Recombinant Lachancea kluyveri Dihydroorotate dehydrogenase (fumarate) (URA1)

What distinguishes L. kluyveri URA1 from other dihydroorotate dehydrogenases?

L. kluyveri URA1 belongs to the Class I-A dihydroorotate dehydrogenases, which are soluble homodimers with one FMN domain per subunit that use fumarate as an electron acceptor. This differs significantly from Class II DHODs (like URA9), which are membrane-associated enzymes that donate electrons to the quinone pool of the mitochondrial respiratory chain . The fundamental biochemical reaction catalyzed by URA1 can be represented as: Dihydroorotate + Fumarate → Orotate + Succinate. This reaction mechanism enables URA1 to function under anaerobic conditions, a critical distinction from Class II DHODs that indirectly require oxygen . The ability to use fumarate instead of oxygen-dependent electron acceptors allows organisms possessing URA1 to maintain pyrimidine prototrophy even in oxygen-limited environments .

How does L. kluyveri URA1 compare structurally and functionally to URA9?

The structural and functional differences between URA1 and URA9 in L. kluyveri are significant and directly impact their biological roles:

URA9 is described as "Dihydroorotate dehydrogenase (quinone), mitochondrial" with a specific amino acid sequence that includes regions for membrane association . In contrast, URA1 is a cytosolic enzyme that contains conserved amino acid residues involved in flavin cofactor binding and an active-site cysteine residue that is strongly conserved in Class I-A DHODs . These structural differences directly impact their functionality, with URA1 enabling pyrimidine synthesis under both aerobic and anaerobic conditions, while URA9 requires the mitochondrial respiratory chain and thus indirectly depends on oxygen .

What evidence supports horizontal gene transfer in the acquisition of URA1?

The presence of Class I-A DHODs like URA1 in Saccharomycotina yeasts is proposed to result from horizontal gene transfer (HGT) from lactic acid bacteria . Several lines of evidence support this hypothesis: First, phylogenetic analyses show that fungal Class I-A DHODs cluster more closely with bacterial homologs than with other fungal DHODs, suggesting a bacterial origin rather than vertical inheritance . Second, the sparse distribution of Class I-A DHODs among fungi (primarily in Saccharomycotina and some Mucoromycotina) is inconsistent with vertical inheritance but aligns with the pattern expected from HGT . Third, the presence of Class I-A DHODs correlates with the ability of these fungi to grow anaerobically without pyrimidine supplementation, suggesting that HGT provided a selective advantage in oxygen-limited environments . The identification of URA1-like sequences in phylogenetically distant fungi like Mucoromycotina further supports the occurrence of HGT events, despite the considerable evolutionary distance between these groups .

How widespread are URA1 orthologs across fungal species?

HMMER searches using L. kluyveri URA1 (LkUra1, Q7Z892) as a query have revealed a surprisingly wide distribution of potential URA1 orthologs across fungal lineages . The search yielded 203 putative Ura1 orthologs, with a distribution pattern more complex than previously thought . Eight of these proteins originated from Mucoromycotina and showed strong homology to well-known Class I-A DHODs from S. cerevisiae, L. kluyveri, and other Saccharomycotina yeasts, despite the significant phylogenetic distance between these groups . Additionally, a large group of putative URA1 orthologs was unexpectedly found in Ascomycota and Basidiomycota . These sequences formed two large clusters, primarily consisting of sequences from either ascomycetes or basidiomycetes exclusively . Only two sequences from basidiomycetes (Exidia glandulosa and Cutaneotrichonsporon oleaginosum) clustered with those from ascomycetes, suggesting distinct evolutionary trajectories for these genes in different fungal lineages .

What are optimal approaches for heterologous expression of L. kluyveri URA1?

For successful heterologous expression of L. kluyveri URA1, researchers should consider the following methodological approaches:

• Vector design: Construct expression vectors containing codon-optimized LkURA1 coding sequences for the host organism, preferably with appropriate promoters (strong constitutive promoters for high expression) .

• Host selection: Saccharomyces cerevisiae ura1Δ strains serve as excellent hosts for functional verification through complementation studies . These strains lack DHOD activity and require uracil supplementation for growth on synthetic medium with glucose (SMD) .

• Transformation protocol: Standard yeast transformation methods such as lithium acetate/PEG transformation for S. cerevisiae provide efficient introduction of URA1 expression constructs .

• Expression verification: Confirm functional expression through:

  • Growth complementation on synthetic media without uracil

  • Western blot analysis if epitope tags are included

  • Enzyme activity assays in cell extracts

• Growth assessment: Compare growth rates of transformants under different conditions (aerobic vs. anaerobic, with/without uracil supplementation) to verify functional expression . Wild-type S. cerevisiae strains typically show specific growth rates of approximately 0.37 h⁻¹ on various media, while ura1Δ strains only grow when supplemented with uracil .

How can URA1 enzyme activity be accurately measured in vitro?

Accurate measurement of URA1 enzyme activity in vitro can be achieved through several complementary approaches:

• Spectrophotometric assays:

  • Monitor dihydroorotate oxidation (decrease in absorbance at 230 nm)

  • Track fumarate reduction (decrease in absorbance at 300 nm)

  • Use coupled assays linking fumarate reduction to NADH oxidation (340 nm)

• Oxygen consumption measurements:

  • While URA1 itself does not consume oxygen, oxygen electrode measurements can help differentiate URA1 activity from oxygen-dependent DHODs .

  • Control experiments with and without oxygen are essential to confirm the oxygen-independent nature of URA1 .

• Substrate specificity testing:

  • Assess activity with different substrates (dihydroorotate, dihydrouracil, dihydrothymine)

  • This helps distinguish URA1 from related enzymes like dihydrouracil oxidase (DHO) .

• Activity verification in cell extracts:

  • Prepare cell extracts from strains expressing URA1

  • Measure enzyme activity under different conditions (aerobic/anaerobic)

  • Quantify substrate conversion and product formation

The search results indicate methods for measuring dihydrouracil-dependent and dihydrothymine-dependent oxygen consumption in cell extracts, which can be adapted for URA1 activity measurements with dihydroorotate as the substrate .

How does URA1 enable anaerobic pyrimidine synthesis in fungi?

URA1 enables anaerobic pyrimidine synthesis through its unique biochemical mechanism that functions independently of oxygen . In most fungi, dihydroorotate dehydrogenases (typically Class II DHODs) donate electrons to the quinone pool of the mitochondrial respiratory chain, which ultimately requires oxygen as the terminal electron acceptor . This dependency makes pyrimidine synthesis oxygen-dependent in these organisms . In contrast, URA1 (a Class I-A DHOD) uses fumarate as an electron acceptor instead of components of the respiratory chain . The reaction catalyzed by URA1 converts dihydroorotate to orotate while reducing fumarate to succinate, completely bypassing the need for oxygen in this critical step of pyrimidine biosynthesis . This capability is evidenced by growth experiments showing that S. cerevisiae strains expressing URA1 can grow anaerobically without pyrimidine supplementation, whereas strains expressing only oxygen-dependent DHODs cannot . The functional significance of URA1 for anaerobic growth was confirmed through experiments with S. cerevisiae reference strains (URA1+) that showed consistent growth rates (~0.37 h⁻¹) under various media conditions, while ura1Δ strains were unable to grow on synthetic media without uracil supplementation .

How can URA1 be utilized as a genetic marker in fungal transformation experiments?

URA1 offers several advantages as a genetic marker for fungal transformation experiments:

• Complementation-based positive selection:

  • URA1 can complement ura1Δ mutations in S. cerevisiae and other fungi

  • This allows for selection of transformants on media lacking uracil

  • The system provides a clean selection method without antibiotics

• Integration verification:

  • PCR and Southern blot analysis can confirm proper integration

  • Functional complementation (growth without uracil) provides phenotypic verification

• Experimental applications:

  • The search results demonstrate the use of URA1-based complementation to study enzyme function in vivo

  • Strains expressing different DHOD variants can be compared on synthetic media with/without uracil supplementation

  • Growth rates can be measured to quantify the efficiency of complementation

• Comparative studies:

  • URA1-based systems can be used to compare the functionality of DHODs from different species

  • The system allows for testing both aerobic and anaerobic functionality

The experimental approach described in the search results demonstrates practical application through systematic testing of strains on different media types (SMD, SMD+ura, SMD+dhu) and measurement of specific growth rates to assess functional complementation .

What structure-function relationships exist between conserved residues in URA1 and its catalytic activity?

The catalytic activity of URA1 is dependent on several key conserved residues that define its structure-function relationships:

• FMN cofactor binding domain:

  • Conserved amino acid residues form hydrogen bonds with the FMN cofactor, positioning it optimally for electron transfer during catalysis .

  • These residues are strongly conserved across Class I-A DHODs, including URA1 from L. kluyveri .

• Active-site cysteine:

  • A strongly conserved cysteine residue is present in all Class I-A DHODs and is critical for catalysis .

  • This cysteine participates directly in the redox chemistry during the catalytic cycle.

• Substrate binding pocket:

  • Residues forming the dihydroorotate binding pocket determine substrate specificity.

  • Alignment of URA1 sequences with related proteins shows conservation of these substrate-binding residues .

• Fumarate binding site:

  • Specific residues that interact with fumarate are essential for URA1's ability to use this molecule as an electron acceptor.

  • These residues distinguish Class I-A DHODs from other classes that use different electron acceptors.

Sequence alignments of URA1 with related proteins confirm the presence of these conserved features across functionally similar enzymes, while highlighting differences with enzymes that catalyze distinct reactions, such as dihydrouracil oxidases .

How can distinguishing between URA1 and other URA1-like proteins improve our understanding of fungal metabolism?

Distinguishing between true URA1 (Class I-A DHOD) proteins and other URA1-like proteins reveals important insights into fungal metabolism and evolution:

• Metabolic diversity identification:

  • Initial searches for URA1 homologs revealed unexpected proteins with sequence similarity but different functions, such as dihydrouracil oxidases (DHO) .

  • These enzymes catalyze different reactions (e.g., DHO: dihydrouracil + O₂ → uracil + H₂O₂) and play distinct metabolic roles .

• Evolutionary insights:

  • The large number of URA1-like proteins in aerobic fungi that also contain Class II DHODs suggests functional diversification following gene duplication or horizontal transfer .

  • These proteins form distinct phylogenetic clusters in ascomycetes and basidiomycetes, indicating separate evolutionary trajectories .

• Functional characterization approaches:

  • Complementation studies in S. cerevisiae ura1Δ strains with different media supplements (uracil, dihydrouracil) can distinguish between true DHODs and other enzymes .

  • Enzyme activity assays measuring oxygen consumption and hydrogen peroxide production help identify DHO activity versus DHOD activity .

• Novel enzyme discovery:

  • The research described in the search results led to the identification of dihydrouracil oxidase genes that were previously unknown .

  • This discovery highlights how investigating URA1-like proteins can reveal new aspects of fungal metabolism .

The experimental approach described in the search results demonstrates how functional testing (growth on different media, enzyme assays) can resolve the true function of URA1-like proteins and expand our understanding of fungal metabolic diversity .