Recombinant Photobacterium profundum Cytidylate kinase (cmk)

What is the primary function of cytidylate kinase in bacterial metabolism?

Cytidylate kinase (CMK) catalyzes the conversion of CMP and dCMP to CDP and dCDP, respectively, using ATP or GTP as phosphate donors. This enzyme plays a crucial role in the pyrimidine nucleotide salvage pathway and is essential for DNA synthesis and repair. In bacteria like P. profundum, CMK is particularly important for maintaining adequate levels of dCTP for DNA replication under varying environmental conditions .

CMK function can be represented by the following reaction:

• CMP + ATP → CDP + ADP

• dCMP + ATP → dCDP + ADP

Some bacterial CMKs show substrate promiscuity, accepting both ATP and GTP as phosphate donors, which may have functional implications in cellular metabolism .

How does CMK differ functionally from other nucleotide kinases?

While eukaryotic CMP/UMP kinases can efficiently convert both UMP and CMP to their respective diphosphate forms, bacterial systems typically employ separate enzymes for these functions. E. coli CMK specifically phosphorylates CMP and dCMP but not UMP, which is instead handled by a separate aspartate kinase system. This specialization represents a key difference in nucleotide metabolism between prokaryotes and eukaryotes .

Interestingly, while E. coli CMK can use either GTP or ATP as the phosphate donor, other bacterial CMKs like the one from Bacillus subtilis cannot use GTP, showing species-specific variations in substrate specificity .

What are the optimal expression systems for recombinant P. profundum CMK?

Based on successful expression strategies for similar proteins, the optimal expression system for recombinant P. profundum CMK typically involves:

• Vector selection: pET expression vectors (particularly pET15b) are effective for CMK expression, allowing for N-terminal 6×His-tag fusion to facilitate purification .

• Host strain selection: E. coli BL21(DE3) strains are commonly used for expression of proteins from marine organisms like P. profundum .

• Expression conditions: Optimal expression is typically achieved at lower temperatures (15-18°C) with moderate IPTG concentrations (0.2-0.5 mM) to mimic the natural cold-adapted environment of P. profundum .

• Media composition: Marine broth supplemented with appropriate antibiotics and sometimes modified with additional glucose (20 mM) and HEPES buffer (pH 7.5, 100 mM) can improve protein expression .

The expression protocol should account for the psychrophilic nature of P. profundum proteins, which may require temperature optimization different from mesophilic proteins.

What purification strategies yield the highest purity and activity of P. profundum CMK?

The most effective purification strategy for recombinant P. profundum CMK combines the following methods:

• Immobilized metal affinity chromatography (IMAC): Using HisTrap columns for the initial capture of His-tagged CMK. Purification can yield approximately 200 mg of purified protein from 60 g of cell paste .

• Buffer composition: Typically containing 50 mM Tris pH 7.4, 2 mM MgCl₂, and 50 mM KCl to maintain stability and activity .

• Concentration method: Using centrifugal filter units with a molecular weight cutoff of approximately 10 kDa (appropriate for the ~25 kDa CMK protein) .

• Size exclusion chromatography: As a polishing step to remove any aggregates or impurities with different molecular weights.

• Storage conditions: The enzyme should be stored at -80°C in a buffer containing glycerol (10-20%) to maintain activity during freeze-thaw cycles.

Verification of purity can be achieved through SDS-PAGE and mass spectrometry, while activity can be confirmed using the enzyme assays described in section 3.1.

How can the recombinant CMK construct be designed to improve stability under high pressure?

When designing recombinant P. profundum CMK constructs for high-pressure studies, consider the following adaptations:

• Domain preservation: If designing truncated versions, ensure that complete domains are preserved, as improper truncation can lead to instability and insolubility .

• Linker regions: Incorporate the native linker regions when expressing individual domains. In BiCMKEngA (a fusion protein containing CMK from Bifidobacterium), constructs that excluded linker regions were insoluble .

• Pressure-adaptive features: Research on P. profundum proteins indicates that they may have specific adaptations for high-pressure environments, including:

  • A higher proportion of flexible surface residues

  • Reduced hydrophobic core packing

  • Stabilizing salt bridges that maintain functionality under pressure

• Expression temperature: Express the protein at lower temperatures (15-18°C) to facilitate proper folding of this psychrophilic enzyme .

• Validation: Test the stability of the purified protein under different pressure conditions using spectroscopic methods (fluorescence, circular dichroism) to ensure it retains its native structure.

What are the standard methods for measuring CMK activity?

There are two primary methods for measuring CMK activity:

• NADH-coupled spectrophotometric assay:

  • Principle: The ADP produced by CMK is coupled to the oxidation of NADH through pyruvate kinase and lactate dehydrogenase reactions.

  • Components: 50 mM Tris pH 7.4, 2 mM MgCl₂, 50 mM KCl, 1 mM phosphoenolpyruvate, 0.2 mM NADH, 0.5 mM ATP, 2 units each of lactate dehydrogenase and pyruvate kinase, and varying concentrations (0.05-0.8 mM) of CMP/dCMP.

  • Measurement: Decrease in absorbance at 340 nm as NADH is oxidized.

  • Limitation: Cannot be used for determining Km for ATP/GTP due to allosteric inhibition of pyruvate kinase by ATP .

• Direct measurement of nucleotide formation:

  • LC-MS/MS analysis of reaction products (CDP/dCDP)

  • HPLC separation of substrate and product

  • Radioactive assays using labeled substrates

• GTPase activity assay (for evaluating alternative phosphate donor usage):

  • Colorimetric malachite green assay to detect released phosphate

  • Components: 100 mM Tris-HCl, 20 mM MgCl₂, 400 mM KCl, pH 7.5, with varying concentrations of GTP (0.02-1 mM) .

What are the typical kinetic parameters of P. profundum CMK compared to other bacterial CMKs?

Based on data from related bacterial CMKs, the expected kinetic parameters for P. profundum CMK would include:

Noteworthy characteristics of bacterial CMKs include:

• Substrate preference: Generally, bacterial CMKs have similar affinities for CMP and dCMP.

• Phosphate donor preferences: P. profundum CMK, like other deep-sea bacterial enzymes, may exhibit flexibility in using both ATP and GTP as phosphate donors, which is found in some bacterial CMKs but not all (e.g., E. coli CMK can use both ATP and GTP, while B. subtilis CMK cannot use GTP) .

• Pressure effects: As P. profundum is a piezophilic organism, its CMK likely shows optimized kinetic parameters at elevated pressure (around 28 MPa) compared to atmospheric pressure, similar to other enzymes from this organism .

• Temperature dependence: Being from a psychrophilic organism, P. profundum CMK likely displays higher catalytic efficiency at lower temperatures (10-15°C) compared to mesophilic homologs .

How does pressure affect the activity and stability of P. profundum CMK?

Studies on proteins from P. profundum provide insights into how pressure might affect its CMK:

• Activity optima: P. profundum enzymes typically display activity optima around 28 MPa, corresponding to the organism's natural habitat depth .

• Conformational changes: High pressure induces specific conformational changes that may actually increase activity within a certain pressure range, rather than simply denaturing the protein. This is evidenced by studies of other P. profundum proteins such as cytochrome P450, which shows pressure-induced transitions with a P₁/₂ of 300-800 bar .

• Pressure adaptation mechanism: The adaptation likely involves:

  • Reduced accessibility of the active site at high pressure

  • Substantial hydration of the protein (characterized by a reaction volume change ΔV around -100 to -200 mL/mol)

  • Displacement of conformational equilibrium toward a "closed" state, even when ligand-free

• Differential gene expression: Proteomic studies of P. profundum grown at atmospheric versus high pressure showed differential expression of key metabolic pathways, suggesting that enzymes like CMK may be regulated in response to pressure .

• Experimental approach: To characterize pressure effects on recombinant P. profundum CMK, high-pressure enzyme assays can be conducted using specialized equipment such as pressure vessels that allow spectroscopic measurements during pressurization.

How does P. profundum CMK compare structurally and functionally with CMK from non-piezophilic organisms?

Comparative analysis between P. profundum CMK and non-piezophilic CMKs reveals several distinctive features:

• Domain organization: Standard bacterial CMKs are typically single-domain proteins of approximately 25 kDa, while some bacteria like Bifidobacterium possess fusion proteins where CMK is fused with other enzymes (e.g., BiCMKEngA, where CMK is fused with EngA, a GTPase) .

• Substrate specificity: While most bacterial CMKs specifically phosphorylate CMP and dCMP, some exhibit promiscuity for phosphate donors. P. profundum CMK likely shows adaptations that optimize function under high pressure conditions, potentially including:

  • Altered substrate binding pocket architecture

  • Modified conformational dynamics that function optimally at high pressure

  • Structural elements that prevent pressure-induced denaturation

• Pressure adaptations: Similar to other P. profundum proteins, CMK likely contains structural features that allow maintained or enhanced activity under pressure, such as:

  • Reduced hydrophobic core packing

  • Increased surface hydration sites

  • Enhanced flexibility of certain regions to accommodate pressure effects

• Amino acid composition: Piezophilic proteins often show specific amino acid substitutions that favor function under pressure, which might be identifiable through sequence alignment of P. profundum CMK with mesophilic homologs.

Are there unique sequence motifs or structural features in P. profundum CMK that may contribute to its function under high pressure?

Based on studies of other pressure-adapted proteins from P. profundum and related organisms, several features may contribute to CMK function under high pressure:

• P. profundum cytochrome P450 research suggests that pressure adaptation involves:

  • A pressure-induced transition to a state with reduced accessibility of the active site

  • Substantial hydration of the protein

  • Reaction volume change (ΔV) around -100 to -200 mL/mol

  • P₁/₂ of 300-800 bar, close to the pressure of habitation of P. profundum

• Likely structural features in pressure-adapted CMK may include:

  • Enhanced hydration networks

  • Specific amino acid substitutions that stabilize the protein under pressure

  • Modified flexible loops that maintain function under compression

  • Altered binding pocket architecture that accommodates substrate binding under pressure

• Proteomic studies have shown that proteins involved in key metabolic pathways are differentially expressed at high pressure versus atmospheric pressure in P. profundum, suggesting specialized adaptations in enzymes like CMK .

• Experimental approaches to identify these features could include:

  • Comparative sequence analysis with non-piezophilic CMKs

  • Structural analysis using X-ray crystallography or cryo-EM under different pressure conditions

  • Site-directed mutagenesis of potential pressure-adaptive residues followed by functional assays

How conserved is the cmk gene across different bacterial species, and what can this tell us about its evolution?

The evolutionary patterns of the cmk gene across bacterial species reveal several important aspects:

• Genomic context: In E. coli, the cmk gene is located immediately upstream of the gene for ribosomal protein S1 (rpsA). This gene arrangement is observed in many bacteria, suggesting functional coupling between CMK activity and translation .

• Essential nature: While cmk deletion strains of E. coli remain viable at 37°C, they exhibit cold sensitivity, suggesting that CMK becomes increasingly important under stress conditions. The pools of CMP and dCMP were elevated approximately 30-fold in a cmk deletion strain .

• Domain fusion events: In some bacteria, particularly within the Actinomycetes phylum (including Bifidobacterium species), CMK is found fused with other enzymes. For example, in Bifidobacterium, CMK is fused with EngA (a GTPase involved in ribosome biogenesis) to form a multifunctional protein (BiCMKEngA) .

• Phylogenetic distribution of fusion proteins: Not all species within the Actinomycetes phylum possess the CMK-EngA fusion. For example, while Bifidobacterium species universally possess this fusion, pathogenic species like Mycobacterium have CMK and EngA as separate proteins, suggesting evolutionary divergence related to lifestyle adaptation .

• Synteny analysis: In Mycobacterium species, although CMK and EngA are separate proteins, their genes are located in close proximity in the genome, suggesting a possible evolutionary precursor state before fusion occurred in other lineages .

This evolutionary context suggests that while CMK function is broadly conserved, its genomic arrangement and integration with other cellular functions show adaptations specific to different bacterial lifestyles and environmental niches.

How might P. profundum CMK be used to study nucleotide metabolism under extreme conditions?

P. profundum CMK represents an excellent model system for studying nucleotide metabolism under extreme conditions:

• Pressure-dependent metabolism studies:

  • Using recombinant P. profundum CMK in high-pressure enzyme assays can provide insights into how nucleotide metabolism adapts to deep-sea conditions

  • Comparative analysis with mesophilic CMKs can reveal mechanistic adaptations to pressure

  • Investigation of substrate specificity under varying pressure conditions can identify pressure-sensitive steps in nucleotide metabolism

• Cold adaptation mechanisms:

  • As P. profundum is both piezophilic and psychrophilic, its CMK can be used to study how nucleotide metabolism adapts to low temperatures

  • Structure-function studies can reveal how enzymatic catalysis is maintained at low temperatures

  • Kinetic analyses at varying temperatures can identify temperature-dependent rate-limiting steps

• Methodological approaches:

  • High-pressure enzyme kinetics using specialized equipment

  • Temperature-dependent activity profiling

  • Structural studies (X-ray crystallography, cryo-EM) under varying conditions

  • Molecular dynamics simulations to model pressure and temperature effects on enzyme dynamics

• Application to extremophile metabolism:

  • Insights from P. profundum CMK can be extended to understand general principles of metabolic adaptation in extremophiles

  • The findings may inform synthetic biology approaches for designing enzymes that function in extreme environments

What insights can structural studies of P. profundum CMK provide for understanding pressure adaptation mechanisms?

Structural studies of P. profundum CMK could reveal several key insights about pressure adaptation mechanisms:

How can recombinant P. profundum CMK be engineered for novel applications in biotechnology?

Recombinant P. profundum CMK offers several opportunities for biotechnological applications:

• Enzyme engineering for extreme conditions:

  • By understanding the pressure adaptation mechanisms of P. profundum CMK, similar modifications could be introduced into other enzymes to enhance their function under pressure

  • The cold-active nature of this enzyme could be exploited for low-temperature biotechnological processes

  • Structure-guided mutagenesis could create CMK variants with enhanced stability or altered substrate specificity

• Nucleotide analog production:

  • CMK's role in converting CMP to CDP could be exploited for the enzymatic synthesis of modified nucleotides

  • The natural substrate promiscuity observed in some bacterial CMKs suggests that P. profundum CMK might accept nucleotide analogs as substrates

  • This could enable the enzymatic production of modified nucleotides for pharmaceutical applications

• Biocatalysis under pressure:

  • P. profundum CMK could serve as a model for developing pressure-enhanced biocatalytic processes

  • High-pressure enzymatic reactions may offer advantages like increased reaction rates, altered stereoselectivity, or reduced side reactions

  • Integration into multi-enzyme cascades for the production of complex nucleotide derivatives

• Methodological approaches:

  • Directed evolution to enhance desired properties

  • Structure-guided rational design of variants with altered substrate specificity

  • High-throughput screening under varying pressure conditions

  • Immobilization strategies for industrial applications

• Potential applications in antiviral research:

  • Studies of prokaryotic viperins have shown the production of modified nucleotides like ddhCTP that have antiviral properties

  • Understanding how CMK interacts with these pathways could inform the development of novel antiviral strategies

What are the main challenges in expressing and purifying active P. profundum CMK, and how can they be overcome?

Working with recombinant P. profundum CMK presents several challenges, with corresponding solutions:

• Challenge: Protein insolubility due to the psychrophilic and piezophilic nature of the enzyme.
  Solutions:

  • Lower expression temperature (15-18°C) to improve proper folding

  • Co-expression with chaperones like GroEL/GroES to assist folding

  • Use of solubility-enhancing fusion tags (SUMO, MBP) in addition to His-tag

  • Optimization of induction conditions (lower IPTG concentration, 0.1-0.2 mM)

• Challenge: Maintaining enzyme stability during purification.
  Solutions:

  • Include stabilizing additives in purification buffers (glycerol 10-20%, reducing agents)

  • Perform all purification steps at 4°C to prevent denaturation

  • Minimize exposure to freeze-thaw cycles by appropriate aliquoting

  • Consider on-column refolding approaches if inclusion bodies form

• Challenge: Domain integrity in multi-domain constructs.
  Solutions:

  • Careful design of constructs to maintain complete domains

  • Include linker regions when expressing individual domains

  • Bioinformatic analysis to identify proper domain boundaries

  • Test multiple construct designs in parallel

• Challenge: Assessing protein activity under high pressure.
  Solutions:

  • Use specialized high-pressure equipment for activity assays

  • Develop indirect assays that can measure product formation after pressure treatment

  • Collaborate with labs specializing in high-pressure biochemistry

  • Establish appropriate controls using mesophilic CMK variants

• Challenge: Low yield due to expression in heterologous hosts.
  Solutions:

  • Codon optimization for E. coli expression

  • Use of strong promoters (T7) and high-copy-number plasmids

  • Media optimization to include components from marine environments

  • Consider cold-adapted expression hosts as alternatives to E. coli

How can researchers effectively measure CMK activity under high-pressure conditions?

Measuring enzyme activity under high pressure requires specialized approaches:

• Specialized equipment options:

  • High-pressure stopped-flow apparatus for kinetic measurements

  • Pressure vessels with optical windows for spectroscopic measurements

  • Custom-designed high-pressure bioreactors for larger-scale reactions

  • Pressure perturbation calorimetry for thermodynamic measurements

• Experimental design considerations:

  • Use of pressure-stable substrates and buffers

  • Establishment of appropriate pressure ranges (typically 0.1-100 MPa for deep-sea enzymes)

  • Temperature control during pressurization

  • Inclusion of appropriate controls (mesophilic enzymes, denatured samples)

• Methodological approaches:

  • Batch processing: Perform reactions under pressure, then release pressure and analyze products

  • Real-time measurements: Use specialized equipment for direct measurement under pressure

  • Indicator systems: Couple CMK activity to color-changing reactions visible through pressure vessel windows

  • Post-pressure analysis: LC-MS or HPLC analysis of reaction products after decompression

• Data analysis considerations:

  • Determine pressure dependence of kinetic parameters (Km, kcat)

  • Calculate activation volumes from pressure-dependent kinetic data

  • Establish pressure-activity profiles to identify optimal pressure conditions

  • Model pressure effects using appropriate equations (e.g., modified Michaelis-Menten equations incorporating pressure terms)

• Experimental examples:

  • P. profundum culture systems have been developed using Pasteur pipettes sealed and incubated at 28 MPa in water-cooled pressure vessels, similar approaches could be adapted for enzyme studies

  • Pressure perturbation studies with spectroscopic monitoring have been used to study other P. profundum proteins like cytochrome P450

What strategies can be used to study the role of P. profundum CMK in DNA repair and replication?

Several experimental approaches can elucidate the role of P. profundum CMK in DNA repair and replication:

• Genetic approaches:

  • Construction of cmk deletion or conditional mutants in P. profundum

  • Complementation studies with wild-type or mutant CMK variants

  • Large-scale transposon mutagenesis to identify genetic interactions with cmk

  • Expression of P. profundum cmk in E. coli cmk deletion strains to assess functional conservation

• Biochemical approaches:

  • In vitro DNA replication and repair assays using purified components

  • Measurement of dNTP pools in cells with altered CMK expression

  • Protein-protein interaction studies to identify repair complexes containing CMK

  • Enzyme activity assays under conditions that mimic DNA damage response

• Cellular localization studies:

  • Fluorescent tagging of CMK to track localization during normal growth and after DNA damage

  • Co-localization studies with known DNA repair factors

  • ChIP-seq or similar approaches to identify genomic regions associated with CMK

  • Live-cell imaging under varying pressure conditions

• Systems biology approaches:

  • Transcriptomic analysis of cells with altered CMK expression

  • Proteomic studies to identify CMK interaction partners

  • Metabolomic analysis focusing on nucleotide pools

  • Integration of multiple data types to model CMK's role in DNA metabolism

• Specific experimental designs:

  • UV damage recovery experiments similar to those performed with CMPK knockdown cells

  • Analysis of DNA synthesis rates and replication fork progression in cells with altered CMK activity

  • Measuring DNA repair efficiency under varying pressure conditions

  • Structure-function studies focusing on the N-terminal domain involved in recruitment to DNA damage sites