Browse
Submit Data
Databases
API
Help

Dataset Information

7 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Expression of a recombinant, 4'-Phosphopantetheinylated, active M. tuberculosis fatty acid synthase I in E. coli.

ABSTRACT:

Background

Fatty acid synthase 1 (FAS I) from Mycobacterium tuberculosis (Mtb) is an essential protein and a promising drug target. FAS I is a multi-functional, multi-domain protein that is organized as a large (1.9 MDa) homohexameric complex. Acyl intermediates produced during fatty acid elongation are attached covalently to an acyl carrier protein (ACP) domain. This domain is activated by the transfer of a 4'-Phosphopantetheine (4'-PP, also termed P-pant) group from CoA to ACP catalyzed by a 4'-PP transferase, termed acyl carrier protein synthase (AcpS).

Methods

In order to obtain an activated FAS I in E. coli, we transformed E. coli with tagged Mtb fas1 and acpS genes encoded by a separate plasmid. We induced the expression of Mtb FAS I following induction of AcpS expression. FAS I was purified by Strep-Tactin affinity chromatography.

Results

Activation of Mtb FAS I was confirmed by the identification of a bound P-pant group on serine at position 1808 by mass spectrometry. The purified FAS I displayed biochemical activity shown by spectrophotometric analysis of NADPH oxidation and by CoA production, using the Ellman reaction. The purified Mtb FAS I forms a hexameric complex shown by negative staining and cryo-EM.

Conclusion

Purified hexameric and active Mtb FAS I is required for binding and drug inhibition studies and for structure-function analysis of this enzyme. This relatively simple and short procedure for Mtb FAS I production should facilitate studies of this enzyme.

SUBMITTER: Baron S

PROVIDER: S-EPMC6152951 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Human fatty acid synthase: assembling recombinant halves of the fatty acid synthase subunit protein reconstitutes enzyme activity.

Project description:Our model of the native fatty acid synthase (FAS) depicts it as a dimer of two identical multifunctional proteins (Mr approximately 272,000) arranged in an antiparallel configuration so that the active Cys-SH of the beta-ketoacyl synthase of one subunit (where the acyl group is attached) is juxtaposed within 2 A of the pantetheinyl-SH of the second subunit (where the malonyl group is bound). This arrangement generates two active centers for fatty acid synthesis and predicts that if we have two appropriate halves of the monomer, we should be able to reconstitute an active fatty acid-synthesizing site. We cloned, expressed, and purified catalytically active thioredoxin (TRX) fusion proteins of the NH2-terminal half of the human FAS subunit protein (TRX-hFAS-dI; residues 1-1,297; Mr approximately 166) and of the C-terminal half (TRX-hFAS-dII-III; residues 1,296-2,504; Mr approximately 155). Adding equivalent amounts of TRX-hFAS-dI and TRX-hFAS-dII-III to a reaction mixture containing acetyl-CoA, malonyl-CoA, and NADPH resulted in the synthesis of long-chain fatty acids. The rate of synthesis was dependent upon the presence of both recombinant proteins and reached a constant level when they were present in equivalent amounts, indicating that the reconstitution of an active fatty acid-synthesizing site required the presence of every partial activity associated with the subunit protein. Analyses of the product acids revealed myristate to be the most abundant with small amounts of palmitate and stearate, possibly because of the way the fused recombinant proteins interacted with each other so that the thioesterase hydrolyzed the acyl group in its myristoyl state. The successful reconstitution of the human FAS activity from its domain I and domains II and III fully supports our model for the structure-function relationship of FAS in animal tissues.

| S-EPMC24928 | biostudies-literature

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

Project description: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 (80.31 mg 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.

| S-EPMC3894691 | biostudies-literature

Structural and Functional Analysis of E. coli Cyclopropane Fatty Acid Synthase.

Project description:Cell membranes must adapt to different environments. In Gram-negative bacteria, the inner membrane can be remodeled directly by modification of lipids embedded in the bilayer. For example, when Escherichia coli enters stationary phase, cyclopropane fatty acid (CFA) synthase converts most double bonds in unsaturated inner-membrane lipids into cyclopropyl groups. Here we report the crystal structure of E. coli CFA synthase. The enzyme is a dimer in the crystal and in solution, with each subunit containing a smaller N-domain that associates tightly with a larger catalytic C-domain, even following cleavage of the inter-domain linker or co-expression of each individual domain. Efficient catalysis requires dimerization and proper linkage of the two domains. These findings support an avidity-based model in which one subunit of the dimer stabilizes membrane binding, while the other subunit carries out catalysis.

| S-EPMC6125206 | biostudies-literature

Structure of Type-I Mycobacterium tuberculosis fatty acid synthase at 3.3?A resolution.

Project description:Tuberculosis (TB) is a devastating and rapidly spreading disease caused by Mycobacterium tuberculosis (Mtb). Therapy requires prolonged treatment with a combination of multiple agents and interruptions in the treatment regimen result in emergence and spread of multi-drug resistant (MDR) Mtb strains. MDR Mtb poses a significant global health problem, calling for urgent development of novel drugs to combat TB. Here, we report the 3.3?Å resolution structure of the ~2 MDa type-I fatty acid synthase (FAS-I) from Mtb, determined by single particle cryo-EM. Mtb FAS-I is an essential enzymatic complex that contributes to the virulence of Mtb, and thus a prime target for anti-TB drugs. The structural information for Mtb FAS-I we have obtained enables computer-based drug discovery approaches, and the resolution achieved by cryo-EM is sufficient for elucidating inhibition mechanisms by putative small molecular weight inhibitors.

| S-EPMC6155276 | biostudies-literature

Characterization of Mycobacterium smegmatis expressing the Mycobacterium tuberculosis fatty acid synthase I (fas1) gene.

Project description:Unlike most other bacteria, mycobacteria make fatty acids with the multidomain enzyme eukaryote-like fatty acid synthase I (FASI). Previous studies have demonstrated that the tuberculosis drug pyrazinamide and 5-chloro-pyrazinamide target FASI activity. Biochemical studies have revealed that in addition to C(16:0), Mycobacterium tuberculosis FASI synthesizes C(26:0) fatty acid, while the Mycobacterium smegmatis enzyme makes C(24:0) fatty acid. In order to express M. tuberculosis FASI in a rapidly growing Mycobacterium and to characterize the M. tuberculosis FASI in vivo, we constructed an M. smegmatis Deltafas1 strain which contained the M. tuberculosis fas1 homologue. The M. smegmatis Deltafas1 (attB::M. tuberculosis fas1) strain grew more slowly than the parental M. smegmatis strain and was more susceptible to 5-chloro-pyrazinamide. Surprisingly, while the M. smegmatis Deltafas1 (attB::M. tuberculosis fas1) strain produced C(26:0), it predominantly produced C(24:0). These results suggest that the fatty acid elongation that produces C(24:0) or C(26:0) in vivo is due to a complex interaction among FASI, FabH, and FASII and possibly other systems and is not solely due to FASI elongation, as previously suggested by in vitro studies.

| S-EPMC421601 | biostudies-literature

The missing piece of the type II fatty acid synthase system from Mycobacterium tuberculosis.

Project description:The Mycobacterium tuberculosis fatty acid synthase type II (FAS-II) system has the unique property of producing unusually long-chain fatty acids involved in the biosynthesis of mycolic acids, key molecules of the tubercle bacillus. The enzyme(s) responsible for dehydration of (3R)-hydroxyacyl-ACP during the elongation cycles of the mycobacterial FAS-II remained unknown. This step is classically catalyzed by FabZ- and FabA-type enzymes in bacteria, but no such proteins are present in mycobacteria. Bioinformatic analyses and an essentiality study allowed the identification of a candidate protein cluster, Rv0635-Rv0636-Rv0637. Its expression in recombinant Escherichia coli strains leads to the formation of two heterodimers, Rv0635-Rv0636 (HadAB) and Rv0636-Rv0637 (HadBC), which also occurs in Mycobacterium smegmatis, as shown by split-Trp assays. Both heterodimers exhibit the enzymatic properties expected for mycobacterial FAS-II dehydratases: a marked specificity for both long-chain (>or=C(12)) and ACP-linked substrates. Furthermore, they function as 3-hydroxyacyl dehydratases when coupled with MabA and InhA enzymes from the M. tuberculosis FAS-II system. HadAB and HadBC are the long-sought (3R)-hydroxyacyl-ACP dehydratases. The correlation between the substrate specificities of these enzymes, the organization of the orthologous gene cluster in different Corynebacterineae, and the structure of their mycolic acids suggests distinct roles for both heterodimers during the elongation process. This work describes bacterial monofunctional (3R)-hydroxyacyl-ACP dehydratases belonging to the hydratase 2 family. Their original structure and the fact that they are essential for M. tuberculosis survival make these enzymes very good candidates for the development of antimycobacterial drugs.

| S-EPMC1976197 | biostudies-literature

Identification of novel protein domains required for the expression of an active dehydratase fragment from a polyunsaturated fatty acid synthase.

Project description:Polyunsaturated fatty acids (PUFAs) are made in some strains of deep-sea bacteria by multidomain proteins that catalyze condensation, ketoreduction, dehydration, and enoyl-reduction. In this work, we have used the Udwary-Merski Algorithm sequence analysis tool to define the boundaries that enclose the dehydratase (DH) domains in a PUFA multienzyme. Sequence analysis revealed the presence of four areas of high structure in a region that was previously thought to contain only two DH domains as defined by FabA-homology. The expression of the protein fragment containing all four protein domains resulted in an active enzyme, while shorter protein fragments were not soluble. The tetradomain fragment was capable of catalyzing the conversion of crotonyl-CoA to β-hydroxybutyryl-CoA efficiently, as shown by UV absorbance change as well as by chromatographic retention of reaction products. Sequence alignments showed that the two novel domains contain as much sequence conservation as the FabA-homology domains, suggesting that they too may play a functional role in the overall reaction. Structure predictions revealed that all domains belong to the hotdog protein family: two of them contain the active site His70 residue present in FabA-like DHs, while the remaining two do not. Replacing the active site His residues in both FabA domains for Ala abolished the activity of the tetradomain fragment, indicating that the DH activity is contained within the FabA-homology regions. Taken together, these results provide a first glimpse into a rare arrangement of DH domains which constitute a defining feature of the PUFA synthases.

| S-EPMC3719089 | biostudies-literature

High-level expression of a recombinant active microbial transglutaminase in Escherichia coli.

Project description:Bacterial transglutaminases are increasingly required as industrial reagents for in vitro modification of proteins in different fields such as in food processing as well as for enzymatic site-specific covalent conjugation of therapeutic proteins to polyethylene glycol to get derivatives with improved clinical performances. In this work we studied the production in Escherichia coli of a recombinant transglutaminase from Streptomyces mobaraensis (microbial transglutaminase or MTGase) as enzymatically active chimeric forms using different expression systems under the control of both lac promoter or thermoinducible phage lambda promoter.Thermoinducible and constitutive expression vectors were constructed expressing Met-MTGase with chimeric LacZ1-8PNP1-20 or LacZ1-8 fusion protein under different promoters. After transformed in competent Escherichia coli K12 strains were fermented in batch and fed-bach mode in different mediums in order to select the best conditions of expression. The two most performing fusion protein systems namely short thermoinducible LacZ1-8Met-MTGase from NP668/1 and long constitutive LacZ1-8PNP1-20Met-MTGase from NP650/1 has been chosen to compare both efficiency of expression and biochemical qualities of the product. Proteins were extracted, purified to homogeneity and verified as a single peak obtained in RP-HPLC. The LacZ1-8PNP1-20Met-MTGase fusion protein purified from NP650/1 exhibited an activity of 15 U/mg compared to 24 U/mg for the shorter fusion protein purified from NP668/1 cell strain.Combining the experimental data on expression levels and specific activities of purified MTGase fusion proteins, the chimeric LacZ1-8Met-MTGase, which displays an enzymatic activity comparable to the wild-type enzyme, was selected as a candidate for producing microbial transglutaminase for industrial applications.

| S-EPMC4570659 | biostudies-literature

Role of fatty acid synthase in oligodendrocyte myelination

Project description:In the central nervous system, oligodendrocytes encase axons with myelin, a highly organized multilayered membrane structure. Myelin allows the rapid propagation of action potentials along the axons, while also supporting their mantainance metabolically. Fatty acids are basic building blocks for both glyco- and phospholipids, key constituents of cell membranes. Moreover, fatty acids can modify proteins via palmitoylation and activate transcriptional networks, e.g. through the PPARs transcription factors. Due to the high demand of membrane oligodendrocytes face to produce myelin, we hypothesized that they strongly rely upon fatty acid synthesis rather than mostly on their intake from the dietary pool. We tested this hypothesis by deleting the enzyme Fatty Acid Synthase specifically from neonatal oligodendrocyte progenitor cells, in vivo in C57Bl/6 olig2Cre FASN floxed mice. We addressed the consequences of this depletion on oligodendrocytes differentation and myelination potential in the central nervous system. In particular, we analyzed by RNA-seq how lack of FASN affected the transcriptome of optic nerves dissected from P14 mutant (olig2Cre FASN lox/lox) versus control (FASN lox/lox) mice.

2019-05-03 | GSE112725 | GEO

In vitro reconstitution and steady-state analysis of the fatty acid synthase from Escherichia coli.

Project description:Microbial fatty acid derivatives are emerging as promising alternatives to fossil fuel derived transportation fuels. Among bacterial fatty acid synthases (FAS), the Escherichia coli FAS is perhaps the most well studied, but little is known about its steady-state kinetic behavior. Here we describe the reconstitution of E. coli FAS using purified protein components and report detailed kinetic analysis of this reconstituted system. When all ketosynthases are present at 1 ?M, the maximum rate of free fatty acid synthesis of the FAS exceeded 100 ?M/ min. The steady-state turnover frequency was not significantly inhibited at high concentrations of any substrate or cofactor. FAS activity was saturated with respect to most individual protein components when their concentrations exceeded 1 ?M. The exceptions were FabI and FabZ, which increased FAS activity up to concentrations of 10 ?M; FabH and FabF, which decreased FAS activity at concentrations higher than 1 ?M; and holo-ACP and TesA, which gave maximum FAS activity at 30 ?M concentrations. Analysis of the S36T mutant of the ACP revealed that the unusual dependence of FAS activity on holo-ACP concentration was due, at least in part, to the acyl-phosphopantetheine moiety. MALDI-TOF mass spectrometry analysis of the reaction mixture further revealed medium and long chain fatty acyl-ACP intermediates as predominant ACP species. We speculate that one or more of such intermediates are key allosteric regulators of FAS turnover. Our findings provide a new basis for assessing the scope and limitations of using E. coli as a biocatalyst for the production of diesel-like fuels.

| S-EPMC3219124 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data