Recombinant Rhodopirellula baltica Type III pantothenate kinase (coaX)

What is Type III pantothenate kinase (coaX) and what is its functional role in Rhodopirellula baltica?

Type III pantothenate kinase (coaX) in Rhodopirellula baltica is an enzyme that catalyzes the phosphorylation of pantothenate (vitamin B5), which represents the first committed step in coenzyme A (CoA) biosynthesis . This enzyme belongs to the type III pantothenate kinase family, which is structurally distinct from type I and type II pantothenate kinases found in other organisms .

In R. baltica, a marine member of the phylum Planctomycetes, this enzyme plays a critical role in cellular metabolism by initiating the synthesis pathway for CoA, which is an essential cofactor involved in numerous metabolic reactions. The R. baltica coaX gene encodes a 273-amino acid protein with a molecular mass of approximately 29.1 kDa . The enzyme is part of the bacterium's metabolic adaptation system, particularly important during its complex life cycle where metabolic adjustments are required during transitions between growth phases .

How does the structure of Type III pantothenate kinase from R. baltica differ from other types, and what are the functional implications?

Type III pantothenate kinases, including that from R. baltica, belong to the acetate and sugar kinase/heat shock protein 70/actin (ASKHA) protein superfamily, which represents a completely different structural fold compared to other types of pantothenate kinases . In contrast, type I pantothenate kinases (exemplified by E. coli coaA) belong to the "P-loop kinase" superfamily, demonstrating a case of convergent evolution where different protein structures evolved to perform the same function .

This structural difference has significant functional implications:

• Substrate affinity: Type III PanKs exhibit an unusually high Km for ATP (in the mM range), which is 30-100 fold higher than other types of PanKs .

• Regulatory mechanisms: Unlike type I and II PanKs, type III enzymes are not inhibited by CoA or its thioesters . This lack of feedback inhibition suggests a different metabolic regulatory strategy in organisms primarily containing this type of PanK.

• Active site configuration: The ASKHA superfamily proteins, including R. baltica coaX, contain highly conserved active site motifs that are critical for catalysis, with several aspartate residues playing crucial roles in the enzyme mechanism .

What expression systems have been successfully used for recombinant production of R. baltica coaX, and what are their comparative advantages?

Multiple expression systems have been successfully employed for the recombinant production of R. baltica Type III pantothenate kinase. According to available data, these include:

For biochemical characterization purposes, E. coli has been the predominant system used. Research indicates that R. baltica enzymes, including coaX, have been successfully expressed in E. coli and characterized in terms of kinetic parameters, substrate specificity, temperature and pH dependence . The E. coli system is particularly suitable for obtaining sufficient quantities for structural studies and enzymatic assays.

For studies requiring post-translational modifications or specific folding conditions, eukaryotic expression systems offer advantages. The choice between these systems should be guided by the specific research questions and downstream applications.

What are the established purification methods for obtaining high-quality recombinant R. baltica coaX for enzymatic studies?

While the search results don't provide specific purification protocols for R. baltica coaX, general methods for recombinant Type III pantothenate kinases can be adapted based on similar enzymes:

For His-tagged R. baltica coaX expressed in E. coli, a typical purification protocol would involve:

• Cell lysis: Bacterial cells expressing the recombinant protein are harvested and lysed using methods such as sonication or French press in a buffer containing 100 mM HEPES (pH 7.6), 20 mM KCl, and 10 mM MgCl₂ .

• Affinity chromatography: The lysate is subjected to nickel-affinity chromatography, with the His-tagged protein binding to the resin while impurities are washed away.

• Size exclusion chromatography: Further purification can be achieved through gel filtration to obtain homogeneous protein preparations.

• Quality assessment: The purified protein should be analyzed by SDS-PAGE for purity assessment, with expected molecular weight around 29.1 kDa .

For enzymatic studies, the active protein preparation should be tested using a coupled assay system. Based on protocols used for similar Type III PanKs, activity can be measured using an ATP-regenerating system coupled to NADH oxidation (measuring absorbance at 340 nm). The reaction mixture typically contains 100 mM HEPES (pH 7.6), 20 mM KCl, 10 mM MgCl₂, 2 mM phosphoenolpyruvate, 0.3 mM NADH, lactate dehydrogenase, pyruvate kinase, and the purified PanK-III protein .

What are the critical residues for catalysis in R. baltica Type III pantothenate kinase, and how can they be investigated through site-directed mutagenesis?

Based on structural and functional studies of Type III pantothenate kinases, three highly conserved aspartate residues are critical for catalysis . Although the specific residues in R. baltica coaX haven't been directly identified in the provided search results, comparative analysis with other Type III PanKs suggests these conserved aspartates are likely crucial.

To investigate catalytic residues in R. baltica coaX, researchers should:

• Perform sequence alignment with characterized Type III PanKs (such as those from H. pylori and T. maritima) to identify conserved aspartate residues.

• Design site-directed mutagenesis experiments to create point mutations (e.g., D→N and D→E substitutions) at these conserved positions.

• Express and purify the mutant proteins using standard protocols.

• Conduct enzymatic assays to determine kinetic parameters (kcat and Km) for both substrates (pantothenate and ATP).

The methodology demonstrated with H. pylori PanK-III can serve as a template, where mutagenesis PCR was used to introduce changes from aspartate to asparagine and glutamate at residues 17, 87, and 102 . Activity assays for mutants should be performed using the coupled enzymatic method measuring NADH oxidation, with reaction mixtures containing 100 mM HEPES (pH 7.6), 20 mM KCl, 10 mM MgCl₂, 2 mM phosphoenolpyruvate, 0.3 mM NADH, 5 U of lactate dehydrogenase, and 2.5 U of pyruvate kinase . For R. baltica coaX specifically, temperature optimization may be necessary considering its marine origin.

How does the expression of R. baltica coaX vary under different growth conditions, and what methodologies can best capture these variations?

R. baltica undergoes metabolic adaptations during its complex life cycle, with significant changes in gene expression patterns across different growth phases . While specific data on coaX expression patterns is not directly provided in the search results, research on R. baltica's life cycle provides insights into methodologies for studying expression variations.

To investigate coaX expression under different growth conditions, researchers should employ:

• Transcriptomic analysis: RNA-seq or microarray methods to monitor changes in coaX expression across growth phases. Previous studies have revealed that R. baltica shows differential expression of metabolic genes, particularly during transition from exponential to stationary phase .

• Proteomics approach: Quantitative proteomics using mass spectrometry to monitor protein levels in correlation with transcriptomic data.

• Growth condition variations: Parameters to vary should include:

  • Nutrient availability (carbon and nitrogen sources)

  • Salinity levels (R. baltica is a marine organism)

  • Temperature ranges

  • Oxygen availability

• Metabolic stress induction: Since pantothenate kinase is involved in CoA biosynthesis, stresses affecting energy metabolism may be particularly informative.

Previous studies have shown that R. baltica adjusts its metabolism significantly during growth phases, with enzymes like glutamate dehydrogenase showing increased expression from exponential to stationary phase . Similar dynamics might be expected for coaX, especially considering its central role in CoA biosynthesis, which is essential for energy metabolism.

What are the structural attributes of R. baltica coaX that could explain its lack of feedback inhibition by CoA, and how might this inform drug development approaches?

Type III pantothenate kinases, including R. baltica coaX, lack the feedback inhibition by CoA and its thioesters that is characteristic of type I and II enzymes . This unique regulatory feature has significant implications for both understanding the enzyme's function and developing potential inhibitors.

Structural attributes that likely contribute to this lack of inhibition include:

• Different protein fold: Type III PanKs belong to the ASKHA superfamily rather than the P-loop kinase superfamily of type I enzymes, resulting in fundamentally different binding site architectures .

• Active site configuration: The binding pocket for ATP and pantothenate in type III PanKs likely differs significantly from the sites in type I and II enzymes that allow for CoA binding.

• Allosteric regulation sites: Type III PanKs may lack the allosteric binding sites that mediate feedback inhibition in other types.

For drug development targeting pathogenic bacteria that exclusively possess type III PanKs, these structural differences present both opportunities and challenges:

• Selective targeting: The structural divergence allows for the design of inhibitors that specifically target type III PanKs without affecting human type II enzymes.

• Mechanism-based inhibition: Understanding the unique catalytic mechanism of type III PanKs could lead to transition-state analogues or other mechanism-based inhibitors.

• Focus on conserved catalytic residues: The three conserved aspartate residues identified as critical for catalysis present potential targets for rational drug design .

• Pantothenate pocket binding: The recent development of PZ-2891, an allosteric PANK activator that occupies the pantothenate pocket and engages the dimer interface of human PANKs , suggests that similar approaches targeting the unique structural features of type III PanKs might be feasible.

How can R. baltica coaX be used as a model to understand the evolution of pantothenate kinases across bacterial phyla?

R. baltica coaX represents an excellent model for studying the evolution of pantothenate kinases due to several factors:

• Evolutionary position: R. baltica belongs to the Planctomycetes, a deep-branching phylum in bacterial evolution , providing insight into ancient metabolic pathways.

• Structural divergence: Type III pantothenate kinases represent a case of convergent evolution, where a different protein fold evolved to perform the same enzymatic function as type I and II PanKs .

• Distribution patterns: Type III PanKs have a wider distribution in the bacterial kingdom than initially anticipated, and some bacteria (like M. tuberculosis) possess genes for both type I and type III enzymes .

Methodological approaches for evolutionary studies should include:

The dual presence of type I and III PanKs in some bacteria raises interesting questions about functional redundancy or specialization that could be addressed through knockout studies in model organisms possessing both enzymes.

What are the implications of R. baltica coaX research for understanding pantothenate kinase-associated neurodegeneration (PKAN) in humans?

Research on R. baltica coaX provides valuable insights for understanding PKAN despite the evolutionary distance between bacterial and human systems:

PKAN is a rare neurological disorder caused by mutations in the human PANK2 gene , leading to movement difficulties, speech problems, and iron accumulation in the brain. Understanding the fundamental mechanisms of pantothenate kinase function across different structural types can inform therapeutic approaches.

Key implications include:

• Structural insights: Although human PANK2 is a type II enzyme while R. baltica possesses a type III enzyme, comparative structural analysis can reveal conserved catalytic principles that transcend structural classification.

• Enzyme activator development: Recent research has developed PZ-2891, an allosteric PANK activator that crosses the blood-brain barrier and can increase CoA levels in mouse brain . Understanding the regulatory mechanisms of diverse PanK types, including the non-inhibited type III enzymes like R. baltica coaX, could suggest novel approaches for PANK2 activation.

• Metabolic compensation strategies: The study of organisms with multiple PanK types could provide insights into metabolic resilience relevant to therapeutic approaches for PKAN that aim to compensate for PANK2 deficiency through activation of other PANK isoforms.

• CoA homeostasis mechanisms: Research into how R. baltica maintains CoA homeostasis without feedback inhibition could provide insights into alternative regulatory mechanisms that might be exploited therapeutically in PKAN patients.

Research suggests that PKAN might involve ferroptosis, a form of cell death associated with iron accumulation and oxidative stress . Studies on CoA metabolism in diverse systems, including R. baltica, could help elucidate the connection between pantothenate kinase function, CoA levels, iron metabolism, and oxidative stress responses.

What novel assay methodologies could be developed to better characterize the kinetic properties of R. baltica coaX under physiologically relevant conditions?

Current assays for Type III pantothenate kinases typically employ coupled enzyme systems monitoring NADH oxidation , but these may not fully capture the enzyme's behavior under physiologically relevant conditions for R. baltica, a marine bacterium with unique metabolic adaptations.

Novel assay methodologies could include:

• Marine-mimetic assay conditions: Developing buffer systems that better simulate the salt concentration, pH, and ion composition of marine environments where R. baltica naturally exists.

• Temperature optimization: R. baltica is a mesophilic organism, but assay conditions should be optimized to match its natural growth temperature range.

• Direct product detection methods:

  • Mass spectrometry-based assays to directly quantify the phosphorylated product

  • NMR methods to monitor reaction progress without coupling to auxiliary enzymes

  • Radiometric assays using γ-³²P-ATP to directly measure product formation

• High-throughput screening platforms:

  • Fluorescence-based assays using environmentally sensitive probes

  • Surface plasmon resonance for real-time binding kinetics

  • Isothermal titration calorimetry for thermodynamic parameters

• Physiological context assays:

  • Assessing enzyme activity in cell extracts under varying growth conditions

  • In-cell activity measurements using cell-permeable substrates or metabolomics approaches

  • Integration with metabolic flux analysis

• Pressure-dependent kinetics: As R. baltica is a marine organism potentially subject to varying hydrostatic pressures, characterizing pressure effects on enzyme kinetics could provide unique insights.