Recombinant Saccharomyces cerevisiae Enoyl reductase TSC13 (TSC13)
Description
Basic Protein Information
TSC13, encoded by the TSC13/YDL015c gene located on chromosome 4 in Saccharomyces cerevisiae, is a membrane-bound protein consisting of 310 amino acid residues . The protein functions as an enoyl reductase that specifically catalyzes the reduction of trans-2-enoyl-CoA intermediates during fatty acid elongation . This enzyme is classified under oxidoreductases, acting specifically on the CH-CH group of donors .
Protein Structure and Localization
TSC13 is a multi-pass membrane protein primarily localized to the endoplasmic reticulum membrane . Interestingly, it shows enrichment at specific cellular junctions, particularly at novel structures marking nuclear-vacuolar junctions . This specialized localization pattern suggests potential regulatory roles beyond its catalytic function in fatty acid metabolism.
The full amino acid sequence of TSC13 has been determined to be: MPITIKSRSKGLRDTEIDLSKKPTLDDVLKKISANNHNISKYRIRLTYKKESKQVPVISE SFFQEEADDSMEFFIKDLGPQISWRLVFFCEYLGPVLVHSLFYYLSTIPTVVDRWHSASS DYNPFLNRVAYFLILGHYGKRLFETLFVHQFSLATMPIFNLFKNCFHYWVLSGLISFGYF GYGFPFGNAKLFKYYSYLKLDDLSTLIGLFVLSELWNFYCHIKLRLWGDYQKKHGNAKIR VPLNQGIFNLFVAPNYTFEVWSWIWFTFVFKFNLFAVLFLTVSTAQMYAWAQKKNKKYHT RRAFLIPFVF .
Production of Recombinant TSC13
Recombinant production of TSC13 typically involves heterologous expression in Escherichia coli systems with N-terminal histidine tags to facilitate purification . The recombinant protein maintains the full 310 amino acid sequence of the native protein and is generally supplied as a lyophilized powder for experimental applications . The expression and purification of functional recombinant TSC13 has enabled detailed mechanistic studies of this essential enzyme.
Role in Fatty Acid Elongation
TSC13 plays a critical role in the microsomal membrane-bound fatty acid elongation system that produces very long-chain fatty acids (VLCFAs) . These VLCFAs, defined as fatty acids with 21 or more carbon atoms, serve as essential precursors for more complex lipids, including ceramides and sphingolipids . The elongation system functions by sequentially adding two-carbon units to palmitate (C16:0) produced by cytosolic fatty acid synthase .
Catalytic Function
Within the fatty acid elongation cycle, TSC13 specifically catalyzes the final reduction step in each elongation cycle . This reaction involves the NADPH-dependent reduction of trans-2-enoyl-CoA intermediates to the corresponding saturated acyl-CoAs . This enzymatic step is essential for completing each round of the elongation cycle, allowing for the subsequent addition of further two-carbon units.
Integration in the Cellular Lipid Network
Beyond its direct role in fatty acid elongation, TSC13 is integrated into broader lipid metabolism networks. The VLCFAs produced through the action of TSC13 are essential components of sphingolipids, which have crucial roles in cell signaling, membrane structure, and stress responses . Additionally, TSC13 is required for normal biogenesis of piecemeal microautophagy of the nucleus during nutrient stress conditions .
Catalytic Mechanism
Recent studies have provided significant insights into the catalytic mechanism of TSC13. Research has identified several critical residues essential for the enzyme's function . Most notably, E91 and Y256 appear to be crucial for catalytic activity, with mutational studies showing substantially impaired function in E91A and Y256A variants .
Based on biochemical analyses and predicted three-dimensional structures, researchers have proposed that Y256 of TSC13 serves as the catalytic residue that supplies a proton to trans-2-enoyl-CoA substrates during the reduction reaction . This mechanistic insight provides a foundation for understanding how the enzyme performs its essential function in fatty acid elongation.
Protein-Protein Interactions
TSC13 operates as part of a multi-enzyme complex in the endoplasmic reticulum. Co-immunoprecipitation studies have demonstrated that TSC13 physically interacts with Elo2p and Elo3p, which are elongases that catalyze the first condensation step of the fatty acid elongation cycle . This physical interaction suggests a coordinated action of the elongation machinery, with the different enzymatic components organized into a functional complex.
The significance of these interactions is highlighted by findings that the activity of fatty acid elongases is reduced in the absence of functional TSC13 . Similarly, TSC13 variants with reduced function, such as the E91A mutant, show diminished interaction with Elo2 and Elo3 proteins, correlating with reduced elongase activity . These observations underscore the importance of protein-protein interactions for the optimal functioning of the entire elongation machinery.
Comparison with Mammalian Homologs
TSC13 has a mammalian homolog called TECR (Trans-2-Enoyl-CoA Reductase) . Comparative studies between TSC13 and TECR have identified conserved catalytic residues, with E94A and Y248A mutants of human TECR corresponding to E91A and Y256A mutants of TSC13, respectively . These mammalian mutants similarly show reduced enzymatic activity, indicating a conserved catalytic mechanism across species.
Recombinant Production Systems
Recombinant TSC13 has been successfully expressed in E. coli expression systems . The standard approach involves using bacterial hosts to express the full-length protein (amino acids 1-310) with an N-terminal histidine tag for purification purposes . The recombinant protein is typically supplied in a Tris/PBS-based buffer containing 6% trehalose at pH 8.0, and is recommended to be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL .
Assay Methods for Activity Assessment
Several experimental approaches have been developed to assess TSC13 activity:
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In vitro fatty acid elongation assays using purified recombinant TSC13 protein to measure the reduction of trans-2-enoyl-CoA substrates .
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Growth complementation analysis in yeast strains with compromised TSC13 function, providing an in vivo assessment of enzymatic activity .
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Measurement of ceramide levels as an indirect indicator of TSC13 function, since ceramides incorporate VLCFAs produced through the elongation cycle .
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Deuterium-sphingosine labeling techniques that allow for tracking the metabolic flow through pathways dependent on TSC13 activity .
These complementary approaches provide a comprehensive toolkit for investigating the function and properties of recombinant TSC13 under various experimental conditions.
Engineering Approaches
As TSC13 is an essential enzyme for yeast viability and cannot be deleted, researchers have explored alternative approaches to mitigate its unwanted side activities while preserving its essential functions . Site saturation mutagenesis has been employed to identify amino acid changes that might reduce the side reaction without compromising the natural function of the enzyme . While some mutations have shown promise in slightly increasing flavonoid production, completely eliminating the side reaction remains challenging .
Potential Applications in Lipid Engineering
Given its central role in VLCFA production, recombinant TSC13 holds potential for applications in lipid engineering. Modulating TSC13 activity could potentially allow for the controlled production of specific VLCFA profiles, with applications in both basic research and industrial biotechnology.
Critical Residues and Their Functions
Recent mutational studies have provided valuable insights into the structure-function relationships in TSC13. Table 1 summarizes key findings related to critical residues and their proposed functions.
Table 1: Critical Residues in TSC13 and Their Proposed Functions
| Residue | Mutation | Effect on Activity | Proposed Function |
|---|---|---|---|
| E91 | E91A | Severely reduced | Critical for catalysis, potentially involved in substrate binding or positioning |
| Y256 | Y256A | Severely reduced | Proposed as the catalytic residue that supplies a proton to trans-2-enoyl-CoA |
| Various | Multiple | Variable reduction | Nine residues identified with reduced activity when mutated to alanine |
Impact of Mutations on Protein Interactions
Mutations in TSC13 not only affect its catalytic activity but also influence its interactions with other components of the fatty acid elongation machinery. The E91A mutation leads to reduced interaction with the elongases Elo2 and Elo3, suggesting that this residue may be involved in protein-protein interactions within the elongation complex .
Evolutionary Conservation
Comparative analysis between yeast TSC13 and mammalian TECR has revealed evolutionary conservation of key catalytic residues. The E91 and Y256 residues in yeast TSC13 correspond to E94 and Y248 in human TECR, and mutations at these positions result in similar functional impairments . This conservation underscores the fundamental importance of these residues for the catalytic mechanism of enoyl reductases across species.
Ongoing Research Challenges
Despite significant advances in understanding TSC13, several research challenges remain. The complete three-dimensional structure of the protein has not yet been experimentally determined, limiting our understanding of its precise catalytic mechanism. Additionally, the specific molecular details of how TSC13 coordinates with other components of the fatty acid elongation machinery still require further elucidation.
Potential Therapeutic Relevance
While this review focuses on the yeast enzyme, research on TSC13 has broader implications. The mammalian homolog TECR has been implicated in neurological disorders, with mutations linked to conditions such as nonsyndromic mental retardation . Understanding the structure-function relationships in TSC13 could therefore provide valuable insights that may inform therapeutic approaches targeting TECR in human disease contexts.
Future Research Directions
Several promising research directions could advance our understanding of TSC13:
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Determination of the high-resolution structure of TSC13 through X-ray crystallography or cryo-electron microscopy.
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Further characterization of the protein-protein interactions within the fatty acid elongation complex.
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Development of more specific inhibitors or modulators of TSC13 activity for both research and potential biotechnological applications.
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Exploration of the evolutionary relationships between enoyl reductases across different species to better understand the conservation and diversification of these important enzymes.
Product Specs
Note: We prioritize shipping the format currently in stock. However, if you have specific format requirements, please indicate them when placing your order, and we will fulfill them accordingly.
Note: All our proteins are shipped with standard blue ice packs. If you require dry ice shipping, please inform us in advance as additional fees may apply.
Generally, the shelf life of liquid form is 6 months at -20°C/-80°C. The shelf life of lyophilized form is 12 months at -20°C/-80°C.
Target Background
- Highly conserved domains of the Tsc13p proteins, likely crucial for enzymatic activity, reside on the cytosolic face of the endoplasmic reticulum, potentially partially embedded within the membrane. PMID: 17463005
KEGG: sce:YDL015C
STRING: 4932.YDL015C
Q&A
What is the biochemical function of Tsc13p in Saccharomyces cerevisiae?
Tsc13p functions as the enoyl reductase that catalyzes the final reduction step in each cycle of very long chain fatty acid (VLCFA) elongation. It specifically reduces trans-2-enoyl-CoAs to acyl-CoAs using NADPH as a cofactor, completing the four-step elongation cycle that adds two carbon units to growing fatty acid chains. This enzymatic activity is essential for the production of VLCFAs, which are critical components of sphingolipids in yeast cell membranes. The TSC13 gene was initially identified in a genetic screen for suppressors of calcium sensitivity in csg2Δ mutants with defective sphingolipid synthesis, highlighting its importance in lipid metabolism .
What phenotypes are associated with TSC13 mutations?
Mutations in TSC13 lead to accumulation of long-chain bases and ceramides containing fatty acids with chain lengths shorter than 26 carbons. These phenotypes become more severe when either ELO2 or ELO3 genes, which encode elongases also required for VLCFA synthesis, are deleted. Compromising malonyl-CoA synthesis (by inactivating acetyl-CoA carboxylase) in tsc13 mutants is lethal, further confirming Tsc13p's essential role in VLCFA synthesis. Temperature sensitivity is another characteristic phenotype, with tsc13 mutants showing growth defects at elevated temperatures (38°C), particularly with mutations at critical residues like E91A .
How does Tsc13p interact with other components of the fatty acid elongation machinery?
Tsc13p physically associates with the elongases Elo2p and Elo3p, as demonstrated by co-immunoprecipitation studies. This interaction suggests that fatty acid elongation enzymes are organized in a functional complex rather than operating as independent entities. The presence of Tsc13p is required for optimal activity of the FA elongases, which catalyze the first step in the elongation cycle. Mutations that disrupt Tsc13p activity, such as E91A, also reduce interaction with Elo2/Elo3, leading to decreased elongase activity. This indicates a coordinated action between these proteins within the elongation complex, with physical interactions potentially facilitating substrate channeling between enzymatic steps .
How can researchers assess Tsc13p enzymatic activity in vitro?
Researchers can evaluate Tsc13p activity through in vitro fatty acid elongation assays using membrane fractions containing the enzyme. A typical protocol involves:
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Isolating membrane fractions from yeast expressing wild-type or mutant Tsc13p
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Incubating these fractions with stearoyl-CoA (C18:0-CoA) and radiolabeled [14C]malonyl-CoA in the presence of NADPH
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Converting the products to free fatty acids via alkaline hydrolysis
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Separating the products using thin-layer chromatography (TLC)
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Quantifying the conversion of trans-2-enoyl-CoAs to acyl-CoAs
In wild-type samples, acyl-CoAs (detected as free fatty acids on TLC) are the predominant products, while cells lacking functional Tsc13p accumulate trans-2-enoyl-CoAs. Alternatively, researchers can use stable isotope-labeled [13C]malonyl-CoA and analyze products by LC-MS/MS for more precise quantification of elongated acyl-CoA species of different chain lengths .
What approaches can be used to generate and characterize TSC13 mutants?
A systematic approach to generating and characterizing TSC13 mutants includes:
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Sequence alignment of Tsc13p orthologs to identify conserved residues (Table 1)
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Site-directed mutagenesis to create alanine substitutions at conserved positions
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Expression of mutant constructs in tsc13Δ cells expressing mammalian ceramide synthase CERS5 (which allows survival without VLCFA production)
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Assessment of mutant function through:
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Growth complementation assays at normal (30°C) and elevated (38°C) temperatures
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In vitro FA elongation assays
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Measurement of ceramide levels
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Deuterium-sphingosine labeling
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Co-immunoprecipitation with elongase partners
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Table 1: Key Conserved Residues in Tsc13p and Their Functional Significance
| Residue | Function | Mutant Phenotype |
|---|---|---|
| Tyr256 | Likely catalytic residue providing proton to substrate | Severe reduction in activity |
| Glu91 | Essential for in vivo function | Substantial impairment in vivo |
| Tyr168 | Important for enzymatic activity | Significant reduction in activity |
| Lys140 | Contributes to catalysis | Reduced activity |
| Tyr92 | Contributes to catalysis | Reduced activity |
| Tyr179 | Contributes to catalysis | Reduced activity |
This systematic mutational analysis has identified residues critical for Tsc13p function, with Tyr256 and Glu91 being particularly important .
What techniques are available for studying Tsc13p subcellular localization?
Tsc13p shows a distinctive localization pattern, being present throughout the endoplasmic reticulum (ER) but highly enriched at nuclear-vacuolar junctions. To study this localization, researchers can employ:
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Fluorescence microscopy with Tsc13p-GFP fusion proteins
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Co-localization studies with markers for the ER, nuclear membrane, and vacuole
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Immunoelectron microscopy for high-resolution localization
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Live-cell imaging to track dynamics of Tsc13p at membrane contact sites
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Fractionation studies to biochemically isolate Tsc13p-enriched membrane domains
These approaches have revealed that Tsc13p marks a novel structure at nuclear-vacuolar junctions, suggesting specialized functions at these membrane contact sites, potentially related to lipid metabolism or trafficking .
Which amino acid residues are critical for Tsc13p catalytic activity?
Through comprehensive mutational analysis, researchers have identified several amino acid residues critical for Tsc13p function:
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Tyr256 - Likely serves as the catalytic residue that donates a proton to trans-2-enoyl-CoAs. The Y256A mutant shows severely impaired activity, producing more trans-2-enoyl-CoAs than acyl-CoAs.
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Glu91 - Essential for in vivo function, with the E91A mutant showing substantial impairment in growth complementation and activity assays.
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Tyr168 - Important for catalytic activity, with the Y168A mutant showing significantly reduced enzyme function.
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Other residues with notable effects when mutated include Y92A, K140A, T155A, N164A, Y179A, and H221A.
These findings are based on analysis of 15 highly conserved residues selected through sequence alignment of Tsc13 orthologs from 13 species. The functional importance of these residues was confirmed through both in vitro activity assays and in vivo phenotypic analysis .
How does the three-dimensional structure of Tsc13p relate to its function?
While a complete crystal structure of Tsc13p is not yet available, structural predictions and analysis of homologous proteins suggest that:
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Tsc13p likely adopts a fold similar to other members of the short-chain dehydrogenase/reductase (SDR) family
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The protein contains NAD(P)H-binding motifs involving conserved Gly residues (Gly79 and Gly94)
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The catalytic Tyr256 residue is positioned to interact directly with the substrate
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Specific regions of the protein mediate interaction with elongases Elo2p and Elo3p
The predicted structural arrangement supports a model where Tyr256 acts as the catalytic residue supplying a proton to trans-2-enoyl-CoAs, completing the reduction reaction. This structural insight provides a framework for understanding the mechanism of action and for designing targeted modifications to alter Tsc13p function .
What is the evolutionary relationship between yeast Tsc13p and human TECR?
Tsc13p has a human ortholog called TECR (trans-2,3-enoyl-CoA reductase), and functional comparison reveals significant conservation:
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Key catalytic residues are conserved between species, with E91 and Y256 in Tsc13p corresponding to E94 and Y248 in human TECR
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Mutational analysis shows that E94A and Y248A mutations in TECR produce defects similar to the corresponding mutations in Tsc13p, with Y248A showing almost no activity
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Both enzymes participate in multiprotein complexes for fatty acid elongation
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Both are involved in sphingolipid metabolism beyond their role in VLCFA synthesis
This evolutionary conservation highlights the fundamental importance of this enzymatic function across eukaryotic species and suggests that insights from the yeast system may be applicable to understanding human VLCFA synthesis and related disorders .
How can researchers modulate Tsc13p activity for heterologous production of phenylpropanoids?
Tsc13p presents a challenge in metabolic engineering for phenylpropanoid production due to an unwanted side reaction:
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Tsc13p can reduce p-coumaroyl-CoA (an intermediate in phenylpropanoid synthesis) to phloretic acid
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This side reaction causes carbon loss from the heterologous flavonoid pathway
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Since TSC13 is essential, it cannot be deleted
Researchers have developed two approaches to address this issue:
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Site saturation mutagenesis to identify amino acid changes that reduce the side activity while preserving the natural function
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Complementation of TSC13 with a plant gene homolog that lacks the side activity
The second approach has proven more effective, essentially eliminating the unwanted side reaction while maintaining phenylpropanoid productivity in fed-batch fermentation. This strategy demonstrates how understanding Tsc13p function can enable targeted modifications for biotechnological applications .
What role does Tsc13p play in sphingolipid metabolism beyond VLCFA synthesis?
Beyond its direct role in VLCFA synthesis, Tsc13p functions in sphingolipid metabolism through:
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Participation in the sphingosine degradation pathway
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Influence on sphingolipid composition by affecting the availability of VLCFAs for ceramide synthesis
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Potential involvement in specialized lipid microdomains at membrane contact sites
The connection to sphingolipid metabolism explains why TSC13 was initially identified in a genetic screen for suppressors of calcium sensitivity in sphingolipid-defective mutants. Tsc13p mutants accumulate abnormal sphingolipid species, including long-chain bases and ceramides with shorter fatty acid chains, which can alter membrane properties and cellular functions dependent on these lipids .
How can Tsc13p be targeted for the development of antifungal compounds?
The essential nature of Tsc13p in fungi and differences from mammalian homologs make it a potential target for antifungal development:
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Selective inhibition of fungal Tsc13p could disrupt VLCFA synthesis without affecting host enzymes
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Critical residues identified through mutational analysis could serve as targets for rational drug design
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The unique localization of Tsc13p at nuclear-vacuolar junctions might allow for specialized targeting strategies
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Compounds that disrupt the interaction between Tsc13p and elongases could selectively impair fungal lipid synthesis
Understanding the structure-function relationships of Tsc13p could facilitate the development of novel antifungals targeting this essential enzyme, potentially addressing the growing problem of resistance to existing antifungal medications .
What are common challenges in expressing and purifying recombinant Tsc13p?
Researchers working with recombinant Tsc13p face several challenges:
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Membrane association - Tsc13p is an integral membrane protein, making solubilization and purification difficult
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Complex formation - Tsc13p functions in a multienzyme complex with Elo2p/Elo3p, and isolation may disrupt essential interactions
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Activity preservation - Maintaining enzymatic activity outside the native membrane environment is challenging
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Protein stability - Membrane proteins often have stability issues when expressed in heterologous systems
Strategies to overcome these challenges include:
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Using membrane-mimicking detergents during purification
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Co-expressing interaction partners
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Working with membrane fractions rather than purified protein
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Creating fusion constructs with solubility-enhancing tags
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Employing nanodiscs or liposomes to reconstitute membrane environment
How can researchers differentiate between direct and indirect effects when studying Tsc13p mutants?
When analyzing Tsc13p mutants, distinguishing direct catalytic effects from indirect impacts on the elongation complex requires:
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Comparative analysis of in vitro activity and in vivo phenotypes:
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Direct catalytic defects should show immediate biochemical consequences
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Indirect effects may manifest progressively or affect specific lipid species
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Assessment of protein-protein interactions:
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Co-immunoprecipitation with Elo2p/Elo3p to determine if mutations affect complex formation
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Analysis of protein stability and localization to rule out general protein folding defects
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Detailed intermediate analysis:
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Profiling of all fatty acid elongation intermediates (3-ketoacyl-CoAs, 3-hydroxyacyl-CoAs, trans-2-enoyl-CoAs)
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Accumulation patterns can indicate the specific step affected by a mutation
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Complementation approaches:
What controls and standards are essential for reliable Tsc13p activity assays?
To ensure reliability in Tsc13p activity assays, researchers should include:
What are promising approaches for structural characterization of the entire fatty acid elongation complex?
Future structural studies of the fatty acid elongation complex could employ:
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Cryo-electron microscopy to visualize the native complex in membrane environments
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Crosslinking mass spectrometry to map interaction interfaces between Tsc13p and elongases
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Hydrogen-deuterium exchange mass spectrometry to identify dynamic regions involved in substrate binding
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Integrative structural biology combining multiple techniques (crystallography, NMR, SAXS) to build comprehensive models
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Molecular dynamics simulations to understand conformational changes during the catalytic cycle
These approaches could reveal how Tsc13p coordinates with elongases, the structural basis for substrate specificity, and potential allosteric regulation within the complex .
How might systems biology approaches enhance our understanding of Tsc13p function?
Systems biology approaches offer new insights into Tsc13p function within the broader context of cellular metabolism:
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Metabolic flux analysis to quantify the impact of Tsc13p mutations on lipid synthesis pathways
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Lipidomics to comprehensively profile changes in membrane composition
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Genetic interaction mapping to identify functional connections with other pathways
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Computational modeling of fatty acid elongation kinetics and regulation
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Integration of transcriptomic, proteomic, and lipidomic data to understand compensatory responses
These approaches could reveal how Tsc13p function is integrated with other aspects of lipid metabolism and cellular physiology, potentially uncovering new regulatory mechanisms and functional relationships .
What implications does understanding Tsc13p have for human disease research?
Research on Tsc13p provides insights with implications for human disease:
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The human ortholog TECR has been implicated in:
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Nonsyndromic mental retardation
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Synaptic lipid metabolism disorders
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Skin barrier dysfunction
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Understanding the catalytic mechanism of Tsc13p/TECR could facilitate:
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Development of targeted therapies for TECR-related disorders
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Biomarker identification for altered VLCFA metabolism
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Genetic counseling for affected families
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The connection to sphingolipid metabolism has relevance for:
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Neurodegenerative disorders
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Cancer biology
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Inflammatory conditions
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Insights from yeast Tsc13p could inform approaches to modulating human TECR activity in disease contexts, potentially through:
