Browse
Submit Data
Databases
API
Help

Dataset Information

24 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Escherichia coli FabG 3-ketoacyl-ACP reductase proteins lacking the assigned catalytic triad residues are active enzymes.

ABSTRACT: The FabG 3-ketoacyl-acyl carrier protein (ACP) reductase of Escherichia coli has long been thought to be a classical member of the short-chain alcohol dehydrogenase/reductase (SDR) family. FabG catalyzes the essential 3-ketoacyl-ACP reduction step in the FAS II fatty acid synthesis pathway. Site-directed mutagenesis studies of several other SDR enzymes has identified three highly conserved amino acid residues, Ser, Tyr, and Lys, as the catalytic triad. Structural analyses of E. coli FabG suggested the triad S138-Y151-K155 to form a catalytically competent active site. To test this hypothesis, we constructed a series of E. coli FabG mutants and tested their 3-ketoacyl-ACP reductase activities both in vivo and in vitro. Our data show that plasmid-borne FabG mutants, including the double and triple mutants, restored growth of E. coli and Salmonella enterica fabG temperature-sensitive mutant strains under nonpermissive conditions. In vitro assays demonstrated that all of the purified FabG mutant proteins maintained fatty acid synthetic ability, although the activities of the single mutant proteins were 20% to 50% lower than that of wildtype FabG. The S138A, Y151F, and K155A residue substitutions were confirmed by tandem mass spectral sequencing of peptides that spanned all three residues. We conclude that FabG is not a classical short-chain alcohol dehydrogenase/reductase, suggesting that an alternative mode of 3-ketoacyl-ACP reduction awaits discovery.

SUBMITTER: Hu Z

PROVIDER: S-EPMC7973133 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Tachyplesin Causes Membrane Instability That Kills Multidrug-Resistant Bacteria by Inhibiting the 3-Ketoacyl Carrier Protein Reductase FabG.

Project description:Tachyplesin is a type of cationic β-hairpin antimicrobial peptide discovered in horseshoe crab approximately 30 years ago that is well known for both its potential antimicrobial activities against multidrug-resistant bacteria and its cytotoxicity to mammalian cells. Though its physical interactions with artificial membranes have been well studied, details of its physiological mechanism of action the physiological consequences of its action remain limited. By using the DNA-binding fluorescent dye propidium iodide to monitor membrane integrity, confocal microscopy to assess the intracellular location of FITC-tagged tachyplesin, and RNA sequencing of the differentially expressed genes in four Gram-negative bacteria (Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa) treated with lethal or sublethal concentrations of tachyplesin, we found that compared with levofloxacin-treated bacteria, tachyplesin-treated bacteria showed significant effects on the pathways underlying unsaturated fatty acid biosynthesis. Notably, RNA levels of the conserved and essential 3-ketoacyl carrier protein reductase in this pathway (gene FabG) were elevated in all of the four bacteria after tachyplesin treatment. In vitro tests including surface plasmon resonance and enzyme activity assays showed that tachyplesin could bind and inhibit 3-ketoacyl carrier protein reductase, which was consistent with molecular docking prediction results. As unsaturated fatty acids are important for membrane fluidity, our results provided one possible mechanism to explain how tachyplesin kills bacteria and causes cytotoxicity by targeting membranes, which may be helpful for designing more specific and safer antibiotics based on the function of tachyplesin.

| S-EPMC5938390 | biostudies-literature

Role of Serine140 in the mode of action of Mycobacterium tuberculosis ?-ketoacyl-ACP Reductase (MabA).

Project description:BACKGROUND: Tuberculosis (TB) still remains one of the most deadly infectious diseases in the world. Mycobacterium tuberculosis ?-ketoacyl-ACP Reductase (MabA) is a member of the fatty acid elongation system type II, providing precursors of mycolic acids that are essential to the bacterial cell growth and survival. MabA has been shown to be essential for M. tuberculosis survival and to play a role in intracellular signal transduction of bacilli. FINDINGS: Here we describe site-directed mutagenesis, recombinant protein expression and purification, steady-state kinetics, fluorescence spectroscopy, and molecular modeling for S140T and S140A mutant MabA enzymes. No enzyme activity could be detected for S140T and S140A. Although the S140T protein showed impaired NADPH binding, the S140A mutant could bind to NADPH. Computational predictions for NADPH binding affinity to WT, S140T and S140A MabA proteins were consistent with fluorescence spectroscopy data. CONCLUSIONS: The results suggest that the main role of the S140 side chain of MabA is in catalysis. The S140 side chain appears to also play an indirect role in NADPH binding. Interestingly, NADPH titrations curves shifted from sigmoidal for WT to hyperbolic for S140A, suggesting that the S140 residue may play a role in displacing the pre-existing equilibrium between two forms of MabA in solution. The results here reported provide a better understanding of the mode of action of MabA that should be useful to guide the rational (function-based) design of inhibitors of MabA enzyme activity which, hopefully, could be used as lead compounds with anti-TB action.

| S-EPMC3519566 | biostudies-literature

Crystallization and X-ray diffraction analysis of the beta-ketoacyl-acyl carrier protein reductase FabG from Aquifex aeolicus VF5.

Project description:The gene product of fabG from Aquifex aeolicus has been heterologously expressed in Escherichia coli. Purification of the protein took place using anion-exchange and size-exclusion chromatography and the protein was then crystallized. Diffraction data were collected to a maximum resolution of 1.8 A and the initial phases were determined by molecular replacement. The A. aeolicus FabG protein is a putative beta-ketoacyl-acyl carrier protein reductase. Structure-function studies of this protein are being performed as part of a larger project investigating naturally occurring deviations from highly conserved residues within the short-chain oxidoreductase (SCOR) family.

| S-EPMC2330128 | biostudies-literature

Insights into ?-ketoacyl-chain recognition for ?-ketoacyl-ACP utilizing AHL synthases.

Project description:Beta-ketoacyl-ACP utilizing enzymes in fatty acid, polyketide and acyl-homoserine lactone biosynthetic pathways are important targets for developing antimicrobial, anticancer and antiparasitic compounds. Published reports on successful isolation of beta-ketoacyl-ACPs in a laboratory remain scarce to date and thus most beta-ketoacyl-ACP utilizing enzymes are routinely characterized using small molecule substrates in lieu of the bonafide 3-oxoacyl-ACPs. We report the systematic investigation into the electronic, geometric and spatial aspects of beta-ketoacyl-chain recognition to develop 3-oxoacyl-ACP substrate mimics for two beta-ketoacyl-ACP utilizing quorum signal synthases.

| S-EPMC6124316 | biostudies-literature

Substrate recognition by β-ketoacyl-ACP synthases.

Project description:β-Ketoacyl-ACP synthase (KAS) enzymes catalyze Claisen condensation reactions in the fatty acid biosynthesis pathway. These reactions follow a ping-pong mechanism in which a donor substrate acylates the active site cysteine residue after which the acyl group is condensed with the malonyl-ACP acceptor substrate to form a β-ketoacyl-ACP. In the priming KASIII enzymes the donor substrate is an acyl-CoA while in the elongating KASI and KASII enzymes the donor is an acyl-ACP. Although the KASIII enzyme in Escherichia coli (ecFabH) is essential, the corresponding enzyme in Mycobacterium tuberculosis (mtFabH) is not, suggesting that the KASI or II enzyme in M. tuberculosis (KasA or KasB, respectively) must be able to accept a CoA donor substrate. Since KasA is essential, the substrate specificity of this KASI enzyme has been explored using substrates based on phosphopantetheine, CoA, ACP, and AcpM peptide mimics. This analysis has been extended to the KASI and KASII enzymes from E. coli (ecFabB and ecFabF) where we show that a 14-residue malonyl-phosphopantetheine peptide can efficiently replace malonyl-ecACP as the acceptor substrate in the ecFabF reaction. While ecFabF is able to catalyze the condensation reaction when CoA is the carrier for both substrates, the KASI enzymes ecFabB and KasA have an absolute requirement for an ACP substrate as the acyl donor. Provided that this requirement is met, variation in the acceptor carrier substrate has little impact on the k(cat)/K(m) for the KASI reaction. For the KASI enzymes we propose that the binding of ecACP (AcpM) results in a conformational change that leads to an open form of the enzyme to which the malonyl acceptor substrate binds. Finally, the substrate inhibition observed when palmitoyl-CoA is the donor substrate for the KasA reaction has implications for the importance of mtFabH in the mycobacterial FASII pathway.

| S-EPMC3361727 | biostudies-literature

Expanding the Catalytic Triad in Epoxide Hydrolases and Related Enzymes.

Project description:Potato epoxide hydrolase 1 exhibits rich enantio- and regioselectivity in the hydrolysis of a broad range of substrates. The enzyme can be engineered to increase the yield of optically pure products as a result of changes in both enantio- and regioselectivity. It is thus highly attractive in biocatalysis, particularly for the generation of enantiopure fine chemicals and pharmaceuticals. The present work aims to establish the principles underlying the activity and selectivity of the enzyme through a combined computational, structural, and kinetic study using the substrate trans-stilbene oxide as a model system. Extensive empirical valence bond simulations have been performed on the wild-type enzyme together with several experimentally characterized mutants. We are able to computationally reproduce the differences between the activities of different stereoisomers of the substrate and the effects of mutations of several active-site residues. In addition, our results indicate the involvement of a previously neglected residue, H104, which is electrostatically linked to the general base H300. We find that this residue, which is highly conserved in epoxide hydrolases and related hydrolytic enzymes, needs to be in its protonated form in order to provide charge balance in an otherwise negatively charged active site. Our data show that unless the active-site charge balance is correctly treated in simulations, it is not possible to generate a physically meaningful model for the enzyme that can accurately reproduce activity and selectivity trends. We also expand our understanding of other catalytic residues, demonstrating in particular the role of a noncanonical residue, E35, as a "backup base" in the absence of H300. Our results provide a detailed view of the main factors driving catalysis and regioselectivity in this enzyme and identify targets for subsequent enzyme design efforts.

| S-EPMC4613740 | biostudies-literature

Gating mechanism of elongating β-ketoacyl-ACP synthases.

Project description:Carbon-carbon bond forming reactions are essential transformations in natural product biosynthesis. During de novo fatty acid and polyketide biosynthesis, β-ketoacyl-acyl carrier protein (ACP) synthases (KS), catalyze this process via a decarboxylative Claisen-like condensation reaction. KSs must recognize multiple chemically distinct ACPs and choreograph a ping-pong mechanism, often in an iterative fashion. Here, we report crystal structures of substrate mimetic bearing ACPs in complex with the elongating KSs from Escherichia coli, FabF and FabB, in order to better understand the stereochemical features governing substrate discrimination by KSs. Complemented by molecular dynamics (MD) simulations and mutagenesis studies, these structures reveal conformational states accessed during KS catalysis. These data taken together support a gating mechanism that regulates acyl-ACP binding and substrate delivery to the KS active site. Two active site loops undergo large conformational excursions during this dynamic gating mechanism and are likely evolutionarily conserved features in elongating KSs.

| S-EPMC7138838 | biostudies-literature

Platensimycin activity against mycobacterial beta-ketoacyl-ACP synthases.

Project description:BackgroundThere is an urgent need for the discovery and development of new drugs against Mycobacterium tuberculosis, the causative agent of tuberculosis, especially due to the recent emergence of multi-drug and extensively-drug resistant strains. Herein, we have examined the susceptibility of mycobacteria to the natural product platensimycin.Methods and findingsWe have demonstrated that platensimycin has bacteriostatic activity against the fast growing Mycobacterium smegmatis (MIC = 14 microg/ml) and against Mycobacterium tuberculosis (MIC = 12 microg/ml). Growth in the presence of paltensimycin specifically inhibited the biosynthesis of mycolic acids suggesting that the antibiotic targeted the components of the mycolate biosynthesis complex. Given the inhibitory activity of platensimycin against beta-ketoacyl-ACP synthases from Staphylococcus aureus, M. tuberculosis KasA, KasB or FabH were overexpressed in M. smegmatis to establish whether these mycobacterial KAS enzymes were targets of platensimycin. In M. smegmatis overexpression of kasA or kasB increased the MIC of the strains from 14 microg/ml, to 30 and 124 microg/ml respectively. However, overexpression of fabH on did not affect the MIC. Additionally, consistent with the overexpression data, in vitro assays using purified proteins demonstrated that platensimycin inhibited Mt-KasA and Mt-KasB, but not Mt-FabH.SignificanceOur results have shown that platensimycin is active against mycobacterial KasA and KasB and is thus an exciting lead compound against M. tuberculosis and the development of new synthetic analogues.

| S-EPMC2707616 | biostudies-literature

Expression of 3-ketoacyl-acyl carrier protein reductase (fabG) genes enhances production of polyhydroxyalkanoate copolymer from glucose in recombinant Escherichia coli JM109.

Project description:Polyhydroxyalkanoates (PHAs) are biologically produced polyesters that have potential application as biodegradable plastics. Especially important are the short-chain-length-medium-chain-length (SCL-MCL) PHA copolymers, which have properties ranging from thermoplastic to elastomeric, depending on the ratio of SCL to MCL monomers incorporated into the copolymer. Because of the potential wide range of applications for SCL-MCL PHA copolymers, it is important to develop and characterize metabolic pathways for SCL-MCL PHA production. In previous studies, coexpression of PHA synthase genes and the 3-ketoacyl-acyl carrier protein reductase gene (fabG) in recombinant Escherichia coli has been shown to enhance PHA production from related carbon sources such as fatty acids. In this study, a new fabG gene from Pseudomonas sp. 61-3 was cloned and its gene product characterized. Results indicate that the Pseudomonas sp. 61-3 and E. coli FabG proteins have different substrate specificities in vitro. The current study also presents the first evidence that coexpression of fabG genes from either E. coli or Pseudomonas sp. 61-3 with fabH(F87T) and PHA synthase genes can enhance the production of SCL-MCL PHA copolymers from nonrelated carbon sources. Differences in the substrate specificities of the FabG proteins were reflected in the monomer composition of the polymers produced by recombinant E. coli. SCL-MCL PHA copolymer isolated from a recombinant E. coli strain had improved physical properties compared to the SCL homopolymer poly-3-hydroxybutyrate. This study defines a pathway to produce SCL-MCL PHA copolymer from the fatty acid biosynthesis that may impact on PHA production in recombinant organisms.

| S-EPMC1183366 | biostudies-literature

Evolution of acyl-ACP-thioesterases and ?-ketoacyl-ACP-synthases revealed by protein-protein interactions.

Project description:The fatty acid synthase (FAS) is a conserved primary metabolic enzyme complex capable of tolerating cross-species engineering of domains for the development of modified and overproduced fatty acids. In eukaryotes, acyl-acyl carrier protein thioesterases (TEs) off-load mature cargo from the acyl carrier protein (ACP), and plants have developed TEs for short/medium-chain fatty acids. We showed that engineering plant TEs into the green microalga Chlamydomonas reinhardtii does not result in the predicted shift in fatty acid profile. Since fatty acid biosynthesis relies on substrate recognition and protein-protein interactions between the ACP and its partner enzymes, we hypothesized that plant TEs and algal ACP do not functionally interact. Phylogenetic analysis revealed major evolutionary differences between FAS enzymes, including TEs and ketoacyl synthases (KSs), in which the former is present only in some species, whereas the latter is present in all, and has a common ancestor. In line with these results, TEs appeared to be selective towards their ACP partners whereas KSs showed promiscuous behavior across bacterial, plant and algal species. Based on phylogenetic analyses, in silico docking, in vitro mechanistic crosslinking and in vivo algal engineering, we propose that phylogeny can predict effective interactions between ACPs and partner enzymes.

| S-EPMC4125210 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

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