Recombinant Saccharomyces bayanus Dihydroorotate dehydrogenase (fumarate) (URA1)

What is Dihydroorotate dehydrogenase (DHOD) and what is its role in the pyrimidine biosynthesis pathway?

Dihydroorotate dehydrogenase (DHOD) catalyzes the oxidation of (S)-dihydroorotate to orotate, which constitutes the fourth step and the only redox reaction in the de novo biosynthesis of UMP, the precursor for all pyrimidine nucleotides .

The reaction proceeds as follows:

$(S)-\text{dihydroorotate} + \text{electron acceptor} \rightarrow \text{orotate} + \text{reduced electron acceptor}$

In Saccharomyces species with family 1A DHOD enzymes like S. cerevisiae and S. bayanus, fumarate serves as the primary electron acceptor, whereas family 2 DHODs in other eukaryotes typically use quinones as electron acceptors . The cytosolic family 1A DHOD enzyme contains FMN as a cofactor, which is essential for its catalytic activity .

Methodology for investigating DHOD activity typically involves spectrophotometric assays that measure either the formation of orotate (which absorbs at 300 nm) or the reduction of the electron acceptor. For S. bayanus DHOD specifically, assays should be designed to monitor fumarate reduction to succinate.

How are DHOD enzymes classified, and which family does S. bayanus URA1 belong to?

DHOD enzymes are classified into two major families based on their structure, subcellular localization, and electron acceptor preferences:

S. bayanus URA1 encodes a Family 1A DHOD that is functionally similar to S. cerevisiae URA1 . This classification has significant implications for experimental design, as Family 1A enzymes require different assay conditions compared to Family 2 enzymes. When working with recombinant S. bayanus DHOD, researchers should use fumarate as the electron acceptor in activity assays and buffer conditions optimized for cytosolic enzymes rather than membrane-bound proteins.

What evolutionary evidence suggests horizontal gene transfer of URA1 in Saccharomyces species?

Comparative genomic and phylogenetic analyses provide compelling evidence that the URA1 gene in Saccharomyces species was acquired through horizontal gene transfer from bacteria:

• Sequence similarity analysis shows that S. cerevisiae and S. bayanus URA1 are more closely related to bacterial family 1A DHODs (particularly from Lactobacillales) than to the typical eukaryotic family 2 DHODs .

• The gene structure of URA1 in Saccharomyces species differs from typical eukaryotic DHOD genes but resembles bacterial homologs .

• Studies with S. kluyveri revealed that this yeast species possesses both a family 1A DHOD (similar to S. cerevisiae) and a family 2 DHOD (typical of eukaryotes), suggesting that the acquisition of family 1A DHOD occurred after the divergence of the Saccharomyces lineage from the Candida albicans lineage .

• The presence of URA1 correlates with the ability of Saccharomyces species to grow anaerobically, suggesting that the horizontally acquired gene provided an adaptive advantage by allowing pyrimidine synthesis without oxygen .

Research methodologies for investigating this evolutionary history include whole genome sequencing, phylogenetic tree construction, and comparative analyses of gene synteny across multiple yeast species.

What is known about the expression and regulation of URA1 in Saccharomyces species?

The expression and regulation of URA1 in Saccharomyces species involve both species-specific and conserved mechanisms:

• In S. cerevisiae, the expression of URA1 is controlled by a nuclear regulatory gene (pprX-1) that constitutively enhances the expression of URA1 as well as URA3 at the transcriptional level .

• The yeast URA1 gene can be expressed in Escherichia coli, but its expression depends on the orientation of the cloned fragment and requires a prokaryotic promoter for transcription initiation .

• In contrast, when URA1 is expressed from a plasmid in S. cerevisiae, its expression does not depend on the orientation of the cloned fragment, indicating that transcription involves a physiological yeast promoter cloned along with the structural part of the gene .

To study URA1 regulation, researchers typically employ techniques such as:

• Northern blot analysis to quantify transcript levels

• Reporter gene assays using URA1 promoter constructs

• Chromatin immunoprecipitation to identify regulatory proteins binding to the URA1 promoter

• Gene deletion studies to identify regulatory factors

What are the biochemical properties of recombinant S. bayanus DHOD?

Recombinant S. bayanus DHOD shares several biochemical properties with its S. cerevisiae homolog but may have distinct characteristics:

• The enzyme exists as a homodimer in vitro, similar to recombinant S. cerevisiae DHOD .

• It contains FMN as a cofactor, which is essential for its catalytic activity .

• The enzyme shows competitive inhibition by the reaction product orotate, with a reported Ki of approximately 7.7 μM for S. cerevisiae DHOD .

• Family 1A DHODs, including S. bayanus DHOD, use fumarate as the physiological electron acceptor, distinguishing them from other DHOD families .

• Like other cytosolic DHODs, S. bayanus DHOD is expected to have optimal activity at neutral to slightly basic pH (7.0-8.0) and moderate temperatures (25-30°C).

When working with recombinant S. bayanus DHOD, researchers should consider these properties for experimental design, particularly for enzyme kinetics studies and inhibitor screening.

What methods are most effective for cloning, expressing, and purifying recombinant S. bayanus DHOD?

Cloning and expressing recombinant S. bayanus URA1 requires careful consideration of several factors to ensure optimal protein production:

Cloning strategy:

• Genomic DNA extraction from S. bayanus using standard yeast protocols

• PCR amplification of the complete URA1 open reading frame using primers designed based on conserved regions

• Restriction enzyme digestion and ligation into an appropriate expression vector

• Verification of the construct by sequencing to confirm the absence of mutations

Expression systems:

• E. coli: When expressing S. bayanus URA1 in E. coli, orientation of the gene in the vector is critical, as improper orientation may result in lack of expression . Consider using vectors with strong prokaryotic promoters (T7, tac) and fusion tags to enhance solubility.

• Yeast expression: For native-like expression, S. cerevisiae expression systems using vectors like pRS416 have been successfully used for URA1 homologs . When expressing in S. cerevisiae, the orientation of the cloned fragment is less critical as transcription involves a physiological yeast promoter .

Purification protocol:

• Affinity chromatography using His-tag or GST-tag depending on the construct design

• Ion exchange chromatography to remove contaminants

• Size exclusion chromatography to obtain the native dimeric form and remove aggregates

• Throughout purification, include FMN (5-10 μM) in all buffers to maintain enzyme stability

Functional validation:
Activity assays measuring the conversion of dihydroorotate to orotate using spectrophotometric methods at 300 nm, or by monitoring fumarate reduction using coupled enzyme assays.

How can researchers characterize the kinetic properties and catalytic mechanism of S. bayanus DHOD?

A comprehensive characterization of S. bayanus DHOD kinetics and catalytic mechanism requires multiple experimental approaches:

Steady-state kinetics:

• Determine Km and kcat values for both dihydroorotate and fumarate using varied substrate concentrations

• Analyze the enzyme's ping-pong mechanism (typical for family 1A DHODs) by varying both substrates systematically

• Determine product inhibition constants for orotate and succinate

Pre-steady-state kinetics:

• Use stopped-flow spectroscopy to measure rates of individual steps in the reaction

• Analyze flavin reduction and oxidation by monitoring absorbance changes at 450 nm

• Determine rate-limiting steps in the catalytic cycle

pH and temperature dependence:

• Measure enzyme activity across pH range (5.5-9.0) to identify catalytic residues

• Determine temperature optima and calculate thermodynamic parameters

Structure-function studies:

• Perform site-directed mutagenesis of key residues (based on homology to characterized DHODs):

  • Lys43 (expected to interact with FMN and stabilize reaction intermediates)

  • Cys130 (expected to function as a general acid/base catalyst)

• Analyze mutant enzymes kinetically to determine the role of each residue

For the catalytic mechanism, studies with L. lactis DHOD (another family 1A enzyme) support a one-site ping-pong mechanism where the first half-reaction involves hydride transfer from dihydroorotate to FMN, yielding orotate and FMNH2, followed by the second half-reaction where fumarate oxidizes FMNH2 back to FMN .

What approaches can be used to determine the three-dimensional structure of S. bayanus DHOD?

Determining the three-dimensional structure of S. bayanus DHOD would provide valuable insights into its function and evolution. Here are methodological approaches:

X-ray crystallography approach:

• Protein preparation:

  • Express recombinant protein with high purity (>95%)

  • Ensure homogeneity through size exclusion chromatography

  • Concentrate to 10-15 mg/mL for crystallization trials

• Crystallization screening:

  • Use commercial sparse matrix screens to identify initial conditions

  • Optimize promising conditions by varying precipitant concentration, pH, and additives

  • Consider co-crystallization with ligands (dihydroorotate, orotate, FMN) to stabilize the protein

• Data collection and structure determination:

  • Collect diffraction data at synchrotron sources for optimal resolution

  • Consider molecular replacement using L. lactis DHOD structure (PDB: 2DOR) as a search model

  • Refine the structure using standard crystallographic protocols

Alternative structural approaches:

• Cryo-electron microscopy:

  • Particularly useful if crystallization proves challenging

  • May require forming larger complexes to increase molecular weight

• NMR spectroscopy:

  • Suitable for analyzing dynamics and ligand binding

  • Limited to smaller domains or fragments due to size constraints

• Small-angle X-ray scattering (SAXS):

  • Provides low-resolution envelope of the protein in solution

  • Useful for confirming quaternary structure and large conformational changes

• Homology modeling:

  • Build preliminary models based on existing structures of family 1A DHODs

  • Validate models through functional studies of predicted critical residues

Structural information would be particularly valuable for understanding the molecular basis of S. bayanus DHOD's specificity for fumarate as an electron acceptor and for designing specific inhibitors.

How can researchers investigate the role of S. bayanus URA1 in anaerobic growth and metabolism?

Investigating the role of S. bayanus URA1 in anaerobic growth requires specialized experimental setups and analytical approaches:

Genetic manipulation approaches:

• Generate URA1 deletion mutants using CRISPR-Cas9 or homologous recombination:

  • Replace URA1 with a selectable marker (e.g., KanMX)

  • Confirm successful deletion by PCR verification

• Complementation studies:

  • Transform ura1Δ strains with vectors expressing URA1 from different species

  • Compare S. bayanus URA1 with S. cerevisiae URA1 and family 2 DHODs

Growth and physiological characterization:

• Anaerobic cultivation:

  • Use anaerobic chambers or sealed vessels with oxygen-scavenging systems

  • Monitor growth by optical density measurements

  • Test growth on different carbon sources to assess metabolic flexibility

• Metabolic flux analysis:

  • Use 13C-labeled substrates to trace metabolic pathways

  • Quantify pyrimidine precursors and products by LC-MS/MS

  • Compare aerobic vs. anaerobic metabolic profiles

Biochemical analysis:

• Enzyme activity assays:

  • Compare DHOD activity under aerobic and anaerobic conditions

  • Assess the ability of different electron acceptors to support activity

• Redox state analysis:

  • Measure NAD+/NADH and FAD/FADH2 ratios

  • Analyze the impact of URA1 deletion on cellular redox balance

Existing research has demonstrated that URA1 is required for anaerobic biosynthesis of uracil in S. cerevisiae, and that the family 2 DHOD from A. gossypii cannot complement uracil auxotrophy under anaerobic conditions . Similar studies with S. bayanus URA1 would provide important comparative data on the evolutionary significance of this horizontally-acquired gene.

What approaches can be used to identify and characterize specific inhibitors of S. bayanus DHOD?

Developing specific inhibitors of S. bayanus DHOD requires a systematic approach combining computational, biochemical, and cellular methods:

Virtual screening and in silico approaches:

• Homology modeling of S. bayanus DHOD based on related family 1A DHOD structures

• Molecular docking of compound libraries to identify potential binding modes

• Molecular dynamics simulations to assess stability of protein-ligand complexes

• Pharmacophore modeling based on known inhibitors of family 1A DHODs

Biochemical screening methods:

• Primary assays:

  • Spectrophotometric assays measuring inhibition of dihydroorotate oxidation

  • Fluorescence-based assays monitoring FMNH2 oxidation

• Mechanism of inhibition studies:

  • Steady-state kinetics with varying substrate and inhibitor concentrations

  • Determine inhibition constants (Ki) and inhibition types (competitive, uncompetitive, or mixed)

• Binding studies:

  • Isothermal titration calorimetry to measure binding affinity and thermodynamics

  • Surface plasmon resonance to determine association and dissociation rates

Structure-activity relationship studies:

• Synthesize analogs of lead compounds

• Test activity against S. bayanus DHOD and related enzymes

• Optimize potency, selectivity, and physicochemical properties

Selectivity profiling:

• Test activity against family 2 DHODs from humans and other organisms

• Assess activity against other flavoenzymes to determine specificity

Studies have shown that benzoates, as pyrimidine ring analogs, are selective inhibitors of cytosolic DHODs like those found in Saccharomyces species . This unique property could be exploited to develop species-specific antifungal agents that target the horizontally-acquired DHOD in S. bayanus without affecting the structurally distinct human enzyme.

How can researchers study the genetic stability and evolutionary implications of the URA1 locus in S. bayanus?

Analyzing the genetic stability and evolutionary significance of URA1 in S. bayanus requires integration of multiple genetic, genomic, and comparative approaches:

Whole genome sequence analysis:

• Compare URA1 sequences across multiple S. bayanus strains to assess intraspecies variation

• Analyze GC content, codon usage, and other sequence features to detect signs of horizontal gene transfer

• Examine synteny and gene neighborhood across Saccharomyces species

Experimental evolution studies:

• Subject S. bayanus to long-term evolution under different selective pressures:

  • Aerobic vs. anaerobic conditions

  • Different carbon sources

  • Presence of pyrimidine precursors or inhibitors

• Sequence URA1 and surrounding regions at various time points to detect mutations

• Perform competitive fitness assays to quantify selection coefficients

Hybrid genetics approaches:

• Create interspecific hybrids between S. bayanus and related species

• Monitor chromosome stability during fermentation and sporulation

• Track inheritance patterns of URA1 during hybrid evolution

Research has shown that in interspecific hybrids (such as between S. cerevisiae and S. uvarum), the S. uvarum Chromosome 14, which contains URA1, showed high instability with 95% of isolates losing the entire chromosome after multiple fermentation cycles . This suggests potential incompatibilities or selective pressures against maintaining both copies of URA1 in hybrid genomes, which has important implications for hybrid stability and evolution.

What methodologies can be applied to engineer S. bayanus DHOD for enhanced activity or novel functionalities?

Engineering S. bayanus DHOD for improved or novel properties requires sophisticated protein engineering approaches:

Rational design strategies:

• Structure-guided mutagenesis:

  • Modify residues in the active site to alter substrate specificity

  • Introduce mutations to enhance thermostability or solubility

  • Engineer the electron acceptor binding site to accommodate alternative acceptors

• Domain swapping:

  • Create chimeric enzymes with domains from family 1B or family 2 DHODs

  • Evaluate functionality of hybrid enzymes under different conditions

Directed evolution approaches:

• Random mutagenesis:

  • Error-prone PCR to generate libraries with random mutations

  • DNA shuffling to recombine beneficial mutations

• Selection systems:

  • Develop growth-based selection in ura1Δ yeast under challenging conditions

  • Use fluorescence-activated cell sorting with activity-based fluorescent probes

• High-throughput screening:

  • Colorimetric assays in microtiter plate format

  • Automated liquid handling for rapid enzyme variant characterization

Computational protein design:

• Use algorithms to predict stabilizing mutations

• In silico evolution to identify mutations that might alter specificity

• De novo active site design for novel catalytic activities

Potential engineering goals:

• Expand substrate range to include modified dihydroorotates

• Engineer the ability to use alternative electron acceptors

• Enhance stability for industrial applications

• Create DHOD variants that function efficiently across wider temperature or pH ranges

This approach could yield engineered enzymes with applications in synthetic biology, metabolic engineering, and biocatalysis. The unique properties of family 1A DHODs, such as their ability to function under anaerobic conditions, make them particularly interesting targets for engineering efforts aimed at creating yeast strains with novel metabolic capabilities.