Recombinant Laccaria bicolor 3-ketoacyl-CoA reductase (LACBIDRAFT_192627)

What is Laccaria bicolor 3-ketoacyl-CoA reductase and what is its function in fatty acid metabolism?

Laccaria bicolor 3-ketoacyl-CoA reductase (LACBIDRAFT_192627) is an enzyme that catalyzes the reduction of the 3-ketoacyl-CoA intermediate formed during each cycle of fatty acid elongation. It is a component of the microsomal membrane-bound fatty acid elongation system that produces very long-chain fatty acids (VLCFAs) from palmitate. These VLCFAs serve as crucial precursors for ceramide and sphingolipids . The enzyme plays a critical role in the fatty acid biosynthesis and degradation pathways in L. bicolor, as part of a genome-wide network of 63 genes involved in fatty acid metabolism that have been annotated and validated through transcript detection .

How does LACBIDRAFT_192627 compare to similar enzymes in other fungi?

Phylogenetic analysis indicates that L. bicolor, along with Ustilago maydis and Coprinopsis cinerea, possesses a vertebrate-like type I fatty acid synthase (FAS) encoded as a single protein. This differs from other basidiomycetes, including the human pathogenic basidiomycete Cryptococcus neoformans, and most ascomycetes, where FAS is composed of two structurally distinct subunits α and β .

Comparisons between 3-ketoacyl-CoA reductases across fungal species reveal conserved functional domains while showing species-specific variations. Commercial recombinant forms of this enzyme are available from multiple fungal sources including Neosartorya fumigata, Ajellomyces capsulata, Candida albicans, Lodderomyces elongisporus, Pyrenophora tritici-repentis, Chaetomium globosum, Ustilago maydis, Coprinopsis cinerea, Aspergillus niger, Cryptococcus neoformans, Debaryomyces hansenii, and Phaeosphaeria nodorum , enabling comparative studies of structure-function relationships.

What are the recommended protocols for expressing recombinant LACBIDRAFT_192627?

Recombinant LACBIDRAFT_192627 can be expressed in various expression systems including E. coli, yeast, baculovirus, or mammalian cells . For E. coli expression, the following methodology is commonly employed:

• Clone the full-length LACBIDRAFT_192627 gene (encoding amino acids 1-338) into an appropriate expression vector with an N-terminal His tag.

• Transform the construct into an E. coli expression strain.

• Induce protein expression under optimized conditions (temperature, IPTG concentration, duration).

• Harvest cells and lyse using appropriate buffer systems.

• Purify the recombinant protein using immobilized metal affinity chromatography (IMAC).

• Perform quality control analysis including SDS-PAGE to confirm purity (>85-90%) .

• Store the purified protein in Tris/PBS-based buffer with 6% trehalose at pH 8.0 .

For long-term storage, it is recommended to add 5-50% glycerol (final concentration) and aliquot for storage at -20°C/-80°C. Repeated freeze-thaw cycles should be avoided, and working aliquots can be stored at 4°C for up to one week .

How can I measure the enzymatic activity of recombinant LACBIDRAFT_192627?

The enzymatic activity of recombinant LACBIDRAFT_192627 can be assessed through the following methodology:

• Preparation of reaction mixture: Combine 0.1 M sodium phosphate (pH 7.0), appropriate co-factors (50 μM NADH, 50 μM NADPH), 1 mM β-mercaptoethanol, the substrate (3-ketoacyl-CoA), and the purified enzyme .

• Assay conditions: The reduction of 3-ketoacyl-CoA to 3-hydroxyacyl-CoA can be monitored spectrophotometrically by measuring the oxidation of NADPH at 340 nm. Alternatively, HPLC or LC-MS methods can be used to directly measure the formation of 3-hydroxyacyl-CoA products .

• Kinetic analysis: Determine kinetic parameters (Km, Vmax) by varying substrate concentrations while keeping enzyme concentration constant. Plot the reaction velocity against substrate concentration and fit to the Michaelis-Menten equation.

• Substrate specificity: Test various chain-length 3-ketoacyl-CoA substrates to determine the enzyme's chain-length preference and specificity.

• Inhibition studies: Evaluate potential inhibitors by including them in the reaction mixture and measuring their effect on enzyme activity.

What reconstitution systems can be used to study LACBIDRAFT_192627 in the context of fatty acid elongation?

To study LACBIDRAFT_192627 in the context of fatty acid elongation, researchers can employ several reconstitution systems:

• Heterologous expression in yeast: Yeast systems can be engineered to express the entire Arabidopsis fatty acid elongase (FAE) complex in place of the endogenous yeast FAE, providing an optimal environment for studying plant and fungal KCSs activities . This approach has been successfully used to characterize the activities of various 3-ketoacyl-CoA synthases (KCS).

• In vitro reconstitution: The complete fatty acid elongation system can be reconstituted in vitro using purified components including malonyl-CoA, acetyl-CoA, NADPH, NADH, holo-ACP, and the four core enzymes of fatty acid elongation: 3-ketoacyl-CoA synthase (KCS), 3-ketoacyl-CoA reductase (KCR), 3-hydroxyacyl-CoA dehydratase (HCD), and enoyl-CoA reductase (ECR) .

• Transient expression systems: The enzyme can be transiently expressed in plant systems such as Nicotiana benthamiana to study its subcellular localization and function. This approach has been used to confirm the endoplasmic reticulum localization of similar enzymes .

How can I investigate the substrate specificity and chain-length preference of LACBIDRAFT_192627?

Investigating the substrate specificity and chain-length preference of LACBIDRAFT_192627 requires sophisticated methodological approaches:

• Profiling acyl-CoA pools: Use liquid chromatography (electrospray ionization-tandem) mass spectrometry with multiple reaction monitoring (MRM) to analyze changes in acyl-CoA profiles when the enzyme is expressed in systems like the TRIPLE Δelo3 yeast strain . This approach allows for detection of changes in levels of specific acyl-CoA species.

• Heterologous expression systems: Express LACBIDRAFT_192627 in yeast strains with specific fatty acid elongation defects (e.g., elo2, elo3 mutants) and analyze the resulting fatty acid profiles using gas chromatography-mass spectrometry (GC-MS) for identification and gas chromatography with flame ionization detection (GC-FID) for quantification .

• Principal component analysis (PCA): Apply PCA to visualize changes in very long-chain fatty acid (VLCFA) contents resulting from the expression of the enzyme, as demonstrated in Figure 2 from reference :

Figure PCA Analysis Results:

• Principal components analysis of fatty acid methyl ester (FAME) profiles can separate strains expressing different enzymes based on their activity

• PC1 and PC2 typically represent 60-70% of the total variance

• Biplots can reveal which specific carbon chain lengths (C20, C22, C24, C26, etc.) are most affected by the enzyme's activity

• In vitro elongation/condensation assays: Use microsomal membranes from cells expressing LACBIDRAFT_192627 to perform in vitro assays with different chain-length substrates .

How does the compartmentalization of fatty acid metabolism affect the function of LACBIDRAFT_192627?

Analysis of fatty acid metabolism in L. bicolor has revealed important insights about subcellular compartmentalization that impacts the function of enzymes like LACBIDRAFT_192627:

How can genomic approaches be used to study the role of LACBIDRAFT_192627 in broader metabolic networks?

Genomic approaches to study LACBIDRAFT_192627 in broader metabolic networks include:

• Genome-wide inventory: The genome sequence of L. bicolor has been explored to construct a genome-wide inventory of genes involved in fatty acid metabolism. Sixty-three genes of the major pathways were annotated and validated by the detection of the corresponding transcripts, with 71% belonging to multigene families of up to five members .

• Transcriptional analysis: Gene expression profiling under different conditions can reveal how LACBIDRAFT_192627 is regulated in response to environmental changes, developmental stages, or metabolic perturbations.

• Comparative genomics: Phylogenetic analysis has shown that L. bicolor shares a vertebrate-like type I fatty acid synthase (FAS) encoded as a single protein with Ustilago maydis and Coprinopsis cinerea, in contrast to most other fungi where FAS is composed of two distinct subunits .

• Functional genomics approaches:

  • RNA interference (RNAi) or CRISPR-Cas9 to create knockdowns or knockouts

  • Overexpression studies to assess gain-of-function phenotypes

  • Complementation studies in yeast mutants lacking the corresponding enzyme

What bioinformatic tools are most effective for analyzing the evolutionary relationships of 3-ketoacyl-CoA reductases across fungal species?

Effective bioinformatic tools for evolutionary analysis of 3-ketoacyl-CoA reductases include:

• Sequence alignment tools: Multiple sequence alignment software such as MUSCLE, CLUSTAL, or T-Coffee can align LACBIDRAFT_192627 with homologs from other fungal species to identify conserved domains and variable regions.

• Phylogenetic analysis software: Programs like MEGA, MrBayes, or PhyML can construct phylogenetic trees to visualize evolutionary relationships. These analyses have revealed that L. bicolor shares a vertebrate-like type I FAS encoded as a single protein with specific other fungi, contrasting with the two-subunit structure found in most fungi .

• Protein domain prediction tools: InterProScan, Pfam, or SMART can identify functional domains within LACBIDRAFT_192627 and compare them across species.

• Structural prediction and comparison: Tools like I-TASSER, SWISS-MODEL, or AlphaFold can predict protein structures and allow for comparative structural analysis across species.

• Genome browsers and comparative genomics platforms: Ensembl Fungi, JGI MycoCosm, or FungiDB facilitate cross-species comparisons of gene order, synteny, and evolutionary conservation.

How can metabolomic approaches complement enzymatic studies of LACBIDRAFT_192627?

Metabolomic approaches provide valuable complementary data to enzymatic studies of LACBIDRAFT_192627:

• Lipid profiling: Comprehensive analysis of fatty acid composition in L. bicolor has identified 19 different fatty acids in the mycelium, including marker fatty acids like palmitvaccenic acid (16:1(11Z)) . Similar profiling in systems where LACBIDRAFT_192627 is manipulated can reveal its specific impact on the lipidome.

• Acyl-CoA profiling: Liquid chromatography-mass spectrometry approaches can quantify the acyl-CoA pool, which in yeast models is typically dominated by C16 species (80.4 ± 3.6% of the total) . Changes in this profile upon expression or manipulation of LACBIDRAFT_192627 can reveal its substrate preferences and metabolic impacts.

• Flux analysis: Stable isotope labeling with 13C-labeled precursors can track the flow of carbon through fatty acid elongation pathways, revealing the in vivo activity and metabolic context of LACBIDRAFT_192627.

• Integrative multi-omics: Combining metabolomic data with transcriptomic, proteomic, and enzymatic data can provide a systems-level understanding of LACBIDRAFT_192627's role in fungal metabolism.

What are the most common issues encountered when working with recombinant LACBIDRAFT_192627 and how can they be addressed?

Common issues with recombinant LACBIDRAFT_192627 and their solutions include:

• Low expression levels:

  • Optimize codon usage for the expression host

  • Test different expression vectors and promoters

  • Evaluate different induction conditions (temperature, inducer concentration, time)

  • Consider fusion partners that enhance solubility (MBP, SUMO, thioredoxin)

• Protein insolubility:

  • Express at lower temperatures (16-20°C)

  • Add solubility enhancers to lysis buffer (glycerol, non-ionic detergents)

  • Consider mild solubilization from inclusion bodies

  • Test different buffer compositions and pH values

• Loss of enzymatic activity:

  • Include stabilizing agents (glycerol, reducing agents like DTT or β-mercaptoethanol)

  • Avoid repeated freeze-thaw cycles

  • Store aliquots at -80°C for long-term storage

  • Include cofactors (NADPH) in storage buffers

  • Consider enzyme immobilization techniques for improved stability

• Purification challenges:

  • Optimize imidazole concentrations in binding and elution buffers

  • Use step-wise elution to improve purity

  • Consider secondary purification steps (ion exchange, size exclusion)

  • Validate protein identity by mass spectrometry or western blotting

How can I address substrate availability issues in enzymatic assays of LACBIDRAFT_192627?

Addressing substrate availability challenges in LACBIDRAFT_192627 assays:

• Chemical synthesis of substrates:

  • Collaborate with synthetic chemists to produce custom 3-ketoacyl-CoA substrates of various chain lengths

  • Consider solid-phase synthesis approaches for CoA-modified compounds

• Enzymatic synthesis of substrates:

  • Generate 3-ketoacyl-CoA substrates using coupled enzymatic reactions

  • For example, use acyl-CoA dehydrogenases and crotonases to generate the appropriate substrates

• Commercial sources:

  • Several specialty biochemical companies now offer CoA thioesters of various chain lengths

  • Consider custom synthesis services for specific substrates

• Substrate mimetics:

  • Develop and validate simplified substrate analogs that retain recognition elements

  • Design fluorogenic substrates that enhance assay sensitivity

• Coupled enzyme assays:

  • Design assays where LACBIDRAFT_192627 is coupled with other enzymes of the fatty acid elongation pathway

  • This approach has been used successfully for similar enzymes, where reactions containing multiple enzymes (e.g., FabH, FabZ, FabI) along with substrates like acetyl-CoA, malonyl-CoA, and holo-ACP were combined

What strategies can be employed to improve the solubility and stability of recombinant LACBIDRAFT_192627 for structural studies?

Strategies to improve solubility and stability for structural studies:

• Protein engineering approaches:

  • Remove flexible regions or disordered termini based on bioinformatic predictions

  • Introduce surface mutations to enhance solubility (e.g., replace hydrophobic residues with hydrophilic ones)

  • Create fusion constructs with highly soluble partners (MBP, SUMO, thioredoxin)

  • Consider construct design guided by comparative analysis with homologous proteins

• Expression optimization:

  • Screen multiple expression hosts (E. coli, yeast, insect cells)

  • Test specialized E. coli strains designed for membrane or difficult proteins

  • Co-express with chaperones or folding modulators

  • Evaluate cell-free expression systems which have been successfully used for this protein

• Purification and stabilization:

  • Include appropriate detergents or lipids to maintain native-like environment

  • Optimize buffer conditions (pH, ionic strength, additives)

  • Add stabilizing ligands or cofactors (NADPH)

  • Consider nanodiscs or other membrane mimetics for maintaining function

  • Implement on-column refolding protocols for proteins recovered from inclusion bodies

• Crystallization strategies:

  • Use crystallization chaperones (antibody fragments, nanobodies)

  • Try in situ proteolysis during crystallization

  • Screen for stabilizing small molecules or substrate analogs

  • Consider lipidic cubic phase crystallization for membrane-associated proteins