Recombinant Meyerozyma guilliermondii 3-ketoacyl-CoA reductase (PGUG_04787)

What is Meyerozyma guilliermondii 3-ketoacyl-CoA reductase and what is its role in metabolism?

Meyerozyma guilliermondii 3-ketoacyl-CoA reductase (PGUG_04787) is an enzyme that catalyzes the reduction of 3-ketoacyl-CoA substrates as part of the fatty acid biosynthesis pathway. Structurally, it belongs to the short-chain dehydrogenase/reductase (SDR) family. The enzyme specifically reduces 3-ketoacyl substrates to 3-hydroxyacyl intermediates using NADPH as a cofactor, representing a critical step in the elongation cycle of fatty acid synthesis. In M. guilliermondii, this enzyme is encoded by the PGUG_04787 gene and consists of 341 amino acids . The enzyme plays a pivotal role in both primary metabolism and potentially in stress response mechanisms, as alterations in fatty acid composition can affect membrane fluidity and cellular resilience to environmental stressors .

What are the structural characteristics of the recombinant PGUG_04787 protein?

The recombinant Meyerozyma guilliermondii 3-ketoacyl-CoA reductase is a full-length protein comprising 341 amino acids. Based on its amino acid sequence (MLRLIDSISDNCTVKTALYGALLLGVYKLTTFALSLVSLVLDLWVLPPVNFAKYGAKKGKWAVITGASDGIGKEYATQLAAKGLNVVLVSRTESKLVALAEEIESKYKVSTKVLAFDVSLDAESSYEDLAATIADLPVTVLVNNVGQSHSIPVPFLETDEKELRNIITINNTATLKITQVVAPKIVHTVASEKKKTRGLILTMGSFGGLLPTPYLATYSGSKAFLQSWSNALSGELQPQGVDVELVISYLVTSAMSKIRRSSASIPNPKAFVKSVLRNVGRRVGAQERFGTTTPYWAHAFMHFGIVNSVGVYSKIANSLNLGMHKSIRSRALKKAARQKKD), it contains characteristic motifs associated with ketoacyl reductases . While specific crystallographic data for the M. guilliermondii enzyme isn't directly available in the search results, comparative analysis with related 3-ketoacyl-CoA reductases suggests it likely adopts a Rossmann fold typical of NAD(P)H-dependent oxidoreductases. The recombinant version includes an N-terminal His-tag to facilitate purification and is expressed in E. coli expression systems .

How does M. guilliermondii 3-ketoacyl-CoA reductase compare to similar enzymes in other organisms?

When compared to homologous enzymes, M. guilliermondii 3-ketoacyl-CoA reductase shares functional similarities with 3-ketoacyl-ACP reductases from other organisms, though with distinct structural characteristics. Unlike the spinach 3-ketoacyl-ACP synthase III, which functions as a homodimer with subunit size of approximately 40,500 Da and native size of about 63,000 Da , the M. guilliermondii reductase likely functions as a monomer based on typical SDR family characteristics.

The M. guilliermondii enzyme exhibits substrate specificity for 3-ketoacyl-CoA intermediates, whereas the spinach synthase demonstrates high specificity for acetyl-CoA and malonyl-ACP . These differences reflect the evolutionary divergence of fatty acid metabolism enzymes across different kingdoms. Additionally, genomic analysis of M. guilliermondii has revealed potential gene copy number variations that may contribute to functional adaptation, suggesting possible enzymatic specialization not observed in plant counterparts .

What genomic features influence the expression and function of PGUG_04787 in M. guilliermondii?

Comprehensive genomic analysis of M. guilliermondii has revealed several factors that may influence PGUG_04787 expression and function. Gene copy number variation (CNV) analysis indicates a direct correlation between gene amplification and competitive fitness, with certain genes showing gains while others exhibit losses . While the search results don't specifically mention PGUG_04787 among these genes, the species demonstrates a pattern of genomic plasticity that likely extends to metabolic enzymes like 3-ketoacyl-CoA reductase.

Gene ontology (GO) analysis of M. guilliermondii has identified several relevant functional categories including enzymes, transcription factors, membrane proteins, and stress-response-related proteins . The 3-ketoacyl-CoA reductase gene would fall within the enzyme category and may be co-regulated with other genes involved in fatty acid metabolism. The organism's ability to adapt to various environmental conditions, including manganese stress, suggests complex regulatory networks governing the expression of metabolic enzymes . Researchers investigating PGUG_04787 should consider these genomic features when designing expression studies or functional analyses.

How can researchers optimize the expression and purification of recombinant PGUG_04787?

Based on the available information, researchers can optimize expression and purification of recombinant PGUG_04787 through the following methodological approach:

Expression System Optimization:

• E. coli is the confirmed successful expression system for this protein

• Consider testing different E. coli strains (BL21, Rosetta, Arctic Express) for optimal expression

• Optimize induction parameters including temperature (typically lower temperatures of 16-20°C improve solubility), IPTG concentration, and induction duration

Purification Protocol:

• Utilize the N-terminal His-tag for initial IMAC (immobilized metal affinity chromatography) purification

• Implement a multi-step purification strategy:

  • IMAC purification (Ni-NTA or Co-NTA columns)

  • Size exclusion chromatography to ensure homogeneity

  • Consider ion exchange chromatography as a polishing step if necessary

Buffer Optimization:

Storage Conditions:
The recombinant protein should be stored according to the manufacturer's recommendations: aliquoted at -20°C/-80°C with 6% trehalose in Tris/PBS-based buffer (pH 8.0) . For working aliquots, storage at 4°C for up to one week is recommended, avoiding repeated freeze-thaw cycles .

What are the implications of M. guilliermondii's antifungal resistance mechanisms for research involving PGUG_04787?

M. guilliermondii has developed significant resistance to conventional antifungals, with reduced sensitivity to amphotericin B, fluconazole, micafungin, and anidulafungin . This resistance has important implications for research involving PGUG_04787:

• Metabolic pathway interconnections: Antifungal resistance mechanisms often involve modifications in membrane lipid composition and fluidity. As 3-ketoacyl-CoA reductase participates in fatty acid metabolism, alterations in its activity may contribute to membrane lipid changes that affect drug permeability or target accessibility. Researchers should investigate potential correlations between PGUG_04787 expression levels and antifungal susceptibility profiles.

• Experimental design considerations: When designing experiments involving M. guilliermondii strains expressing recombinant PGUG_04787, researchers should account for potential interference from antifungal agents in culture media or experimental treatments. Control experiments should include appropriate drug-free conditions to establish baseline enzyme activity.

• Therapeutic target potential: The increasing incidence of M. guilliermondii infections and their reduced sensitivity to conventional antifungals highlights the need for alternative therapeutic targets. PGUG_04787, as a metabolic enzyme, may represent a novel target for antifungal development. Researchers could explore inhibitor screening assays using the recombinant enzyme to identify compounds that selectively inhibit fungal 3-ketoacyl-CoA reductase without affecting human homologs.

What enzymatic assays are recommended for characterizing the activity of recombinant PGUG_04787?

Several enzymatic assays can be employed to characterize the activity of recombinant PGUG_04787, with careful consideration of its function as a 3-ketoacyl-CoA reductase:

Spectrophotometric NADPH Oxidation Assay:
This is the primary method for measuring reductase activity, monitoring the decrease in absorbance at 340 nm as NADPH is oxidized to NADP+ during the reduction of 3-ketoacyl-CoA substrates.

Reaction Setup:

Reaction kinetics should be monitored continuously for 5-10 minutes at 25-30°C. Calculate activity using the extinction coefficient of NADPH (ε₃₄₀ = 6,220 M⁻¹ cm⁻¹).

Substrate Specificity Analysis:
Test the enzyme's activity with different chain-length 3-ketoacyl-CoA substrates (C4 to C18) to determine chain-length preference and construct a substrate specificity profile. Based on related enzymes, the M. guilliermondii 3-ketoacyl-CoA reductase likely exhibits preferences similar to other ketoreductases involved in fatty acid synthesis .

Inhibition Studies:
Evaluate potential inhibitors such as N-ethylmaleimide or sodium arsenite, which have been shown to inhibit related enzymes . This can provide insights into active site chemistry and potential regulatory mechanisms.

How should researchers approach experimental design when investigating PGUG_04787's role in M. guilliermondii stress response?

Investigating PGUG_04787's role in M. guilliermondii stress response requires a multi-faceted experimental approach:

• Gene Expression Analysis Under Stress Conditions:

  • Expose M. guilliermondii cultures to various stressors (oxidative, thermal, osmotic, nutrient limitation)

  • Quantify PGUG_04787 expression using RT-qPCR relative to housekeeping genes

  • Compare expression profiles across different stress conditions and time points

• Generation of PGUG_04787 Knockout/Knockdown Strains:

  • Develop gene deletion mutants or RNA interference constructs targeting PGUG_04787

  • Confirm knockdown/knockout efficiency at protein and transcript levels

  • Assess growth rates, morphology, and stress survival compared to wild-type strains

• Metabolic Profiling:

  • Analyze fatty acid composition and lipid profiles in wild-type vs. mutant strains

  • Quantify changes in fatty acid saturation and chain length under stress conditions

  • Correlate metabolic changes with stress resistance phenotypes

• Complementation Studies:

  • Reintroduce functional PGUG_04787 into knockout strains under native or inducible promoters

  • Assess restoration of wild-type phenotypes to confirm specificity of observed effects

  • Test heterologous expression of PGUG_04787 in other yeast species to evaluate functional conservation

• Subcellular Localization Analysis:

  • Use fluorescently-tagged PGUG_04787 to track protein localization under normal and stress conditions

  • Perform subcellular fractionation followed by Western blotting to confirm localization patterns

  • Investigate potential relocalization or complex formation during stress response

Given M. guilliermondii's demonstrated resilience to manganese stress and its modulation of protein expression, particularly in genes related to DNA repair and oxidoreductase activity , special attention should be given to oxidative stress conditions when designing these experiments.

What bioinformatic approaches can be employed to analyze the evolutionary relationships of PGUG_04787?

Comprehensive bioinformatic analysis of PGUG_04787 evolutionary relationships should incorporate the following methodological approaches:

• Sequence-Based Phylogenetic Analysis:

  • Retrieve 3-ketoacyl-CoA reductase homologs from diverse fungal species, particularly within the Meyerozyma guilliermondii species complex (M. guilliermondii, M. carpophila, M. caribbica)

  • Include bacterial and plant homologs for broader evolutionary context

  • Construct multiple sequence alignments using MUSCLE or MAFFT algorithms

  • Generate maximum likelihood or Bayesian phylogenetic trees with appropriate substitution models

  • Assess node support through bootstrap analysis or posterior probabilities

• Protein Structure Prediction and Comparison:

  • Generate three-dimensional structure models using AlphaFold2 or similar tools

  • Compare predicted structures with experimentally determined structures of homologous enzymes

  • Identify conserved catalytic residues and substrate-binding regions

  • Analyze potential structural adaptations specific to M. guilliermondii

• Genome Context Analysis:

  • Examine syntenic relationships of PGUG_04787 across related genomes

  • Identify conservation or rearrangement of neighboring genes

  • Investigate potential horizontal gene transfer events or gene duplications

• Selection Pressure Analysis:

  • Calculate dN/dS ratios to detect signatures of positive, negative, or relaxed selection

  • Identify specific amino acid sites under selective pressure

  • Correlate selection patterns with functional domains or catalytic sites

• Gene Clustering Based on Expression Profiles:

  • Utilize available transcriptomic data to identify co-expressed genes

  • Construct gene co-expression networks to identify functional modules

  • Compare expression patterns across different stress conditions and growth phases

This multi-faceted approach will provide insights into the evolutionary history of PGUG_04787, its functional constraints, and potential adaptive specializations within the M. guilliermondii lineage, particularly in the context of its role in the species complex's remarkable adaptability to various environmental conditions .

What are the key catalytic residues in PGUG_04787 and how do they contribute to enzymatic function?

Based on comparative analysis with related ketoacyl reductases and the amino acid sequence provided , the key catalytic residues in PGUG_04787 likely include:

Catalytic Triad:
The enzyme likely contains a conserved catalytic triad typical of short-chain dehydrogenase/reductase (SDR) family proteins:

• Serine (S): Acts as a stabilizing residue for substrate positioning

• Tyrosine (Y): Functions as the catalytic base, abstracting a proton during the reduction reaction

• Lysine (K): Stabilizes the cofactor NADPH and lowers the pKa of the tyrosine residue

From the amino acid sequence (MLRLIDSISDNCTVKTALYGALLLGVYKLTTFALSLVSLVLDLWVLPPVNFAKYGAKKGKWAVITGASDGIGKEYATQLAAKGLNVVLVSRTESKLVALAEEIESKYKVSTKVLAFDVSLDAESSYEDLAATIADLPVTVLVNNVGQSHSIPVPFLETDEKELRNIITINNTATLKITQVVAPKIVHTVASEKKKTRGLILTMGSFGGLLPTPYLATYSGSKAFLQSWSNALSGELQPQGVDVELVISYLVTSAMSKIRRSSASIPNPKAFVKSVLRNVGRRVGAQERFGTTTPYWAHAFMHFGIVNSVGVYSKIANSLNLGMHKSIRSRALKKAARQKKD) , the key catalytic tyrosine is likely within the conserved YxxxK motif.

Cofactor Binding Residues:

• The glycine-rich motif (TGxxxGxG) near the N-terminus forms part of the Rossmann fold for NADPH binding

• This region typically contains the sequence WAVITGASDGIGK in the M. guilliermondii enzyme

• Additional residues, including asparagine and arginine residues, likely form hydrogen bonds with the adenine ribose and nicotinamide portions of NADPH

Substrate Binding Pocket:

• Hydrophobic residues create a pocket that accommodates the acyl chain of the substrate

• The pocket size and shape determine substrate specificity and chain-length preference

• Specific residues within this pocket interact with the thioester carbonyl group of the 3-ketoacyl-CoA substrate

The catalytic mechanism likely involves:

• NADPH binding in the Rossmann fold region

• 3-ketoacyl-CoA substrate binding in the adjacent pocket

• Hydride transfer from NADPH to the C3 carbonyl carbon of the substrate

• Proton donation from the catalytic tyrosine to form the 3-hydroxyacyl-CoA product

• Release of products (NADP+ and 3-hydroxyacyl-CoA)

How does the structure-function relationship of PGUG_04787 compare with mammalian 3-ketoacyl-CoA reductases?

The structure-function relationship of PGUG_04787 reveals both similarities and significant differences when compared to mammalian 3-ketoacyl-CoA reductases:

Similarities:

• Catalytic Mechanism: Both fungal and mammalian enzymes likely utilize a similar catalytic mechanism involving NADPH-dependent reduction of 3-ketoacyl substrates.

• Core Structural Elements: Both contain the characteristic Rossmann fold for nucleotide binding and a substrate-binding domain.

• Conserved Catalytic Residues: The presence of a catalytic triad (Ser-Tyr-Lys) is likely conserved across species boundaries for this enzyme class.

Key Differences:

• Quaternary Structure: While the specific quaternary structure of PGUG_04787 isn't explicitly stated in the search results, related fungal reductases often function as monomers or homodimers. In contrast, mammalian 3-ketoacyl-CoA reductases frequently form part of multi-enzyme complexes, particularly in the elongation of very long-chain fatty acids.

• Substrate Specificity Profile:

  Feature	M. guilliermondii PGUG_04787	Mammalian 3-ketoacyl-CoA reductases
  Chain length preference	Likely diverse range based on fungal metabolism	Often specialized for specific chain length ranges
  Cellular localization	Primarily cytosolic	Endoplasmic reticulum membrane-associated
  Integration with other enzymes	Likely independent enzyme	Often part of fatty acid synthase complex or elongase system

• Regulatory Mechanisms: Mammalian enzymes are typically subject to complex regulatory mechanisms including hormonal control and feedback inhibition, while fungal enzymes may respond more directly to environmental conditions and stress factors .

• Inhibitor Sensitivity: Fungal and mammalian reductases often display differential sensitivity to inhibitors, which has potential implications for antifungal drug development targeting PGUG_04787.

These differences reflect the evolutionary divergence between fungal and mammalian fatty acid metabolism, as well as the specialized adaptations of M. guilliermondii to its ecological niche, including its remarkable resilience to various environmental stressors .

What post-translational modifications might affect PGUG_04787 activity and how can they be detected?

Several post-translational modifications (PTMs) potentially affect PGUG_04787 activity, with methodological approaches for their detection:

Potential PTMs Affecting Activity:

• Phosphorylation

  • Impact: May regulate enzyme activity, subcellular localization, or protein-protein interactions

  • Detection methods:

    • Mass spectrometry (MS)-based phosphoproteomic analysis

    • Phospho-specific antibodies (if available)

    • Phos-tag SDS-PAGE for mobility shift detection

  • Experimental approach: Compare phosphorylation patterns under different growth conditions or stress responses

• Acetylation

  • Impact: May affect catalytic activity or protein stability

  • Detection methods:

    • MS-based acetylome analysis

    • Anti-acetyllysine antibodies

    • HDAC inhibitor treatment to enhance detection

• Ubiquitination/SUMOylation

  • Impact: May regulate protein turnover or localization

  • Detection methods:

    • Immunoprecipitation followed by ubiquitin/SUMO-specific Western blotting

    • MS-based ubiquitome/SUMOylome analysis

    • Expression of tagged ubiquitin/SUMO constructs

• Disulfide Bond Formation

  • Impact: May affect protein folding and stability

  • Detection methods:

    • Non-reducing vs. reducing SDS-PAGE

    • Mass spectrometry under non-reducing conditions

    • Targeted cysteine mutagenesis

Integrated Methodological Workflow:

• In silico PTM Prediction:

  • Use computational tools (NetPhos, UbPred, SUMOplot) to identify potential modification sites

  • Compare predictions across homologous proteins from related species

• PTM-Enriched Proteomics:

  • Perform targeted enrichment of modified peptides (e.g., TiO2 for phosphopeptides)

  • Analyze using high-resolution MS/MS

  • Quantify changes in modification status under different conditions

• Site-Directed Mutagenesis:

  • Generate mutants mimicking constitutive modification (e.g., S→D for phosphorylation) or preventing modification (e.g., S→A)

  • Compare enzymatic activities of wild-type and mutant proteins

  • Assess changes in protein stability, localization, or interaction partners

• In vitro Modification Assays:

  • Treat purified recombinant PGUG_04787 with specific kinases, acetylases, or other modifying enzymes

  • Measure changes in enzymatic activity

  • Confirm modification by western blotting or MS

Given M. guilliermondii's ability to modulate its protein expression in response to environmental stressors , investigation of stress-induced PTMs may provide valuable insights into regulatory mechanisms controlling PGUG_04787 activity during adaptive responses.

How can PGUG_04787 be utilized in biotechnological applications for fatty acid synthesis?

Recombinant PGUG_04787 offers several promising biotechnological applications for fatty acid synthesis:

• Biocatalysis for Stereoselective Reduction Reactions

  • The enzyme likely catalyzes the reduction of 3-ketoacyl-CoA to (3R)-3-hydroxyacyl-CoA with high stereoselectivity

  • This property can be exploited for the production of chirally pure 3-hydroxy fatty acids, which serve as valuable building blocks for pharmaceuticals, cosmetics, and specialty chemicals

  • Implementation methodology:

    • Immobilize purified PGUG_04787 on suitable matrices to enable recycling

    • Optimize reaction conditions (pH, temperature, cofactor regeneration)

    • Scale up to bioreactor systems for continuous production

• Metabolic Engineering of Microbial Cell Factories

  • Heterologous expression of PGUG_04787 in microbial hosts can alter fatty acid profiles

  • Potential applications include:

    • Production of medium-chain fatty acids (MCFAs) for biofuels

    • Synthesis of hydroxylated fatty acids for polymer applications

    • Modification of membrane lipid composition for stress tolerance

  • Implementation methodology:

    • Express PGUG_04787 in industrial microorganisms (E. coli, S. cerevisiae)

    • Combine with other pathway engineering strategies (e.g., thioesterase expression)

    • Optimize fermentation conditions for target product synthesis

• Enzyme Evolution for Novel Substrate Specificities

  • PGUG_04787 can serve as a starting point for directed evolution experiments

  • Goals may include:

    • Broadening substrate range to accept non-natural ketones

    • Enhancing thermostability for industrial processes

    • Modifying cofactor preference (NADH vs. NADPH)

  • Implementation methodology:

    • Generate mutation libraries using error-prone PCR or DNA shuffling

    • Develop high-throughput screening assays for desired properties

    • Characterize improved variants and elucidate structure-function relationships

• Development of Biosensors for Fatty Acid Metabolites

  • Engineer PGUG_04787 as a component of biosensing systems

  • Applications include:

    • Real-time monitoring of fatty acid synthesis in bioprocesses

    • Detection of fatty acid metabolites in diagnostic applications

    • Screening for enzyme inhibitors in drug discovery

  • Implementation methodology:

    • Couple enzyme activity to fluorescent or colorimetric readouts

    • Develop whole-cell biosensors for high-throughput applications

    • Optimize sensitivity and specificity through protein engineering

The cold adaptation properties observed in other enzymes from M. guilliermondii suggest that PGUG_04787 might possess unique characteristics suitable for biotechnological applications at lower temperatures, potentially reducing energy requirements for industrial processes.