GO:0141148 enoyl-[acyl-carrier-protein] reductase (NADPH) activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0141148 defines the NADPH-dependent enoyl-[acyl-carrier-protein] reductase activity that reduces a 2,3-saturated acyl-[ACP] to a (2E)-enoyl-[ACP], producing NADP+ and H+.
• This activity is a key regulatory step in bacterial fatty acid biosynthesis and is the target of several antibacterial compounds.
• Multiple enzyme families carry this activity, including FabI (NADH/NADPH dual-specific), FabK, FabV, and a unique cyclopropanating enoyl-ACP reductase.
• In humans, the enoyl-ACP reductase domain is part of the multifunctional fatty acid synthase (FASN), and recent structural work has captured acyl carrier protein shuttling during catalysis.
• The reaction is essential for membrane lipid synthesis and is being explored for biofuel and bioalkane production in cyanobacteria.
• Dysregulation of the human FASN enoyl-ACP reductase step is linked to metabolic and proliferative diseases, making it a target for therapeutic intervention.
Description
Enoyl-[acyl-carrier-protein] reductase (NADPH) activity, encoded by GO:0141148, catalyzes the reduction of a 2,3-saturated acyl-[ACP] to a (2E)-enoyl-[ACP] using NADPH as the electron donor, releasing NADP+ and H+. This activity is a central component of the type II fatty acid synthase (FAS-II) pathway in bacteria and is also present within the multifunctional type I FASN in humans. The reaction is a key regulatory step in fatty acid biosynthesis and is essential for membrane lipid homeostasis. Because of its essentiality in bacteria and its role in human metabolism, this activity has attracted intense interest as a target for antibiotics and as a potential node for metabolic engineering. Researchers studying this activity aim to understand its catalytic mechanism, its regulation, and its potential as a drug target.
enoyl-[acyl-carrier-protein] reductase (NADPH) activity At A Glance
| GO ID | GO:0141148 |
|---|---|
| GO term | enoyl-[acyl-carrier-protein] reductase (NADPH) activity |
| Ontology | molecular_function |
| Synonym | (none) |
| Major function | Reduction of a 2,3-saturated acyl-[ACP] to a (2E)-enoyl-[ACP] using NADPH |
| Reaction direction | a 2,3-saturated acyl-[ACP] + NADP+ = a (2E)-enoyl-[ACP] + H+ + NADPH |
| Cofactor | NADPH (some enzymes also accept NADH) |
| Pathway context | Fatty acid biosynthesis (FAS-II in bacteria, FASN in humans) |
| Representative enzymes | FabI, FabK, FabV, and the enoyl-ACP reductase domain of FASN |
What Is GO:0141148?
GO:0141148 describes the catalysis of the reaction: a 2,3-saturated acyl-[ACP] + NADP+ = a (2E)-enoyl-[ACP] + H+ + NADPH. In other words, the enzyme transfers a hydride from NADPH to the beta-carbon of the acyl chain, creating a trans double bond at the 2,3-position while oxidizing NADPH to NADP+. This activity is specific for acyl chains attached to an acyl-carrier protein (ACP) and uses NADPH as the preferred cofactor, although some enzymes in this class can also accept NADH.
Why Is enoyl-[acyl-carrier-protein] reductase (NADPH) activity Important in Cell Biology?
GO:0141148 is critical because it catalyzes a rate-limiting step in fatty acid biosynthesis, a process required for membrane biogenesis and cell viability. In bacteria, this activity is the target of several classes of antibiotics, and resistance mechanisms often involve mutations in the corresponding enzymes. In humans, the enoyl-ACP reductase activity within FASN is essential for de novo lipogenesis, which is upregulated in many cancers and metabolic disorders. Understanding this activity therefore has direct implications for antimicrobial drug discovery, cancer therapy, and metabolic engineering.
• Essential for bacterial membrane lipid synthesis and cell survival.
• Validated target for antibacterial drug discovery, including against Mycobacterium tuberculosis.
• Represents a key regulatory node in fatty acid biosynthesis.
• Involved in bioalkane production in cyanobacteria, relevant for renewable fuels.
• Human FASN enoyl-ACP reductase domain is linked to cancer and metabolic diseases.
• Enzyme families with this activity include FabI, FabK, FabV, and cyclopropanating enzymes.
• Mutations in the enzyme can confer resistance to inhibitors, informing drug design.
• Structural studies reveal dynamic acyl carrier protein shuttling during catalysis.
• Activity can be regulated by cofactor availability and product inhibition.
• Provides a model system for studying enzyme mechanism and protein-protein interactions.
What Happens During enoyl-[acyl-carrier-protein] reductase (NADPH) activity?
Substrate Binding and Acyl-ACP Recognition
In simple terms: The enzyme grabs the fatty acid chain that is attached to a carrier protein.
The enoyl-ACP reductase binds to its substrate, a 2,3-saturated acyl-[ACP], through interactions with the acyl carrier protein (ACP) and the acyl chain. Structural studies of the human FASN enoyl-ACP reductase domain have captured the ACP shuttling between catalytic centers, highlighting the dynamic nature of substrate delivery. In bacterial FabI, the enzyme forms a ternary complex with the acyl-ACP substrate and the NADPH cofactor.
Hydride Transfer and Double Bond Formation
In simple terms: The enzyme uses NADPH to remove two hydrogen atoms from the fatty acid chain, creating a double bond.
The catalytic mechanism involves a hydride transfer from NADPH to the beta-carbon of the acyl chain, followed by protonation of the alpha-carbon, resulting in the formation of a trans-2,3 double bond. This reaction is stereospecific and requires the correct orientation of the substrate and cofactor. The enzyme's active site residues, such as tyrosine and lysine, facilitate this process.
Product Release and Cofactor Recycling
In simple terms: After the reaction, the enzyme releases the unsaturated fatty acid and the used cofactor.
Following catalysis, the (2E)-enoyl-[ACP] product is released, and NADP+ dissociates from the enzyme. The enzyme can then bind a new NADPH molecule to start another catalytic cycle. In some organisms, the enzyme can also use NADH, albeit with lower efficiency.
Enzyme Families and Variations
In simple terms: Different bacteria use different versions of this enzyme, which affects how drugs work.
The enoyl-ACP reductase activity is found in several distinct enzyme families: FabI (the canonical bacterial enzyme), FabK (a flavoprotein), FabV (found in Vibrio cholerae), and a unique cyclopropanating enzyme. Each family has distinct structural features and cofactor preferences, which impact inhibitor specificity. For example, FabV from Vibrio cholerae defines a new class of enoyl-ACP reductases with a different mechanism.
Key Genes Involved in GO:0141148 enoyl-[acyl-carrier-protein] reductase (NADPH) activity
The following genes encode enzymes that possess enoyl-[acyl-carrier-protein] reductase (NADPH) activity or are directly involved in its function.
| Gene | Major Role | Research Relevance |
|---|---|---|
| fabI | Enoyl-ACP reductase in E. coli; accepts NADH and NADPH | Model enzyme for mechanism and inhibitor studies |
| fabK | Enoyl-ACP reductase in Streptococcus pneumoniae; flavoprotein | Target for antibacterial development |
| fabV | Enoyl-ACP reductase in Vibrio cholerae; new class | Expands understanding of enzyme diversity |
| FASN | Human fatty acid synthase; contains enoyl-ACP reductase domain | Target for cancer and metabolic disease |
| acpP | Acyl carrier protein; delivers substrate to the enzyme | Essential for substrate shuttling |
| fabD | Malonyl-CoA:ACP transacylase; upstream of enoyl-ACP reductase | Provides substrate for the pathway |
| fabH | Beta-ketoacyl-ACP synthase III; initiates fatty acid synthesis | Upstream enzyme in FAS-II |
| fabB | Beta-ketoacyl-ACP synthase I; elongates fatty acids | Downstream of enoyl-ACP reductase |
| fabF | Beta-ketoacyl-ACP synthase II; elongates fatty acids | Downstream of enoyl-ACP reductase |
| fabG | Beta-ketoacyl-ACP reductase; reduces keto group | Upstream of enoyl-ACP reductase |
| fabA | Beta-hydroxyacyl-ACP dehydratase; forms enoyl-ACP | Produces substrate for enoyl-ACP reductase |
| fabZ | Beta-hydroxyacyl-ACP dehydratase; forms enoyl-ACP | Produces substrate for enoyl-ACP reductase |
| inhA | Enoyl-ACP reductase in Mycobacterium tuberculosis | Target of isoniazid and novel inhibitors |
| kasA | Beta-ketoacyl-ACP synthase in M. tuberculosis | Component of mycolic acid synthesis |
| kasB | Beta-ketoacyl-ACP synthase in M. tuberculosis | Component of mycolic acid synthesis |
| acpM | Acyl carrier protein in M. tuberculosis | Delivers substrate to InhA |
| ndh | NADH dehydrogenase; affects NADPH/NADH balance | Indirect regulator of enoyl-ACP reductase |
How Is enoyl-[acyl-carrier-protein] reductase (NADPH) activity Regulated?
The activity of enoyl-[acyl-carrier-protein] reductase is regulated at multiple levels. In E. coli, FabI is inhibited by palmitoyl-CoA, a long-chain acyl-CoA, suggesting feedback regulation by end products of fatty acid synthesis. The enzyme's activity also depends on the availability of NADPH and NADH, which are influenced by central metabolism. In Mycobacterium tuberculosis, InhA is activated by the transcriptional regulator FadR and is the target of the prodrug isoniazid, which requires activation by KatG. Additionally, in cyanobacteria, the expression of enoyl-ACP reductase genes is coordinated with other fatty acid synthesis genes to meet the demand for membrane lipids and bioalkane production.
enoyl-[acyl-carrier-protein] reductase (NADPH) activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| inhA | Tuberculosis, isoniazid resistance | M. tuberculosis knockout or point-mutant strains |
| fabI | Bacterial infections, triclosan resistance | E. coli or S. aureus knockout and overexpression |
| fabK | Streptococcus pneumoniae infections | S. pneumoniae fabK deletion mutants |
| fabV | Vibrio cholerae infections | V. cholerae fabV knockout |
| FASN | Cancer, obesity, NAFLD | Human cancer cell lines with FASN knockout or knockdown |
Tuberculosis and Antibiotic Resistance
Mycobacterium tuberculosis enoyl-ACP reductase InhA is the primary target of isoniazid, a first-line anti-tuberculosis drug. Mutations in inhA or its promoter lead to isoniazid resistance, and novel 4-aminoquinolines have been developed to inhibit InhA and overcome resistance. This highlights the clinical importance of enoyl-ACP reductase as a drug target.
Bacterial Infections and FabI Inhibitors
FabI is essential in many bacterial pathogens, including Staphylococcus aureus and Escherichia coli, and is the target of broad-spectrum antibiotics such as triclosan. However, some bacteria use alternative enzymes like FabK or FabV, which are not inhibited by FabI-targeting drugs, leading to intrinsic resistance. Understanding these variations is crucial for developing new antibiotics.
Cancer and Metabolic Disorders
Human fatty acid synthase (FASN) contains an enoyl-ACP reductase domain, and its overexpression is observed in many cancers, including breast, prostate, and colon cancer. Inhibitors of FASN enoyl-ACP reductase activity are being explored as anticancer agents. Additionally, FASN is linked to obesity and non-alcoholic fatty liver disease, making this activity relevant to metabolic disorders.
Biofuel and Industrial Applications
Cyanobacterial enoyl-ACP reductases are part of pathways engineered for bioalkane production, offering a sustainable route to biofuels. Modulating this activity can enhance yields of alkanes and other fatty acid-derived products.
From enoyl-[acyl-carrier-protein] reductase (NADPH) activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| What is the essentiality of fabI in E. coli? | CRISPR knockout of fabI (conditional) |
| How does InhA mutation confer isoniazid resistance? | Point mutation knock-in in M. tuberculosis |
| Can FabK complement FabI deficiency? | Knock-in of fabK into E. coli fabI mutant |
| What is the role of FASN enoyl-ACP reductase in cancer? | Overexpression of FASN in cancer cell lines |
| How does FabV contribute to Vibrio cholerae virulence? | fabV knockout in V. cholerae |
| Can cyanobacterial enoyl-ACP reductase enhance bioalkane production? | Overexpression in Synechococcus |
How to Study the enoyl-[acyl-carrier-protein] reductase (NADPH) activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| NADPH oxidation assay | Enzyme activity | Inhibitor screening |
| X-ray crystallography | Three-dimensional structure | Mechanism and drug design |
| CRISPR knockout | Gene essentiality | Target validation |
| Lipidomics | Fatty acid composition | Pathway flux analysis |
| Isothermal titration calorimetry | Binding affinity | Cofactor and inhibitor binding |
| Site-directed mutagenesis | Residue function | Catalytic mechanism |
| High-throughput screening | Inhibitor potency | Drug discovery |
Enzymatic Assays for Enoyl-ACP Reductase Activity
Direct measurement of enoyl-ACP reductase activity is typically performed using spectrophotometric assays that monitor the oxidation of NADPH at 340 nm. These assays use purified enzyme and acyl-ACP substrates, and can be adapted for high-throughput screening of inhibitors. For enzymes with dual cofactor specificity, both NADH and NADPH can be tested.
Structural Biology and Mechanism
X-ray crystallography and cryo-electron microscopy have been used to determine the structures of enoyl-ACP reductases from bacteria and humans, revealing the active site architecture and conformational changes during catalysis. These studies guide the design of specific inhibitors and help understand resistance mutations.
Genetic and CRISPR Screens
CRISPR knockout and knockdown screens can identify genes that are essential for bacterial growth or that modulate sensitivity to enoyl-ACP reductase inhibitors. In human cells, CRISPR screens can uncover synthetic lethal interactions with FASN inhibition.
Metabolic and Lipidomics Profiling
Mass spectrometry-based lipidomics can quantify changes in fatty acid profiles upon modulation of enoyl-ACP reductase activity, providing insights into pathway flux and compensatory mechanisms. This approach is valuable for engineering strains for biofuel production.
How CRISPR Can Be Used to Study GO:0141148 enoyl-[acyl-carrier-protein] reductase (NADPH) activity
Knockout
CRISPR knockout of enoyl-ACP reductase genes such as fabI in E. coli or inhA in M. tuberculosis can be used to study gene essentiality and to validate drug targets. Conditional knockouts are often necessary for essential genes.
Point Mutation
Point mutations in enoyl-ACP reductase genes, such as those found in isoniazid-resistant M. tuberculosis, can be introduced using CRISPR base editing or homology-directed repair to study resistance mechanisms and to test new inhibitors.
Knock-in
Knock-in of alternative enoyl-ACP reductase genes (e.g., fabK into E. coli) can be used to study functional complementation and to understand species-specific differences in drug susceptibility.
Overexpression
CRISPR activation (CRISPRa) or plasmid-based overexpression of enoyl-ACP reductase genes can be used to investigate the effects of increased enzyme levels on fatty acid synthesis, membrane composition, and biofuel production.
How EDITGENE Supports enoyl-[acyl-carrier-protein] reductase (NADPH) activity Research
Researchers studying enoyl-[acyl-carrier-protein] reductase (NADPH) activity-related genes often need to determine whether a candidate gene is causally involved in fatty acid synthesis, drug resistance, or metabolic disease. EDITGENE provides a comprehensive suite of CRISPR-based services to accelerate this research, from gene knockout to precise point mutations and overexpression.
Contact EDITGENE today to design your custom CRISPR model for enoyl-[acyl-carrier-protein] reductase (NADPH) activity research.
Frequently Asked Questions About enoyl-[acyl-carrier-protein] reductase (NADPH) activity
What is enoyl-[acyl-carrier-protein] reductase (NADPH) activity?
It is a molecular function defined by GO:0141148 that catalyzes the reduction of a 2,3-saturated acyl-[ACP] to a (2E)-enoyl-[ACP] using NADPH, producing NADP+ and H+.
What genes are involved in enoyl-[acyl-carrier-protein] reductase (NADPH) activity?
Key genes include fabI, fabK, fabV, inhA, and FASN, which encode enzymes with this activity.
Why is enoyl-[acyl-carrier-protein] reductase important for bacteria?
It is essential for fatty acid biosynthesis and membrane lipid formation, making it a target for antibiotics.
How is enoyl-[acyl-carrier-protein] reductase regulated?
It is regulated by feedback inhibition (e.g., palmitoyl-CoA), cofactor availability, and transcriptional control.
What diseases are associated with enoyl-[acyl-carrier-protein] reductase?
Tuberculosis, other bacterial infections, cancer, and metabolic disorders are linked to this activity.
What is the difference between FabI, FabK, and FabV?
They are distinct enzyme families with different structures and cofactor preferences, affecting drug susceptibility.
Can enoyl-[acyl-carrier-protein] reductase be used for biofuel production?
Yes, cyanobacterial enoyl-ACP reductases are part of engineered pathways for bioalkane production.
How can CRISPR be used to study enoyl-[acyl-carrier-protein] reductase?
CRISPR knockout, point mutation, knock-in, and overexpression can be used to study gene function and drug resistance.
What methods measure enoyl-[acyl-carrier-protein] reductase activity?
Spectrophotometric NADPH oxidation assays, structural biology, and lipidomics are commonly used.
Is enoyl-[acyl-carrier-protein] reductase a good drug target?
Yes, it is a validated target for antibacterial and anticancer drug development.
Conclusion
Enoyl-[acyl-carrier-protein] reductase (NADPH) activity (GO:0141148) is a fundamental enzymatic activity in fatty acid biosynthesis with broad implications for bacterial physiology, human metabolism, and biotechnology. Its essentiality in bacteria and its role in human FASN make it a prime target for therapeutic intervention and metabolic engineering. Continued research using CRISPR-based models and advanced biochemical methods will further illuminate its mechanism and regulation, paving the way for new drugs and biotechnological applications.
References
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- 3. Paz JD et al.. 2023. Novel 4-aminoquinolines: Synthesis, inhibition of the Mycobacterium tuberculosis enoyl-acyl carrier protein reductase, antitubercular activity, SAR, and preclinical evaluation.. Eur J Med Chem 245(Pt 1):114908 PMID: 36435016
- 4. Arai M et al.. 2018. Cyanobacterial Enzymes for Bioalkane Production.. Adv Exp Med Biol 1080:119-154 PMID: 30091094
- 5. Massengo-Tiassé RP et al.. 2008. Vibrio cholerae FabV defines a new class of enoyl-acyl carrier protein reductase.. J Biol Chem 283(3):1308-1316 PMID: 18032386
- 6. Bergler H et al.. 1996. The enoyl-[acyl-carrier-protein] reductase (FabI) of Escherichia coli, which catalyzes a key regulatory step in fatty acid biosynthesis, accepts NADH and NADPH as cofactors and is inhibited by palmitoyl-CoA.. Eur J Biochem 242(3):689-94 PMID: 9022698
- 7. Khare D et al.. 2015. Structural Basis for Cyclopropanation by a Unique Enoyl-Acyl Carrier Protein Reductase.. Structure 23(12):2213-2223 PMID: 26526850
- 8. Schultz K et al.. 2025. Snapshots of acyl carrier protein shuttling in human fatty acid synthase.. Nature 641(8062):520-528 PMID: 39979457
