Recombinant Candida glabrata Dihydroorotate dehydrogenase (quinone), mitochondrial (URA9)

What is dihydroorotate dehydrogenase (URA9) and what is its role in Candida glabrata?

Dihydroorotate dehydrogenase (DHOD) is an essential enzyme involved in the de novo pyrimidine biosynthesis pathway, catalyzing the conversion of dihydroorotate to orotate, which is a crucial step in the formation of uridine monophosphate (UMP). In Candida glabrata, this enzyme is encoded by the URA9 gene and belongs to the Class-II DHOD family, which typically requires a quinone electron acceptor for functionality. The enzyme plays a vital role in nucleic acid synthesis and cellular growth, making it essential for the survival and virulence of this opportunistic fungal pathogen. Recent research has demonstrated that URA9 in C. glabrata has evolved specific characteristics that enable the organism to grow under various oxygen conditions, which contributes to its adaptability in different host environments during infection . Unlike some other yeasts that rely strictly on aerobic respiration for DHOD function, C. glabrata appears to have mechanisms that allow pyrimidine synthesis even under oxygen-limited conditions, which may explain part of its success as a human pathogen.

How does URA9 differ between Candida glabrata and other fungal species?

The URA9 enzyme in Candida glabrata exhibits distinct characteristics compared to orthologous enzymes in other fungal species, particularly in terms of electron acceptor utilization and subcellular localization. While most eukaryotic Class-II DHODs are tightly coupled to the respiratory chain in mitochondria, some fungal species have evolved variations that allow for greater metabolic flexibility. For instance, comparative studies have shown that URA9 orthologs from obligately anaerobic fungi like Anaeromyces robustus (ArUra9) use free flavins (FAD and FMN) as electron acceptors rather than quinones, enabling pyrimidine synthesis in the absence of oxygen . In contrast, the URA9 enzyme from the facultative anaerobe Schizosaccharomyces japonicus (SjURA9) has developed a different adaptation, though expression of this ortholog in Saccharomyces cerevisiae results in loss of respiratory function . Phylogenetic analysis clearly separates bacterial and fungal URA9 orthologs, with the fungal enzymes following the expected fungal phylogeny patterns . These differences highlight the evolutionary adaptations in pyrimidine metabolism across fungal lineages, with C. glabrata's URA9 representing an important adaptation for its pathogenic lifestyle.

What are the key structural features of URA9 that contribute to its function?

The URA9 enzyme from Candida glabrata possesses several key structural features that are critical for its catalytic activity and biological function. Like other Class-II DHODs, the enzyme contains a flavin mononucleotide (FMN) cofactor in its active site, which participates in the electron transfer during the oxidation of dihydroorotate to orotate. The protein typically has a mitochondrial targeting sequence that directs it to the inner mitochondrial membrane, where it can interact with the respiratory chain components. Research on URA9 orthologs has revealed that anaerobically functional versions often feature conserved amino acid substitutions that modify their electron acceptor preferences and functionality under oxygen-limited conditions . For example, GFP-tagging experiments have demonstrated that while SjUra9 and DbUra9 (from Dekkera bruxellensis) localize to mitochondria when expressed in S. cerevisiae, ArUra9 lacks a mitochondrial targeting sequence and localizes to the cytosol instead . This cytosolic localization correlates with ArUra9's ability to use free flavins rather than membrane-bound quinones as electron acceptors, representing a structural adaptation to anaerobic environments. Understanding these structural determinants is crucial for comprehending how C. glabrata URA9 functions in different host microenvironments.

How can researchers express and purify recombinant URA9 from Candida glabrata?

Researchers seeking to express and purify recombinant Candida glabrata URA9 typically employ heterologous expression systems, with Escherichia coli or Saccharomyces cerevisiae being the most common host organisms. When using S. cerevisiae as an expression host, researchers often utilize a ura1Δ strain to eliminate the native DHOD activity and facilitate functional complementation studies, as demonstrated in comparative studies of various URA9 orthologs . The gene encoding C. glabrata URA9 can be PCR-amplified from genomic DNA and cloned into an appropriate expression vector containing a strong promoter (such as TEF1 or CYC1) and a selection marker (typically HIS3, LEU2, or URA3 for S. cerevisiae) . For protein purification, the addition of an affinity tag such as polyhistidine (His6) or glutathione S-transferase (GST) to either the N- or C-terminus of the protein enables efficient purification using affinity chromatography. Researchers must carefully consider the subcellular localization of the enzyme, as the presence of mitochondrial targeting sequences may affect the folding and solubility of the recombinant protein in bacterial expression systems. For functional studies, it is essential to ensure proper incorporation of the FMN cofactor, which may require supplementation during expression or reconstitution during purification.

What growth conditions and media are optimal for studying URA9 function in Candida glabrata?

For investigating URA9 function in Candida glabrata, researchers should consider several key growth conditions that influence enzyme activity and expression. Standard rich media like YPD (yeast extract, peptone, dextrose) are suitable for routine cultivation, but synthetic defined media are preferred for controlled experiments, particularly when studying pyrimidine auxotrophy. When evaluating URA9 functionality, synthetic media lacking uracil (SC-Ura) forces cells to rely on de novo pyrimidine synthesis, making URA9 function essential for growth . To study the enzyme's performance under different oxygen conditions, researchers should compare growth in aerobic (with shaking in baffled flasks) versus anaerobic environments (using an anaerobic chamber or by purging vessels with nitrogen and sealing them) . Temperature is typically maintained at 30°C for optimal growth of C. glabrata, though testing at 37°C may be relevant for understanding behavior during human infection. Carbon source variation (glucose, glycerol, lactate) can help elucidate respiratory versus fermentative metabolism connections to URA9 function, with respiratory carbon sources enhancing the phenotypic differences between wild-type and URA9-deficient strains. For genetic studies, researchers may need to supplement media with uracil (typically 50-80 mg/L) to support growth of URA9-deficient strains during the construction and verification phases.

How can CRISPR-Cas9 be optimized for studying URA9 in Candida glabrata?

Optimizing CRISPR-Cas9 for manipulating URA9 in Candida glabrata requires careful consideration of several technical aspects to achieve high editing efficiency while minimizing off-target effects. Researchers have developed a robust CRISPR-Cas9 system specifically tailored for C. glabrata that consists of three main components: a recombinant strain constitutively expressing Cas9, an online program for selecting efficient guide RNAs (sgRNAs), and a plasmid for expressing the selected sgRNAs . For maximal efficiency in gene disruption via non-homologous end joining (NHEJ), the Cas9 enzyme should be expressed under the control of the C. glabrata CYC1 promoter, while sgRNAs yield better results when expressed from the RNA polymerase III promoter RNAH1 . When designing sgRNAs targeting URA9, researchers should select sequences with high on-target efficiency and minimal off-target potential, typically 20 nucleotides in length followed by an NGG PAM sequence. Experimental data indicates that most NHEJ repair events in C. glabrata lead to the insertion of a single nucleotide (predominantly T) at the cutting site, which can be leveraged to design frameshift mutations . For homologous recombination approaches, the CRISPR-Cas9 system significantly enhances recombination efficiency, allowing researchers to use shorter homology arms (20-200 bp) compared to the standard 500 bp typically required for efficient recombination in C. glabrata . This optimization not only simplifies experimental procedures but also enables more complex genetic manipulations of URA9 and related genes.

How can researchers resolve inconsistent results when studying URA9 activity in different experimental systems?

Resolving inconsistent results when studying URA9 activity across different experimental systems requires systematic troubleshooting of multiple biological and technical variables that can affect enzyme function. One common source of variation is the expression level of URA9, which can be addressed by quantifying transcript and protein levels using RT-qPCR and Western blotting respectively, followed by normalization of activity data to expression levels. Researchers should also consider the influence of promoter choice when expressing recombinant URA9, as studies show significant functional differences when Cas9 and sgRNAs are expressed from S. cerevisiae versus C. glabrata promoters . The cofactor status of the enzyme represents another critical variable, as incomplete incorporation of FMN could lead to partially active enzyme populations; purification protocols should therefore include steps to ensure full cofactor saturation. Oxygen levels must be carefully controlled and monitored during experiments, as URA9 activity is inherently sensitive to oxygen availability and may show variable performance in microaerobic conditions that can inadvertently develop in certain culture vessels or locations within a culture . When comparing URA9 orthologs from different species, researchers should account for potential differences in optimal temperature, pH, and ionic strength for enzyme activity. Laboratory evolution during experiments can also lead to inconsistent results, as demonstrated by the emergence of adaptive mutations in FUM1 (encoding fumarase) in strains expressing DbURA9 . Finally, researchers should standardize assay conditions, particularly electron acceptor availability, which can be accomplished by adding defined concentrations of quinones or flavins to reaction mixtures.

What are the most accurate methods for measuring URA9 activity in vitro and in vivo?

Accurate measurement of URA9 activity requires tailored approaches for both in vitro biochemical assays and in vivo functional assessments. For in vitro analysis, the gold standard is a spectrophotometric assay that directly monitors the conversion of dihydroorotate to orotate by following the reduction of electron acceptors at their respective absorption wavelengths (typically 300 nm for ubiquinone or 450-500 nm for flavins). This assay should be performed using purified enzyme with defined concentrations of substrate and electron acceptors under controlled temperature and pH conditions. Alternative in vitro approaches include coupled enzyme assays that link DHOD activity to a more easily detectable readout, or radiometric assays using 14C-labeled dihydroorotate for highest sensitivity. For in vivo assessment, the gold standard is complementation analysis in a URA1Δ S. cerevisiae strain (lacking endogenous DHOD activity) or a URA9 knockout C. glabrata strain, measuring growth rates in media lacking uracil under both aerobic and anaerobic conditions . More sophisticated in vivo approaches include metabolic flux analysis using 13C-labeled glucose to trace the flow of carbon through the pyrimidine biosynthesis pathway, or transcriptomic analysis to identify compensatory mechanisms activated in response to URA9 manipulation. Researchers can also employ fluorescent biosensors that report on pyrimidine nucleotide pools as indirect measures of URA9 activity within living cells. Each method has distinct advantages and limitations, so combining multiple approaches typically provides the most comprehensive assessment of URA9 function.

How can evolutionary approaches enhance our understanding of URA9 function in Candida glabrata?

Evolutionary approaches offer powerful tools for understanding the functional constraints, adaptability, and regulatory networks governing URA9 in Candida glabrata. Laboratory evolution experiments, where strains with altered URA9 function are subjected to selective pressures, can reveal compensatory mechanisms and genetic interactions. For instance, researchers observed that C. glabrata strains expressing DbURA9 initially showed delayed anaerobic growth, but adapted through mutations in FUM1 (encoding fumarase), highlighting an unexpected metabolic connection between the TCA cycle and pyrimidine biosynthesis . Comparative genomics across Candida species with different ecological niches and oxygen requirements can identify conserved and divergent features of URA9, revealing which aspects are most critical for function versus those that represent species-specific adaptations. Phylogenetic analysis of URA9 orthologs across fungi has already demonstrated clear separation between bacterial and fungal DHOD enzymes, with the fungal tree topology following established fungal phylogeny . Researchers can employ ancestral sequence reconstruction to resurrect and characterize ancestral forms of URA9, providing insights into the evolutionary trajectory of the enzyme and identifying key mutations that enabled adaptation to anaerobic growth. Site-directed mutagenesis guided by evolutionary conservation analysis can pinpoint functionally critical residues, particularly those involved in electron acceptor recognition and catalysis. Additionally, experimental evolution of C. glabrata under fluctuating oxygen conditions can reveal the adaptive potential of URA9 and associated pathways, potentially identifying novel regulatory mechanisms that could serve as targets for antifungal development.

What research tools and model systems are most effective for comparative studies of URA9 across different Candida species?

Comparative studies of URA9 across Candida species benefit from a strategic combination of research tools and model systems that enable detailed functional, structural, and evolutionary analyses. Heterologous expression in Saccharomyces cerevisiae ura1Δ strains provides a well-controlled system for functional complementation assays, allowing researchers to directly compare the ability of different URA9 orthologs to support pyrimidine prototrophy under various conditions . This system can be enhanced with fluorescent protein tagging to simultaneously assess subcellular localization and correlation with function. For detailed biochemical characterization, recombinant expression and purification systems using E. coli or insect cells allow researchers to produce sufficient quantities of different URA9 variants for enzymatic assays, thermal stability measurements, and structural studies. Genome editing technologies, particularly CRISPR-Cas9 systems optimized for Candida species, enable precise gene replacements where the native URA9 gene can be swapped with orthologs from other species to assess functionality in the natural cellular context . For evolutionary analyses, whole-genome sequencing combined with phylogenetic tools can reveal selection pressures and evolutionary trajectories across different lineages. Structural biology approaches, including X-ray crystallography and cryo-electron microscopy, provide atomic-level insights into how sequence differences translate to structural adaptations that enable functional diversity. Systems biology tools such as metabolomics and fluxomics help characterize the integration of different URA9 variants into cellular metabolism. Finally, infection models ranging from invertebrates (Drosophila melanogaster) to mammals enable assessment of how URA9 variations impact virulence and host adaptation across Candida species .

What can the study of URA9 teach us about adaptation to different ecological niches in pathogenic fungi?

The study of URA9 provides a compelling window into how pathogenic fungi adapt to diverse ecological niches through modifications of central metabolic pathways. The remarkable functional diversity of URA9 orthologs across fungal species demonstrates how a single enzyme can evolve to maintain essential pyrimidine biosynthesis under vastly different environmental conditions, from fully aerobic to strictly anaerobic habitats. This adaptability is particularly relevant for understanding how opportunistic pathogens like Candida glabrata colonize different host microenvironments, which can vary dramatically in oxygen availability, nutrient composition, and immune pressure. The evolution of alternative electron acceptor mechanisms in URA9 represents a sophisticated adaptation that enables metabolic flexibility without requiring wholesale rewiring of pyrimidine biosynthesis pathways . The observation that expression of certain URA9 orthologs in S. cerevisiae results in loss of respiratory function suggests complex tradeoffs between maintaining pyrimidine synthesis and other metabolic processes, potentially explaining why some pathogens favor fermentative metabolism even in oxygen-rich environments . The subcellular relocalization of URA9 seen in some species, from the conventional mitochondrial membrane to the cytosol, exemplifies how spatial reorganization of metabolic enzymes can create new functional capabilities . From an evolutionary perspective, the phylogenetic distribution of different URA9 variants provides insights into the ancestral state of fungal metabolism and the sequence of adaptations that enabled colonization of new niches, including the human host. Together, these lessons from URA9 research illustrate how metabolic adaptation contributes to the remarkable ecological diversity of fungi and their success as human pathogens.