Recombinant Neurospora crassa Phosphoenolpyruvate carboxykinase [ATP] (pck-1), partial

What is Phosphoenolpyruvate carboxykinase (PEPCK) and what is its role in Neurospora crassa?

Phosphoenolpyruvate carboxykinase (PEPCK) catalyzes the conversion of oxaloacetate to phosphoenolpyruvate, a critical step in gluconeogenesis. In Neurospora crassa, PEPCK encoded by the pck-1 gene is an ATP-dependent enzyme (EC 4.1.1.49) that enables the organism to synthesize glucose from non-carbohydrate carbon sources. This enzyme is particularly important during growth on alternative carbon sources when glucose is unavailable, allowing N. crassa to utilize organic acids and amino acids for energy production. The enzyme is subject to carbon catabolite repression (CCR), indicating its role in the cell's adaptive response to changing nutrient conditions .

How does the expression system for recombinant N. crassa PEPCK affect protein yield and activity?

The expression system significantly impacts both yield and activity of recombinant N. crassa PEPCK. When designing expression protocols, researchers should consider:

Expression Host Selection:

• E. coli BL21(DE3): Offers high expression levels but may produce inclusion bodies requiring refolding

• Yeast systems (P. pastoris): Provides post-translational modifications more similar to native N. crassa

• Homologous expression in N. crassa: Ensures proper folding but with typically lower yields

Methodology for Optimal Expression:

• Temperature optimization (typically 16-25°C for slower expression with improved folding)

• Induction conditions (IPTG concentration of 0.1-0.5 mM for E. coli systems)

• Co-expression with molecular chaperones to enhance proper folding

• Addition of metal cofactors (Mn²⁺ or Mg²⁺) to stabilize the enzyme during expression

Activity assessments should be performed using spectrophotometric assays that couple PEPCK activity to NADH oxidation, allowing for quantitative measurement of enzyme functionality across different expression conditions.

What purification strategies yield the highest purity and activity for recombinant PEPCK?

Multi-step purification approaches yield the highest purity and activity for recombinant N. crassa PEPCK:

Recommended Purification Protocol:

Maintaining enzyme stability throughout purification requires inclusion of glycerol (10%) and reducing agents (DTT or β-mercaptoethanol) in all buffers. Activity assays should be performed after each purification step to monitor retention of enzymatic function. The final preparation should be stored with stabilizing agents to prevent loss of activity during storage.

How do different carbon sources affect PEPCK regulation in Neurospora crassa?

Carbon source availability significantly influences PEPCK expression and activity in N. crassa through carbon catabolite repression (CCR). This regulation ensures metabolic efficiency by prioritizing preferred carbon sources:

• Under glucose-rich conditions, transcription of the pck-1 gene is repressed through CCR mechanisms involving the transcription factor CreA/CRE-1 .

• When preferred carbon sources are depleted, this repression is relieved, allowing PEPCK expression to support gluconeogenesis from alternative substrates.

• Fructose has been observed to affect carbon-dependent morphology in N. crassa, suggesting interactions between carbon sensing and developmental pathways that may indirectly influence PEPCK regulation .

Experimental studies of carbon source effects typically employ media containing different sole carbon sources (glucose, glycerol, acetate, or amino acids) followed by analysis of PEPCK expression at both transcriptional (RT-qPCR) and protein (Western blot) levels.

What are the key differences between ATP-dependent and GTP-dependent PEPCK enzymes at the structural and functional levels?

ATP-dependent PEPCKs (EC 4.1.1.49) found in N. crassa differ substantially from GTP-dependent PEPCKs (EC 4.1.1.32) found in mammals:

Structural Comparisons:

• N. crassa PEPCK likely shares structural features with other ATP-dependent PEPCKs, such as the dimeric organization observed in T. cruzi PEPCK .

• Each monomer typically folds into two complex mixed α/β domains with the active site located in a deep cleft between domains .

• ATP-dependent PEPCKs contain a characteristic nucleotide-binding domain with distinct conformational changes upon ATP binding.

Functional Differences:

• Kinetic parameters (Km, Vmax) differ between ATP and GTP-utilizing enzymes

• Metal ion requirements: Both require divalent cations (Mg²⁺ or Mn²⁺), but with different affinities and effects on catalysis

• Allosteric regulation mechanisms differ significantly between the two types

These differences make ATP-dependent PEPCK a potential target for antifungal drug development, similar to how the T. cruzi PEPCK has been considered a target for anti-parasitic drugs due to its differences from the human GTP-dependent enzyme .

How can site-directed mutagenesis be used to investigate catalytic mechanisms of N. crassa PEPCK?

Site-directed mutagenesis provides powerful insights into PEPCK catalytic mechanisms through systematic modification of key residues:

Methodological Approach:

• Identify conserved catalytic residues through sequence alignment with characterized PEPCKs

• Design mutagenesis primers with appropriate base substitutions

• Perform PCR-based mutagenesis using established protocols (QuikChange or similar)

• Express and purify mutant proteins using identical conditions to wild-type

• Conduct comparative kinetic analyses using coupled spectrophotometric assays

Key Residues for Investigation:

• Metal-binding residues (typically aspartate and histidine residues)

• Nucleotide-binding residues in the P-loop region

• Substrate-binding residues that interact with oxaloacetate or phosphoenolpyruvate

• Residues involved in domain movement during catalysis

The impact of mutations should be assessed through comprehensive kinetic analyses including determination of kcat, Km, and kcat/Km values for all substrates. Structural studies using crystallography or hydrogen-deuterium exchange mass spectrometry can provide additional insights into how mutations alter protein conformation and dynamics.

What challenges exist in crystallizing recombinant N. crassa PEPCK for structural studies?

Crystallizing recombinant N. crassa PEPCK presents several technical challenges that must be addressed systematically:

• Conformational Heterogeneity: PEPCK likely undergoes significant conformational changes during catalysis, similar to the ATP-induced transition observed in E. coli PEPCK . This conformational flexibility can impede crystal formation.

• Protein Stability Issues: Purified PEPCK may exhibit limited stability, particularly in the absence of stabilizing ligands or cofactors.

• Buffer Optimization: As observed with T. cruzi PEPCK, the crystallization conditions, particularly ion concentration (e.g., sulfate), can significantly affect enzyme conformation .

Strategies to Overcome Crystallization Challenges:

• Co-crystallization with substrates, products, or substrate analogs to stabilize specific conformations

• Surface entropy reduction through targeted mutagenesis of surface residues

• Truncation of disordered regions that may interfere with crystal packing

• Screening diverse precipitants and additives, particularly those that mimic physiological ligands

• Exploration of crystallization at different temperatures and pH values

How does PEPCK interact with other metabolic enzymes in N. crassa carbon metabolism networks?

PEPCK functions within a complex metabolic network in N. crassa, with multiple interactions that affect carbon flux:

• TCA Cycle Integration: PEPCK works in concert with malate dehydrogenase, which supplies oxaloacetate from malate. This interaction forms a critical node connecting the TCA cycle and gluconeogenesis.

• Glycolysis/Gluconeogenesis Interface: PEPCK activity must be coordinated with phosphofructokinase and fructose-1,6-bisphosphatase activities to prevent futile cycling.

• PKA-Mediated Regulation: The cAMP-dependent protein kinase A (PKA) pathway affects carbon-source-dependent processes in N. crassa , potentially influencing PEPCK through direct phosphorylation or regulation of transcription factors.

• CRE-1 Regulatory Network: As a key carbon catabolite repression regulator, CRE-1 affects the expression of carbon-metabolizing enzymes, including PEPCK, creating a regulatory network that responds to carbon source availability .

Research approaches to study these interactions include metabolic flux analysis using isotope-labeled substrates, protein-protein interaction studies (co-immunoprecipitation, yeast two-hybrid), and systems biology approaches combining transcriptomics, proteomics, and metabolomics data.

How can recombinant N. crassa PEPCK contribute to studies of fungal adaptation to different nutrient environments?

Recombinant N. crassa PEPCK serves as an excellent model for investigating fungal metabolic adaptation:

• Comparative Enzymology: By characterizing the kinetic parameters of PEPCK across different fungal species, researchers can identify adaptations specific to different ecological niches.

• Evolution of Metabolic Regulation: Analysis of PEPCK regulatory mechanisms across fungal lineages provides insights into the evolution of metabolic control systems.

• Environmental Response Studies: Examining how PEPCK expression and activity change in response to different carbon sources, stress conditions, or developmental stages reveals mechanisms of fungal adaptation.

Experimental Approaches:

What is the relationship between PEPCK and fungal development in N. crassa?

The relationship between PEPCK and fungal development in N. crassa likely involves complex interactions between metabolism and developmental signaling:

• Carbon Metabolism During Different Life Stages: PEPCK activity may be differentially regulated during vegetative growth, conidiation, and sexual development to accommodate changing metabolic demands.

• Connection to Polarity and Morphogenesis: Carbon source-dependent polarity changes in N. crassa involve PKA and CRE-1 , which may indirectly affect PEPCK regulation and impact hyphal morphogenesis.

• Stress Response Integration: PEPCK regulation may be integrated with stress response pathways, particularly those responding to nutrient limitation during development.

Research approaches should include:

• Analysis of PEPCK expression and activity across different developmental stages

• Creation of conditional pck-1 mutants to examine stage-specific requirements

• Investigation of potential interactions between pck-1 and known developmental regulators

• Metabolomic analysis of wild-type and pck-1 mutant strains during development to identify metabolic signatures associated with developmental transitions