii) it induces increased thermostability of the isolated N-terminal domain; (iii) it stimulates the allosteric interaction of MDM2 with the DNA-binding domain of p53; and (iv) it stimulates a novel protein–protein interaction with the E2-ubiquitin complex in the absence of substrate p53 that, in turn, increases hydrolysis of the E2-ubiquitin thioester bond. These data also suggest a new strategy to disrupt MDM2 function by targeting the E2-ubiquitin discharge reaction.

A multi-scale computational study on the mechanism of Streptococcus pneumoniae Nicotinamidase (SpNic).

Nicotinamidase (Nic) is a key zinc-dependent enzyme in NAD metabolism that catalyzes the hydrolysis of nicotinamide to give nicotinic acid. A multi-scale computational approach has been used to investigate the catalytic mechanism, substrate binding and roles of active site residues of Nic from Streptococcus pneumoniae (SpNic). In particular, density functional theory (DFT), molecular dynamics (MD) and ONIOM quantum mechanics/molecular mechanics (QM/MM) methods have been employed. The overall mechanism occurs in two stages: (i) formation of a thioester enzyme-intermediate (IC2) and (ii) hydrolysis of the thioester bond to give the products. The polar protein environment has a significant effect in stabilizing reaction intermediates and in particular transition states. As a result, both stages effectively occur in one step with Stage 1, formation of IC2, being rate limiting barrier with a cost of 53.5 kJ·mol−1 with respect to the reactant complex, RC. The effects of dispersion interactions on the overall mechanism were also considered but were generally calculated to have less significant effects with the overall mechanism being unchanged. In addition, the active site lysyl (Lys103) is concluded to likely play a role in stabilizing the thiolate of Cys136 during the reaction.

A Kinetic Isotope Effect and Isotope Exchange Study of the Nonenzymatic and the Equine Serum Butyrylcholinesterase-Catalyzed Thioester Hydrolysis

Formylthiocholine (FTC) was synthesized and found to be a substrate for nonenzymatic and butyrylcholinesterase (BChE)-catalyzed hydrolysis. Solvent (D2O) and secondary formyl-H kinetic isotope effects (KIEs) were measured by an NMR spectroscopic method. The solvent (D2O) KIEs are (D2O)k = 0.20 in 200 mM HCl, (D2O)k = 0.81 in 50 mM HCl, and (D2O)k = 4.2 in pure water. The formyl-H KIEs are (D)k = 0.80 in 200 mM HCl, (D)k = 0.77 in 50 mM HCl, (D)k = 0.75 in pure water, (D)k = 0.88 in 50 mM NaOH, and (D)(V/K) = 0.89 in the BChE-catalyzed hydrolysis in MES buffer at pH 6.8. Positional isotope exchange experiments showed no detectable exchange of (18)O into the carbonyl oxygen of FTC or the product, formate, under any of the above conditions. Solvent nucleophile-O KIEs were determined to be (18)k = 0.9917 under neutral conditions, (18)k = 1.0290 (water nucleophile) or (18)k = 0.989 (hydroxide nucleophile) under alkaline conditions, and (18)(V/K) = 0.9925 for BChE catalysis. The acidic, neutral, and BChE-catalyzed reactions are explained in terms of a stepwise mechanism with tetrahedral intermediates. Evidence for a change to a direct displacement mechanism under alkaline conditions is presented.

Expression of dehydratase domains from a polyunsaturated fatty acid synthase increases the production of fatty acids in Escherichia coli

Increasing the production of fatty acids by microbial fermentation remains an important step toward the generation of biodiesel and other portable liquid fuels. In this work, we report an Escherichia coli strain engineered to overexpress a fragment consisting of four dehydratase domains from the polyunsaturated fatty acid (PUFA) synthase enzyme complex from the deep-sea bacterium, Photobacterium profundum. The DH1-DH2-UMA enzyme fragment was excised from its natural context within a multi-enzyme PKS and expressed as a stand-alone protein. Fatty acids were extracted from the cell pellet, esterified with methanol and quantified by GC-MS analysis. Results show that the E. coli strain expressing the DH tetradomain fragment was capable of producing up to a 5-fold increase (80.31mg total FA/L culture) in total fatty acids over the negative control strain lacking the recombinant enzyme. The enhancement in production was observed across the board for all the fatty acids that are typically made by E. coli. The overexpression of the DH tetradomain did not affect E. coli cell growth, thus showing that the observed enhancement in fatty acid production was not a result of effects associated with cell density. The observed enhancement was more pronounced at lower temperatures (3.8-fold at 16°C, 3.5-fold at 22°C and 1.5-fold at 30°C) and supplementation of the media with 0.4% glycerol did not result in an increase in fatty acid production. All these results taken together suggest that either the dehydration of fatty acid intermediates are a limiting step in the E. coli fatty acid biosynthesis machinery, or that the recombinant dehydratase domains used in this study are also capable of catalyzing thioester hydrolysis of the final products. The enzyme in this report is a new tool which could be incorporated into other existing strategies aimed at improving fatty acid production in bacterial fermentations toward accessible biodiesel precursors.