Recombinant Saccharomyces cerevisiae 3-ketoacyl-CoA reductase (IFA38)

What is IFA38 and what is its function in Saccharomyces cerevisiae?

IFA38 (YBR159W) is a very-long-chain 3-oxoacyl-CoA reductase, also known as microsomal beta-keto-reductase, that belongs to the short-chain dehydrogenases/reductases (SDR) family. It plays a crucial role in fatty acid metabolism, particularly in the elongation of very long-chain fatty acids (VLCFAs) . The enzyme contains an oleate response element (ORE) sequence in its promoter region, suggesting regulation by fatty acid availability. IFA38 mutants exhibit reduced VLCFA synthesis and accumulate high levels of dihydrosphingosine, phytosphingosine, and medium-chain ceramides, indicating its importance in lipid homeostasis .

Which cellular pathways involve IFA38 in S. cerevisiae?

IFA38 is primarily involved in the fatty acid elongation pathway, working in concert with several other enzymes. This pathway is distinct from but related to the β-oxidation pathway, which is the predominant route for fatty acid metabolism in S. cerevisiae . IFA38 functions specifically in the reductive step of the fatty acid elongation cycle, converting 3-ketoacyl-CoA to 3-hydroxyacyl-CoA. This enzyme is part of a complex that includes elongases and other reductases that collectively synthesize very long-chain fatty acids essential for membrane structure and function .

What protein interactions are important for IFA38 function?

IFA38 exhibits strong functional interactions with several proteins involved in fatty acid elongation. The most significant interactions include:

• TSC13 (interaction score: 0.999) - A very-long-chain enoyl-CoA reductase that catalyzes the last step in each cycle of very long chain fatty acid elongation .

• PHS1 (interaction score: 0.998) - An essential 3-hydroxyacyl-CoA dehydratase of the ER membrane involved in elongation of very long-chain fatty acids .

• ELO2 (interaction score: 0.986) - A fatty acid elongase involved in sphingolipid biosynthesis that acts on fatty acids of up to 24 carbons in length .

These interactions highlight the coordinated enzymatic activity required for proper fatty acid elongation and metabolism in S. cerevisiae.

What are the optimal conditions for expressing recombinant IFA38 in S. cerevisiae?

The optimal expression of recombinant IFA38 in S. cerevisiae depends on several factors including temperature, pH, and media composition. Based on response surface methodology (RSM) studies of similar enzymes, maximum enzymatic activity can be achieved at approximately 37°C and pH 5.9 . It's important to note that temperatures above 50°C may result in reduced enzymatic activity, even at the optimal pH range of 5-6 .

How can I measure the enzymatic activity of recombinant IFA38?

Enzymatic activity of recombinant IFA38 can be measured through several approaches:

• Spectrophotometric assay: Monitor the oxidation of NADPH to NADP+ at 340 nm, as IFA38 uses NADPH as a cofactor in the reduction of 3-ketoacyl-CoA to 3-hydroxyacyl-CoA.

• Substrate depletion assay: Measure the consumption of 3-ketoacyl-CoA substrates using HPLC or LC-MS/MS techniques.

• Product formation assay: Quantify the 3-hydroxyacyl-CoA products formed using analytical techniques such as gas chromatography.

When designing these assays, it's important to consider factors that influence enzymatic activity, including enzyme concentration, substrate concentration, incubation time, temperature, and pH . For optimal results, consider using a response surface methodology (RSM) approach to determine the precise conditions that maximize IFA38 activity in your specific experimental setup.

What expression systems are most effective for producing recombinant IFA38?

For heterologous expression of IFA38, several systems can be considered:

When selecting an expression system, consider that membrane-associated or peroxisomal enzymes from S. cerevisiae may require specific cellular environments to fold properly and maintain activity, as observed with FOX2 .

How does IFA38 function differ from similar enzymes in other organisms?

While IFA38 in S. cerevisiae functions as a 3-ketoacyl-CoA reductase in the fatty acid elongation pathway, there are notable differences when compared to similar enzymes in other organisms:

• Compared to Yarrowia lipolytica: Unlike S. cerevisiae, Y. lipolytica fatty acyl-CoA synthetases have dual functions, both activating fatty acids and transporting them. Y. lipolytica also shows differences in compartmentalization of fatty acid activation, with long-chain fatty acids activated by ACS I, while medium and short-chain fatty acids enter peroxisomes directly for activation by ACS II before β-oxidation .

• Compared to mammalian systems: Human homologs of the fatty acid elongation system exhibit greater complexity and tissue-specific expression patterns. The human homolog of TSC13 (TECR), which interacts strongly with IFA38, has been implicated in nonsyndromic mental retardation, highlighting the broader significance of these pathways in higher organisms .

These differences underscore the importance of considering evolutionary context when studying IFA38 and potentially extrapolating findings to other systems.

What are the effects of IFA38 mutations on cellular lipid composition and metabolism?

Mutations in IFA38 have significant consequences for cellular lipid composition and metabolism:

• Reduced VLCFA synthesis: IFA38 mutants exhibit decreased production of very long-chain fatty acids, demonstrating the enzyme's essential role in fatty acid elongation .

• Sphingolipid abnormalities: Mutants accumulate high levels of dihydrosphingosine, phytosphingosine, and medium-chain ceramides, indicating disruption of sphingolipid biosynthesis pathways .

• Metabolic compensation: When key enzymes in fatty acid metabolism are disrupted, cells may attempt to compensate through alternative pathways. For instance, in the absence of efficient β-oxidation, cells might redirect fatty acids toward lipid synthesis pathways .

These alterations in lipid profiles not only affect membrane composition but may also influence various cellular processes including signaling pathways, stress responses, and organelle function.

How can IFA38 be engineered to improve specific biotechnological applications?

Engineering IFA38 for biotechnological applications can be approached through several strategies:

• Protein engineering for altered substrate specificity: Directed evolution or rational design approaches can be employed to modify the substrate binding pocket of IFA38, potentially allowing it to process non-native substrates for production of novel fatty acid derivatives.

• Metabolic engineering of IFA38 expression: Modifying regulatory elements such as the oleate response element (ORE) in the promoter region could allow for more controlled expression patterns . Additionally, considering that β-oxidation can be reversed to synthesize fatty acids with less energy expenditure than de novo synthesis, engineering IFA38 to function optimally in such a reversed pathway could be valuable .

• Cofactor optimization: Engineering IFA38 for altered cofactor preference or improved efficiency could enhance productivity. Research has shown that NADH availability is a key factor affecting β-oxidation efficiency, with optimization of the acetyl-CoA and NADH supply significantly improving the production of medium-chain fatty acids (MCFAs) .

When implementing these engineering strategies, it's important to consider the complex metabolic context in which IFA38 operates, particularly its interactions with other enzymes like TSC13, PHS1, and ELO2 .

What analytical methods are most effective for studying IFA38 activity and its products?

Several analytical methods can be employed to study IFA38 activity and its products:

• Chromatographic methods:

  • Gas chromatography (GC) with flame ionization detection (FID) or mass spectrometry (MS) detection is particularly useful for analyzing fatty acid profiles and can be used to track changes in fatty acid composition resulting from IFA38 activity or mutation .

  • High-performance liquid chromatography (HPLC) coupled with MS can be used to analyze CoA derivatives and intermediates in the fatty acid elongation pathway.

• Radiotracer studies:

  • Radiolabeled fatty acid substrates (such as oleic acid) can be used to track fatty acid utilization and metabolism, as demonstrated in studies of acyl-CoA synthetase activity .

• Enzyme kinetics assays:

  • Spectrophotometric assays tracking NADPH oxidation can provide detailed kinetic parameters for IFA38, including Km, Vmax, and catalytic efficiency.

  • Response surface methodology (RSM) can be employed to optimize assay conditions by systematically varying parameters such as temperature and pH .

The choice of analytical method should be guided by the specific research question and the nature of the information sought.

How does temperature and pH affect recombinant IFA38 stability and activity?

Temperature and pH are critical factors affecting the stability and activity of recombinant IFA38:

Temperature effects:

• Enzymatic activity typically increases with temperature up to an optimal point, after which thermal denaturation leads to activity loss.

• For similar enzymatic systems, activity has been shown to peak around 37°C, with significant decreases at temperatures above 50°C .

pH effects:

• The optimal pH for IFA38 activity likely falls in the range of 5-6, based on studies of similar enzymatic systems .

• pH affects both substrate binding and catalytic activity by influencing the ionization state of key amino acid residues in the active site.

The interaction between temperature and pH is also important to consider. As shown in optimization studies, at a fixed pH between 5 and 6, increases in temperature initially improve activity until reaching an optimum (around 37°C), after which further temperature increases lead to decreased activity . This illustrates the importance of simultaneously considering multiple parameters when optimizing recombinant enzyme expression and activity.

How does IFA38 activity coordinate with the β-oxidation pathway in S. cerevisiae?

While IFA38 primarily functions in fatty acid elongation, its activity is intricately connected to the β-oxidation pathway in S. cerevisiae:

What role does IFA38 play in sphingolipid biosynthesis in yeast?

IFA38 plays a significant role in sphingolipid biosynthesis in yeast, primarily through its involvement in very long-chain fatty acid (VLCFA) production:

• VLCFA provision: By participating in fatty acid elongation, IFA38 helps generate the VLCFAs that are essential components of complex sphingolipids in yeast cell membranes .

• Impact of IFA38 mutations: Mutants lacking functional IFA38 accumulate high levels of dihydrosphingosine, phytosphingosine, and medium-chain ceramides, indicating disruption of normal sphingolipid metabolism .

• Interaction with sphingolipid biosynthesis enzymes: IFA38 shows functional connections with other enzymes involved in sphingolipid biosynthesis. For example, ELO2, which strongly interacts with IFA38, is directly involved in sphingolipid biosynthesis and acts on fatty acids of up to 24 carbons in length .

• Regulatory integration: The presence of an oleate response element (ORE) in the IFA38 promoter suggests that its expression can be modulated by fatty acid availability, potentially allowing coordination between fatty acid metabolism and sphingolipid biosynthesis .

This involvement in sphingolipid biosynthesis makes IFA38 particularly important for maintaining membrane integrity and function in S. cerevisiae.

What are the current limitations in our understanding of IFA38 function?

Despite significant advances in characterizing IFA38, several limitations remain in our understanding:

• Structural insights: Detailed structural information about IFA38, particularly in complex with substrates or interacting proteins, is limited. Such information would facilitate structure-based engineering approaches and improved understanding of substrate specificity.

• Regulatory networks: While the presence of an oleate response element (ORE) in the promoter region has been identified , the complete regulatory network controlling IFA38 expression and activity remains to be fully elucidated.

• Metabolic integration: A comprehensive understanding of how IFA38 activity is integrated with other cellular processes beyond fatty acid metabolism, such as stress responses or cell cycle regulation, is still developing.

• Post-translational modifications: Information about potential post-translational modifications that might regulate IFA38 activity or stability is limited.

Addressing these knowledge gaps represents important directions for future research on this enzyme.

What emerging technologies might advance research on recombinant IFA38?

Several emerging technologies show promise for advancing research on recombinant IFA38:

• CRISPR-Cas9 genome editing: Precise genomic modifications can facilitate detailed structure-function studies of IFA38 in its native context, potentially revealing new insights into its regulation and function.

• Single-cell lipidomics: Emerging technologies for single-cell analysis of lipid composition could reveal cell-to-cell variability in how IFA38 function affects lipid profiles.

• Protein engineering platforms: High-throughput directed evolution approaches, potentially coupled with machine learning algorithms for protein design, could accelerate the development of IFA38 variants with enhanced or altered functions.

• Metabolic flux analysis: Advanced techniques for measuring metabolic fluxes could provide dynamic information about how IFA38 activity influences fatty acid and lipid metabolism in real-time.

• Cryo-electron microscopy: This technique could potentially reveal the structure of IFA38 in complex with its interaction partners, providing insights into the molecular basis of functional relationships observed in protein interaction studies .

These technological advances, individually or in combination, have the potential to significantly enhance our understanding of IFA38 and facilitate its application in both basic research and biotechnology.