Recombinant Saccharomyces cerevisiae Squalene monooxygenase (ERG1)

What is Saccharomyces cerevisiae squalene monooxygenase (ERG1) and what is its function?

Squalene monooxygenase (also known as squalene epoxidase) is encoded by the ERG1 gene in Saccharomyces cerevisiae and catalyzes a critical step in the ergosterol biosynthesis pathway. This enzyme converts squalene to 2,3-oxidosqualene by introducing an epoxide group, representing a rate-limiting step in sterol biosynthesis. The ERG1 gene encodes a 55,190-Da peptide consisting of 496 amino acids . This enzyme is particularly important as it serves as a target for antifungal drugs such as allylamines (e.g., terbinafine), making it both biochemically and pharmacologically significant in fungal research .

How was the ERG1 gene initially isolated and characterized?

The ERG1 gene was initially cloned by screening a gene library prepared from an allylamine-resistant (AlR) Saccharomyces cerevisiae mutant. Researchers transformed a sensitive strain with this library and selected for AlR colonies. The squalene epoxidase activity tested in cell-free extracts from one of these transformants demonstrated resistance to the allylamine derivative terbinafine, confirming that the recombinant plasmid carried an allelic form of the ERG1 gene .

Nucleotide sequencing revealed one open reading frame coding for a 55,190-Da peptide of 496 amino acids. Through Southern hybridization experiments, researchers were able to localize the ERG1 gene on yeast chromosome 15 . This approach exemplifies how combining functional selection with molecular characterization can successfully identify genes encoding important enzymes.

What is the structural organization of the Erg1 protein?

The Erg1 protein contains multiple functional domains critical for its enzymatic activity. Based on homology modeling with p-hydroxybenzoate hydroxylase (PHBH) from Pseudomonas fluorescens, the Erg1 protein contains:

• Two flavin adenine dinucleotide (FAD) binding domains (FADI and FADII)

• One nucleotide binding (NB) site

How do mutations in the ERG1 gene affect antifungal susceptibility?

Mutations in the ERG1 gene can significantly alter susceptibility to antifungal compounds, particularly allylamines such as terbinafine. Research has identified several key amino acid substitutions affecting drug sensitivity:

In vitro mutagenesis of the ERG1 gene has yielded alleles that confer either increased terbinafine sensitivity or complete loss of function (lethal phenotype) when expressed in erg1-knockout strains . Most critical mutations affect conserved FAD/nucleotide binding sites. The G25S, D335X, and G210A substitutions in the FADI, FADII, and NB sites respectively render the enzyme variants completely non-functional, while G30S and L37P variants exhibit decreased enzymatic activity accompanied by a sevenfold increase in sensitivity to terbinafine .

What regulatory mechanisms control ERG1 expression?

ERG1 expression is regulated as part of the broader ergosterol biosynthesis pathway, with several transcription factors playing key roles. Although the search results don't provide direct information about ERG1 regulation specifically, they offer insights into the regulation of other ERG genes that likely apply to ERG1 as well:

• The oxygen-responsive repressor Rox1p plays a significant role in regulating ERG gene expression. ROX1 deletion resulted in 2.5- to 16-fold-lower susceptibilities to azoles and terbinafine, indicating increased expression of ergosterol biosynthesis genes .

• Expression patterns show an inverse correlation between ERG11 (another ergosterol biosynthesis gene) and ROX1 expression. In untreated cultures, ERG11 was maximally expressed in mid-log phase with decreased expression in late log phase, while ROX1 showed the inverse pattern .

• In azole-treated cultures, ERG11 upregulation was preceded by a decrease in ROX1 RNA levels, suggesting that transcriptional regulation of ROX1 is an important determinant of ERG gene expression .

This regulatory relationship likely extends to ERG1, suggesting that Rox1p may be a key negative regulator of ERG1 expression as well, particularly under different oxygen conditions or in response to antifungal treatment.

What experimental approaches are effective for studying ERG1 function in vivo?

Several experimental approaches have proven effective for studying ERG1 function in vivo:

• Gene knockout and complementation studies: Creating erg1-knockout strains (such as KLN1) and complementing with wild-type or mutant ERG1 alleles allows for assessment of gene function and phenotypic effects . This approach reveals whether specific mutations affect enzyme activity, antifungal susceptibility, or cell viability.

• PCR-based random mutagenesis: Random mutagenesis of the ERG1 gene by PCR amplification followed by transformation and selection in appropriate yeast strains can identify functional variants with altered properties . This technique has been successful in identifying mutations that confer altered terbinafine sensitivity.

• Aerobic growth complementation assays: Since ERG1 is essential for aerobic growth, complementation of growth defects on YPD agar plates provides a straightforward method to assess ERG1 functionality .

• Antifungal susceptibility testing: Testing transformants for growth on media containing antifungal agents (e.g., 10 μg/ml terbinafine) allows for identification of mutations affecting drug sensitivity .

These approaches can be combined with molecular analyses such as DNA sequencing to correlate specific genetic changes with functional outcomes.

What methods are most effective for creating site-directed mutations in the ERG1 gene?

For creating specific mutations in the ERG1 gene, several methodologies have proven effective:

• PCR-based site-directed mutagenesis: Researchers have successfully used PCR amplification with mutagenic primers to introduce specific nucleotide changes into the ERG1 gene . This approach allows for precise alterations at defined positions within conserved domains.

• Plasmid-based expression systems: Cloning the ERG1 gene into centromere vectors like pRS315 facilitates both mutagenesis and subsequent expression in yeast . These vectors maintain stable low copy numbers, providing expression levels close to physiological conditions.

• Transformation and selection protocol: The protocol developed by Gietz et al. has been effectively used for transforming mutant constructs into S. cerevisiae strains like KLN1 . This involves:

  • Preparation of competent yeast cells

  • Transformation with purified plasmid DNA

  • Selection for transformants based on complementation of growth defects

  • Secondary screening for altered antifungal susceptibility

• Functional validation: Confirming the effects of mutations through complementation assays and drug susceptibility testing provides functional validation of the mutagenesis approach .

For optimal results, targeting conserved domains (FAD binding sites, nucleotide binding sites) based on homology models has proven particularly informative in structure-function studies of the Erg1 protein.

How can recombinant ERG1 be expressed and purified for biochemical studies?

While the search results don't provide specific details on ERG1 expression and purification, we can infer approaches based on similar recombinant protein systems:

What analytical methods are suitable for measuring ERG1 enzymatic activity?

Several analytical approaches can be employed to measure ERG1 enzymatic activity:

• Cell-free extract assays: Measuring squalene epoxidase activity in cell-free extracts from transformed yeast strains has been successfully used to evaluate the effects of mutations on enzyme function .

• Inhibition assays: Assessing the inhibitory effects of allylamine derivatives like terbinafine on wild-type versus mutant ERG1 enzymes provides valuable information about structure-function relationships and drug resistance mechanisms .

• Chromatographic analysis: HPLC or GC-MS methods can be used to directly measure the conversion of squalene to 2,3-oxidosqualene, allowing for quantitative assessment of enzyme kinetics.

• Coupled enzyme assays: Developing coupled enzyme systems where the activity of ERG1 is linked to a detectable signal (e.g., fluorescence, absorbance) can facilitate high-throughput analysis of enzyme activity.

• In vivo ergosterol quantification: Indirectly assessing ERG1 function by measuring total ergosterol content in cells expressing different ERG1 variants.

How can researchers resolve conflicting data on ERG1 inhibition mechanisms?

When faced with conflicting data regarding ERG1 inhibition mechanisms:

• Standardize experimental conditions: Ensure that all comparisons are made under identical conditions, including:

  • Enzyme source and preparation

  • Substrate concentrations

  • Buffer composition and pH

  • Temperature and incubation times

  • Inhibitor preparation and storage

• Consider strain background effects: Genetic background can significantly impact drug susceptibility profiles. The same mutation may produce different phenotypes in different strain backgrounds due to compensatory mechanisms or genetic interactions .

• Verify protein expression levels: Western blot analysis to confirm that observed differences are not simply due to varying expression levels of the ERG1 variants being compared.

• Employ multiple inhibition assessment methods: Combine:

  • In vitro enzymatic assays

  • Whole-cell growth inhibition assays

  • Molecular dynamics simulations based on structural models

• Investigate time-dependent effects: Some inhibition mechanisms may show time-dependent characteristics that could explain apparent discrepancies between studies using different experimental timeframes.

What approaches help distinguish between direct ERG1 inhibition and indirect effects on enzyme activity?

Distinguishing direct inhibition from indirect effects requires multi-faceted approaches:

• Purified enzyme studies: Working with purified recombinant ERG1 allows for direct assessment of inhibitor binding and enzymatic inhibition without confounding cellular factors.

• Domain-specific mutations: Creating mutations in putative inhibitor binding sites can help confirm the direct binding mechanism of inhibitors. If such mutations alter inhibitor efficacy without affecting enzyme activity, this supports a direct interaction model .

• Cellular metabolite profiling: Analyzing the sterol profile in treated cells can help distinguish between direct ERG1 inhibition and effects on other pathway components.

• Transcriptional response analysis: Monitoring the expression of ERG1 and related genes (like the ROX1 repressor) following inhibitor treatment can reveal whether observed effects involve transcriptional regulation mechanisms .

• In silico docking studies: Computational approaches using homology models of ERG1 can predict inhibitor binding modes and guide experimental verification .

What emerging technologies might enhance our understanding of ERG1 structure and function?

Several emerging technologies hold promise for advancing ERG1 research:

• Cryo-electron microscopy: This technique could potentially resolve the complete structure of ERG1, providing insights beyond what homology modeling with p-hydroxybenzoate hydroxylase has revealed .

• CRISPR-Cas9 genome editing: This approach allows for precise modification of the ERG1 gene in its native chromosomal context, enabling studies of ERG1 function without the complications of plasmid-based expression.

• Quantitative proteomics: MS-based approaches to monitor changes in the fungal proteome in response to ERG1 inhibition or mutation could reveal new insights into compensatory mechanisms and pathway regulation.

• Single-cell analysis technologies: These methods could help understand the heterogeneity in ERG1 expression and activity within populations, which may contribute to antifungal resistance phenomena.

• Synthetic biology approaches: Engineering synthetic regulatory circuits controlling ERG1 expression could provide new tools for studying its regulation and potentially developing novel antifungal strategies.

How might research on ERG1 contribute to addressing antifungal resistance?

Understanding the molecular mechanisms of ERG1 function and inhibition has significant implications for addressing antifungal resistance:

• Rational drug design: Detailed knowledge of the ERG1 structure and inhibitor binding modes can guide the development of new antifungal compounds that might overcome existing resistance mechanisms .

• Combination therapy strategies: Understanding how ERG1 inhibition interacts with other antifungal mechanisms could inform effective drug combinations that prevent resistance development.

• Resistance surveillance: Molecular characterization of ERG1 mutations in clinical isolates could serve as biomarkers for predicting treatment outcomes and guiding antifungal selection.

• Understanding regulatory networks: Elucidating how transcription factors like Rox1p regulate ERG1 expression may reveal new targets for intervention to prevent adaptive responses that lead to resistance .

• Cross-species comparative studies: Comparing ERG1 structure and function across fungal species could identify conserved vulnerabilities that might be exploited for broad-spectrum antifungal development.