Recombinant Photobacterium profundum Dihydroorotate dehydrogenase (quinone) (pyrD)

What is Dihydroorotate dehydrogenase (quinone) in Photobacterium profundum and what is its functional significance?

Dihydroorotate dehydrogenase (quinone) (EC 1.3.5.2), encoded by the pyrD gene in Photobacterium profundum, is a class 2 enzyme that catalyzes the fourth step in de novo pyrimidine biosynthesis. The reaction involves the oxidation of (S)-dihydroorotate to orotate using quinone as an electron acceptor:

(S)-dihydroorotate + quinone → orotate + quinol

This enzyme plays a critical role in nucleotide metabolism, which is particularly important for P. profundum's adaptation to deep-sea environments where it experiences high hydrostatic pressure (up to 90 MPa). The enzyme's significance extends beyond basic metabolism, as it represents an important adaptation mechanism that allows P. profundum to maintain proper nucleotide balance under pressure conditions that would typically impair growth in non-piezophilic organisms .

How does Photobacterium profundum pyrD differ structurally from homologous genes in mesophilic bacteria?

While the search results don't provide specific structural comparisons of P. profundum pyrD with mesophilic equivalents, we can infer likely differences based on general piezophilic adaptations:

• P. profundum pyrD likely contains amino acid substitutions that confer structural stability under high pressure conditions.

• Research on other P. profundum genes suggests that pressure-adapted enzymes often show modifications in regions associated with conformational flexibility .

• The pyrD gene in P. profundum may exhibit altered codon usage patterns optimized for expression under high pressure and low temperature conditions characteristic of the deep sea.

• Unlike mesophilic bacteria, P. profundum's pyrD may have co-evolved with specific pressure-sensing regulatory elements that modulate its expression in response to environmental pressure changes .

Comparative genomic studies have shown that P. profundum demonstrates significant metabolic diversity and redundancy compared to mesophilic counterparts, a feature that may extend to its pyrimidine biosynthesis pathway .

What are the optimal growth conditions for Photobacterium profundum relevant to pyrD expression studies?

P. profundum SS9 grows optimally under the following conditions:

These growth parameters are important considerations when designing expression studies for recombinant pyrD . Notably, when growing P. profundum at atmospheric pressure rather than its optimal pressure, the organism experiences stress that alters its metabolic profile and gene expression patterns. This is evidenced by the up-regulation of several proteins involved in the oxidative phosphorylation pathway at atmospheric pressure (0.1 MPa), while proteins involved in the glycolysis/gluconeogenesis pathway are up-regulated at high pressure (28 MPa) .

For cloning and genetic manipulation, researchers typically maintain P. profundum at atmospheric pressure (0.1 MPa) and 15°C, using appropriate antibiotic selection, which makes laboratory work more practical .

What expression systems and strategies are most effective for producing recombinant Photobacterium profundum Dihydroorotate dehydrogenase?

Based on research with other P. profundum proteins and DHODH from other organisms, the following expression strategies have proven effective:

Heterologous Expression in E. coli:

• Vector selection: pET29a and similar T7-based expression vectors have been successfully used for recombinant P. profundum proteins .

• E. coli strains: BL21(DE3) or Rosetta strains are recommended to address potential codon bias issues.

• Induction conditions: Low-temperature induction (15-20°C) for 16-24 hours with 0.5-1.0 mM IPTG generally yields better results for proteins from psychrophilic organisms .

Expression optimization protocol:

• Transform expression plasmid into E. coli cells

• Grow cultures to OD600 of 0.6-1.2 at 37°C

• Cool cultures to 15-20°C before induction

• Induce with 0.5 mM IPTG

• Continue expression for 16-24 hours at 15-20°C

• Harvest cells by centrifugation at 4,000-6,000 × g for 15 minutes

For challenging cases where traditional E. coli expression yields inactive enzyme, alternative approaches include:

• Native host expression using conjugative plasmid transfer to P. profundum

• Cell-free protein synthesis systems

• Codon optimization of the pyrD gene for improved expression in E. coli

When designing recombinant constructs, inclusion of a C-terminal His-tag has shown good results for purification while maintaining enzymatic activity, as demonstrated with other P. profundum proteins .

What purification and characterization methods are recommended for recombinant P. profundum Dihydroorotate dehydrogenase?

Purification Protocol:

• Cell lysis: Sonication in buffer containing 50 mM Tris-HCl (pH 8.0), 500 mM NaCl, 1 mM TCEP, and 0.5% TritonX-100

• Initial purification: Ni-NTA affinity chromatography for His-tagged constructs

• Further purification: Size exclusion chromatography in buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 10% glycerol

• For insoluble protein: Solubilization in 7-8 M guanidine hydrochloride or urea followed by refolding via dialysis against buffer containing stabilizing agents like L-arginine (0.5 M) and glycerol (10%)

Characterization Methods:

• Enzymatic activity assay: The standard assay uses DCIP (2,6-dichloroindophenol) as an artificial electron acceptor and monitors the decrease in absorbance at 600 nm. Reaction conditions: 50 mM Tris-HCl (pH 8.0), 150 mM KCl, 0.1% Triton X-100, 10% glycerol, 1 mM L-dihydroorotate, 0.1 mM DCIP, and 0.1 mM decylubiquinone .

• Pressure effects: For evaluating high-pressure effects on enzyme activity, specialized high-pressure vessels coupled with spectrophotometric or fluorescence-based assays are employed.

• Thermostability analysis: Differential scanning fluorimetry or thermal shift assays can determine the enzyme's thermal stability profile.

• Kinetic parameters determination: Vary substrate concentrations (dihydroorotate: 3.1-1600 μM; quinone: 1.2-600 μM) to determine Km and Vmax under different pressure conditions .

How does high hydrostatic pressure affect the structure and function of P. profundum pyrD-encoded DHODH?

While the search results don't provide specific data on pressure effects on P. profundum DHODH, insights can be drawn from studies on other P. profundum proteins and general principles of protein adaptation to high pressure:

• Structural adaptations: P. profundum proteins often display increased flexibility in certain regions while maintaining rigidity in catalytic domains to function efficiently under high pressure. This balance of structural elements likely applies to DHODH as well.

• Metabolic context: At high pressure (28 MPa), P. profundum up-regulates several proteins involved in the glycolysis/gluconeogenesis pathway, suggesting an altered metabolic flux that may affect pyrimidine biosynthesis .

• Protein hydration changes: Studies on other P. profundum enzymes, such as cytochrome P450, have shown that high pressure affects protein hydration and potentially alters the equilibrium between open and closed conformations . These hydration changes may be critical for substrate binding and catalysis in DHODH.

• Pressure as a sensing mechanism: P. profundum potentially uses pressure as a sensing mechanism to modulate enzyme activity and metabolic pathways. The pyrD gene product may respond directly to pressure changes or be regulated through pressure-responsive signaling pathways .

• Comparative analysis: Experiments comparing the activity of P. profundum DHODH with homologs from non-piezophilic organisms under various pressures would help elucidate specific pressure adaptations.

What genetic approaches can be used to study pyrD function in Photobacterium profundum?

Several genetic approaches have been successfully applied to study gene function in P. profundum and can be adapted for pyrD studies:

• Insertional inactivation: Internal fragments of pyrD can be amplified by PCR and cloned into suicide vectors like pMUT100. These constructs can be mobilized into P. profundum via biparental conjugation using E. coli donor strains carrying helper plasmids .

• Gene deletion via homologous recombination: Construct deletion vectors containing flanking regions of pyrD and a counterselectable marker (sacB). After conjugation and selection, screen for sucrose-resistant colonies that have undergone double crossover events .

Protocol for pyrD disruption:

• Amplify an internal fragment of pyrD

• Clone into pMUT100 using standard molecular techniques

• Mobilize into P. profundum SS9 by biparental conjugation using E. coli MC1061 containing pRK24 and pRL528 helper plasmids

• Select exconjugants on 2216 agar containing rifampicin and kanamycin at 15°C

• Confirm disruption by PCR and sequencing

• Transposon mutagenesis: Mini-Tn5 transposons have been successfully used for large-scale mutagenesis in P. profundum with minimal insertion bias, allowing for random gene disruption and phenotypic screening .

• Complementation analysis: Express wild-type pyrD in trans to verify gene mutation-growth phenotype relationships, particularly under varying pressure conditions .

• Reporter gene fusions: Fuse pyrD to reporter genes like lacZ to monitor expression under different environmental conditions, similar to approaches used for other pyr genes .

What is known about the role of pyrD in P. profundum's adaptation to deep-sea environments?

While the search results don't specifically address pyrD's role in deep-sea adaptation, several insights can be inferred:

• Metabolic adaptation: Proteomic analyses have shown that P. profundum alters its metabolic profile in response to pressure changes. At high pressure (28 MPa), proteins involved in glycolysis/gluconeogenesis are up-regulated, while at atmospheric pressure (0.1 MPa), oxidative phosphorylation proteins are up-regulated . This metabolic plasticity suggests that pyrimidine biosynthesis, including pyrD function, may be integrated into these pressure-responsive metabolic networks.

• Chromosomal function: Large-scale mutagenesis studies have identified that genes involved in chromosomal structure and function are particularly important for pressure adaptation in P. profundum . As pyrD encodes an enzyme critical for nucleotide biosynthesis, it likely contributes to maintaining DNA replication and repair processes under high-pressure conditions.

• Growth phenotypes: By analogy with recD, which is required for high-pressure growth in P. profundum, pyrD may be similarly essential for growth under deep-sea conditions . The pyrD gene product could be involved in pressure-specific metabolic adjustments that enable cellular function at elevated pressures.

• Signal transduction: P. profundum employs various sensory and regulatory mechanisms to adapt to pressure and temperature changes . The regulation of pyrD expression may be integrated into these signaling networks, allowing for fine-tuned responses to environmental conditions.

How do the kinetic parameters of P. profundum DHODH compare with those from mesophilic and thermophilic organisms?

While specific kinetic parameters for P. profundum DHODH are not provided in the search results, we can construct a comparative framework based on studies of DHODH from other organisms:

The kinetic parameters of P. profundum DHODH would likely show:

• Temperature dependence: As a psychrotolerant organism, P. profundum DHODH would maintain activity at lower temperatures compared to mesophilic equivalents.

• Pressure effects: Unique among the compared enzymes, P. profundum DHODH would demonstrate optimal activity at high pressures (~28 MPa).

• Substrate affinity adaptations: Potentially altered substrate binding affinities that optimize function in the deep-sea environment.

Methods for determining these parameters would be similar to those used for other DHODHs, with the addition of high-pressure experimental setups .

What inhibitors are effective against P. profundum Dihydroorotate dehydrogenase and how might they differ from inhibitors of human DHODH?

Based on studies of DHODH inhibitors from various organisms, we can predict effective inhibitor classes for P. profundum DHODH and how they might differ from human DHODH inhibitors:

Potential inhibitor classes:

• Quinolone derivatives: Alkylquinolones have been established as inhibitors of bacterial DHODH, including E. coli DHODH . These compounds might show selective inhibition of P. profundum DHODH.

• Ubiquinone analogues: As competitive inhibitors of the quinone binding site, these would likely be effective against P. profundum DHODH but might show different binding affinities compared to human DHODH due to structural differences in the quinone binding pocket.

• Dihydroorotate analogues: Competitive inhibitors of the substrate binding site could be developed based on the unique structural features of P. profundum DHODH.

Expected differences from human DHODH inhibitors:

• Binding pocket architecture: P. profundum DHODH likely has structural adaptations for high-pressure environments that alter inhibitor binding characteristics.

• Selectivity profile: Inhibitors targeting the N-terminal domain, which differs significantly between human and bacterial DHODHs, could provide selectivity for P. profundum DHODH .

• Pressure-dependent inhibition: The efficacy of inhibitors against P. profundum DHODH may vary with pressure, with some showing enhanced inhibition at high pressure while others might be less effective.

An experimental approach to identify effective inhibitors would include:

• Structure-based virtual screening against homology models of P. profundum DHODH

• High-throughput screening of compound libraries using the enzyme activity assay described in question 5

• Evaluation of inhibitor efficacy under varying pressure conditions