Recombinant Carbamoyl-phosphate synthase small chain (carA)

What is the structure and function of Carbamoyl-phosphate synthase small chain (carA)?

Carbamoyl-phosphate synthase small chain (carA) is the glutaminase subunit of the carbamoyl-phosphate synthase enzyme complex. In E. coli, this 40 kDa subunit (also called GLN) hydrolyzes glutamine and transfers the resulting ammonia to the larger synthetase subunit (carB) . The CarA subunit associates with the CarB subunit to form heterodimers and tetramers . This association is essential for the enzymatic function of synthesizing carbamoyl phosphate, which serves as a precursor for both pyrimidine nucleotides and arginine biosynthesis.

The carA gene is part of the carAB operon, which is subject to complex regulatory control. In E. coli, this operon is regulated by transcription factors including ArgR (arginine repressor), which binds to the P2 promoter region in response to arginine levels . This regulation ensures appropriate production of carbamoyl phosphate based on cellular needs for both arginine and pyrimidine synthesis.

Which expression systems are most effective for producing recombinant carA?

Several expression systems have proven effective for recombinant carA production, with the choice depending on experimental goals:

E. coli Expression System:

• Host strains: BL21(DE3) is commonly used for recombinant protein expression as demonstrated in several studies .

• Vector systems: pET vectors (such as pET21c) provide strong T7 promoter-driven expression inducible with IPTG .

• Expression conditions: Typical induction with 0.5-1.0 mM IPTG, growth at 30-37°C.

Baculovirus/Insect Cell System:

• Particularly useful for complex proteins like human CPS1, which has been successfully expressed in this system .

• Allows proper folding and post-translational modifications.

• Yields highly purified, active enzyme suitable for crystallization and structural studies .

Advantages of E. coli vs. Baculovirus systems for carA expression:

What are the key considerations for purifying recombinant carA?

Purification of recombinant carA requires attention to several critical factors:

• Protein Stability: The enzyme may require specific buffer conditions with stabilizing agents. Glycerol has been shown to partially replace the essential activator N-acetyl-L-glutamate (NAG) in CPS1, which may be relevant for stabilizing recombinant carA during purification .

• Purification Strategy:

  • Affinity tags: His-tags facilitate one-step purification as demonstrated in some recombinant CPS expression systems .

  • Size exclusion chromatography: Useful for separating different oligomeric states (dimers/tetramers) .

  • Ion exchange chromatography: Helps remove contaminants based on charge differences.

• Activity Preservation: Maintain enzyme activity during purification by:

  • Including protease inhibitors to prevent degradation

  • Using appropriate cofactors or stabilizing agents

  • Monitoring pH and temperature carefully

• Oligomeric State Assessment: Chemical cross-linking and size exclusion chromatography can confirm the expected homodimeric/tetrameric structure of the purified protein .

How can I verify the activity of recombinant carA?

Verification of recombinant carA activity can be approached through several complementary methods:

• Glutaminase Activity Assay:

  • Measure the hydrolysis of glutamine to glutamate and ammonia

  • Monitor ammonia production using colorimetric assays

  • Track glutamate formation using HPLC or enzymatic coupled assays

• Coupled Enzyme Assays:

  • Co-express or combine with purified carB to measure complete CPS activity

  • Monitor ATP consumption or carbamoyl phosphate formation

  • Steady-state kinetics can reveal apparent affinity for substrates (Km values)

• Structural Verification:

  • Size exclusion chromatography to confirm proper oligomeric state

  • Circular dichroism to assess secondary structure

  • Thermal shift assays to evaluate protein stability

One study of a small CPSase from M. smithii (MS-s) demonstrated that the recombinant enzyme catalyzed ATP-dependent partial reactions similar to full-length CPSases and exhibited high apparent affinity for ATP and ammonia . Similar approaches can be applied to verify recombinant carA activity.

What are common challenges in working with recombinant carA?

Researchers commonly encounter several challenges when working with recombinant carA:

• Protein Solubility: carA may form inclusion bodies in bacterial expression systems, particularly at high expression levels. Strategies to overcome this include:

  • Lowering expression temperature (18-25°C)

  • Using solubility-enhancing tags (SUMO, MBP, etc.)

  • Co-expression with chaperones

• Protein Stability: Recombinant carA may show limited stability after purification. Research has shown upregulation of cellular stress proteins (DnaK, HtpG, catalase-peroxidase) during overexpression of certain recombinant proteins, indicating cellular stress responses that might affect protein quality .

• Activity Reconstitution: As carA functions in complex with carB, reconstituting full enzymatic activity may require:

  • Co-expression of both subunits

  • Carefully controlled reconstitution of the complex in vitro

  • Inclusion of appropriate cofactors and substrates

• Expression Toxicity: Overexpression of recombinant proteins can trigger stress responses in host cells. As observed in a study of mini-spidroin expression, cellular stress proteins showed increased expression (>12-fold) alongside the recombinant protein .

How do mutations in carA affect enzyme activity and substrate binding?

Studying mutations in carA provides critical insights into structure-function relationships. Based on human CPS1 studies, which share functional similarities with bacterial carA, several approaches can be utilized:

• Site-Directed Mutagenesis Approach:

  • Target conserved active site residues identified through sequence alignment

  • Develop a systematic mutation strategy targeting catalytic residues, substrate binding sites, and allosteric sites

  • Express and purify mutant proteins for comparative functional analysis

• Functional Impact Assessment:
  Studies on human CPS1 mutations revealed diverse effects that can guide research on bacterial carA:

  • Some mutations decrease enzyme stability

  • Others specifically hamper catalysis

  • Some particularly affect allosteric regulation

  • Many polymorphisms may have no detectable effects

• Structural Analysis of Mutations:

  • Molecular modeling can predict structural impacts of mutations

  • Comparing the model with wild-type structures gives insights into altered binding or catalytic mechanisms

  • Ramachandran plot analysis can identify problematic mutations affecting protein folding

Research on CPS from M. smithii demonstrated that despite only 20% sequence identity with E. coli domains, ten out of twelve critical active site residues were identical or had highly conservative substitutions (K/R or L/I), highlighting the importance of these residues for function .

What structural domains are critical for carA function and how can they be characterized?

The functional characterization of carA structural domains requires integrated approaches:

• Domain Identification Methods:

  • Sequence alignment with homologous proteins

  • Secondary structure prediction

  • Limited proteolysis to identify domain boundaries

  • Expression of isolated domains to test independent functions

• Critical Functional Domains:
  Analysis of CPSase structures reveals several key regions:

  • Glutamine binding site in the glutaminase domain

  • Ammonia channel for substrate transfer to carB

  • Interface regions mediating interactions with carB

  • Regulatory domains responding to allosteric effectors

• Domain Characterization Techniques:

  • X-ray crystallography has been successfully used for human CPS1 after recombinant expression, enabling structural studies

  • Molecular modeling approaches, even with relatively low sequence identity (20%), can provide valuable insights into tertiary structure

  • Hydrogen/deuterium exchange mass spectrometry can identify flexible regions and binding interfaces

  • SAXS (Small Angle X-ray Scattering) for low-resolution domain arrangement in solution

The interaction between carA and carB subunits is particularly important, as the association forms heterodimers and tetramers that are essential for function. Chemical cross-linking coupled with mass spectrometry can map these interaction surfaces .

How does carA interact with carB to form a functional CPS complex?

The interaction between carA and carB subunits is crucial for CPS function and can be investigated through various approaches:

• Physical Interaction Studies:

  • Chemical cross-linking followed by mass spectrometry to identify interaction surfaces

  • Co-immunoprecipitation to verify complex formation

  • Surface plasmon resonance to measure binding kinetics and affinity

  • FRET (Förster Resonance Energy Transfer) to study interactions in real-time

• Functional Communication:
  CPSase function involves a sophisticated mechanism where:

  • carA hydrolyzes glutamine to produce ammonia

  • Ammonia is channeled to carB through an intramolecular tunnel

  • carB catalyzes the synthesis of carbamate from ATP, bicarbonate, and ammonia

  • A second ATP-dependent reaction in carB produces carbamoyl phosphate

• Interface Mutation Analysis:

  • Site-directed mutagenesis targeting interface residues can disrupt or enhance interactions

  • The impact on complex formation and enzyme activity can reveal critical interaction points

  • Compensatory mutations can restore function lost through primary mutations

Studies on E. coli CPSase have shown that the SYN subunit (carB) consists of two homologous domains with nearly identical tertiary fold and active site residues, thought to have evolved by ancestral duplication and fusion . This structural arrangement facilitates the coordinated sequential reactions catalyzed by the enzyme complex.

What techniques can be used to study the kinetics of carA-mediated reactions?

Advanced kinetic analysis of carA requires sophisticated techniques and careful experimental design:

• Steady-State Kinetics:

  • Measurement of glutaminase activity under varying substrate concentrations

  • Determination of Michaelis-Menten parameters (Km, Vmax, kcat)

  • Analysis of allosteric regulation through Hill coefficient determination

• Pre-Steady-State Kinetics:

  • Stopped-flow spectroscopy to capture rapid reaction phases

  • Quenched-flow techniques to analyze reaction intermediates

  • Temperature-jump methods to study conformational changes

• Coupled Assays for Complete CPS Activity:
  The small CPSase from M. smithii synthesizes carbamoyl phosphate from ATP, bicarbonate, and ammonia, catalyzing the same ATP-dependent partial reactions observed for full-length CPSases . Similar assay approaches can be applied to carA studies:

  Parameter	Value for M. smithii CPSase	Typical Range for bacterial carA
  Km for ATP	Low (high apparent affinity)	0.1-1.0 mM
  Km for ammonia	Low (high apparent affinity)	0.1-5.0 mM
  Km for bicarbonate	Variable	1.0-10.0 mM
  Temperature optimum	Species-dependent	25-50°C
  pH optimum	Slightly alkaline	7.5-8.5

• Isotope Exchange Studies:

  • Use of isotopically labeled substrates (15N-glutamine, 13C-bicarbonate)

  • Analysis of reaction intermediates and mechanism through isotope tracing

  • Determination of rate-limiting steps in the reaction sequence

How does carA differ across bacterial species, and what are the implications for research?

Comparative analysis of carA across bacterial species reveals important evolutionary and functional insights:

How can I design experiments to investigate the regulatory mechanisms of carA expression?

Investigating the regulation of carA expression requires sophisticated experimental approaches:

• Promoter Analysis:

  • The carAB operon in E. coli contains multiple promoters (P1 and P2) with complex regulation

  • Reporter gene assays (using LacZ, GFP, or luciferase) can measure promoter activity under different conditions

  • Electrophoretic mobility shift assays (EMSA) can identify protein-DNA interactions at regulatory sites

  • DNase I footprinting can precisely map transcription factor binding sites

• Transcriptional Regulation Mechanisms:
  In E. coli, the P2 promoter of the carAB operon is regulated by the arginine repressor ArgR :

  • ArgR binding to the P2 promoter region is mutually exclusive with RNA polymerase binding

  • Transcription from the P1 promoter can proceed even in the presence of excess arginine

  • ArgR-mediated repression of P2 is stronger when P1 initiation is silenced

• Environmental Response Analysis:

  • qRT-PCR to measure carA transcript levels under different conditions

  • Microarray or RNA-seq for global transcriptional response analysis

  • ChIP-seq to identify transcription factor binding across the genome

  • Metabolomic analysis to correlate carA expression with metabolite levels

• Mathematical Modeling:

  • Develop kinetic models of carA regulation incorporating feedback mechanisms

  • Use systems biology approaches to integrate carA regulation into larger metabolic networks

  • Apply machine learning to identify complex regulatory patterns from experimental data

Understanding these regulatory mechanisms is critical because in E. coli and most other Gram-negative bacteria, CP is produced by a single enzyme (CPSase) encoded by the carAB operon, creating the need for complex control of CP production for both arginine and pyrimidine synthesis .

What advanced analytical techniques are most effective for studying recombinant carA?

Several sophisticated analytical techniques provide powerful insights into recombinant carA structure and function:

• Structural Analysis Methods:

  • X-ray crystallography: Successfully used for human CPS1 after recombinant expression in baculovirus/insect cell systems

  • Cryo-electron microscopy: Provides high-resolution structural information without crystallization

  • Hydrogen/deuterium exchange mass spectrometry: Maps solvent accessibility and conformational changes

  • NMR spectroscopy: Provides atomic-level insights into protein dynamics in solution

• Proteomic Approaches:

  • Quantitative shotgun proteomics: Can identify proteins associated with carA synthesis, as demonstrated in studies of recombinant protein expression

  • 2D gel electrophoresis: After recombinant protein expression, can visualize global proteomic changes

  • Protein spots can be visualized with silver staining and analyzed using densitometry software like Melanie II

• Mass Spectrometry Applications:

  • Native MS: Analyze intact protein complexes and determine stoichiometry

  • Cross-linking MS: Identify interaction surfaces between subunits

  • Top-down proteomics: Characterize full-length proteins and post-translational modifications

  • Ion mobility MS: Provide insights into protein conformation and dynamics

• Biophysical Characterization:

  • Circular dichroism: Assess secondary structure and stability

  • Differential scanning calorimetry: Determine thermal stability

  • Analytical ultracentrifugation: Analyze oligomeric state and complex formation

  • Surface plasmon resonance: Measure binding kinetics and affinity

A combined approach using multiple analytical techniques provides the most comprehensive understanding of recombinant carA structure, function, and interactions.

How can I improve yield and solubility of recombinant carA?

Optimizing recombinant carA production requires systematic approach to expression conditions:

• Expression System Optimization:

  • Test multiple E. coli strains (BL21(DE3), Rosetta, Arctic Express, etc.)

  • Evaluate different expression vectors and promoter strengths

  • Consider codon optimization for the host organism

  • Explore alternative expression systems for difficult proteins

• Culture Conditions:

  • Optimize growth temperature (typically lowering to 16-25°C improves solubility)

  • Test various induction conditions (IPTG concentration, induction timing)

  • Evaluate media composition (rich vs. minimal, supplementation with cofactors)

  • Consider auto-induction media for gradual protein expression

• Co-expression Strategies:

  • Co-express with chaperones (DnaK, GroEL/ES, trigger factor)

  • Consider co-expression with carB to promote complex formation

  • Express with protein partners that enhance solubility

• Fusion Tags and Solubility Enhancers:

  • Test various solubility-enhancing tags (MBP, SUMO, TrxA, GST)

  • Evaluate different affinity tags for purification (His, FLAG, Strep)

  • Optimize tag placement (N-terminal vs. C-terminal)

  • Include efficient tag removal strategies (TEV protease, etc.)

Evidence from studies on recombinant protein expression indicates that cellular stress responses may be triggered during overexpression, as shown by increased levels of chaperone proteins DnaK, HtpG, and catalase-peroxidase . Managing this stress response may be key to optimizing yields.

What approaches can resolve activity loss during purification of recombinant carA?

Maintaining carA activity throughout purification requires attention to several critical factors:

• Buffer Optimization:

  • Test buffers with different pH values (typically 7.0-8.5)

  • Include stabilizing agents (glycerol has shown stabilizing effects for CPS1)

  • Add cofactors or substrate analogs to stabilize active conformation

  • Consider detergents for membrane-associated forms

• Protecting Against Proteolysis and Oxidation:

  • Add protease inhibitor cocktails during cell lysis and early purification steps

  • Include reducing agents (DTT, β-mercaptoethanol, TCEP) to prevent oxidation

  • Maintain low temperature throughout purification

  • Minimize handling time and exposure to air

• Purification Strategy Refinement:

  • Evaluate different chromatography methods in various sequences

  • Optimize elution conditions to minimize exposure to harsh chemicals

  • Consider on-column refolding for proteins recovered from inclusion bodies

  • Test gentle elution methods (gradient vs. step elution)

• Activity Preservation Strategies:

  • Add stabilizing ligands during purification

  • Store enzyme with appropriate cofactors

  • Optimize protein concentration for storage (too high may promote aggregation)

  • Evaluate various storage conditions (liquid nitrogen, -80°C, -20°C with glycerol)

Research on human CPS1 demonstrated that glycerol can partially replace the essential activator N-acetyl-L-glutamate (NAG) , suggesting that similar approaches might help stabilize recombinant carA during purification and storage.

How can I troubleshoot unexpected oligomeric states of recombinant carA?

Addressing unexpected oligomerization of recombinant carA requires systematic investigation:

• Oligomeric State Analysis:

  • Size exclusion chromatography to determine predominant species

  • Native PAGE to separate different oligomeric forms

  • Chemical cross-linking to capture transient interactions

  • Analytical ultracentrifugation for detailed characterization

• Factors Affecting Oligomerization:

  • Protein concentration (higher concentrations often promote oligomerization)

  • Buffer composition (salt concentration, pH, additives)

  • Presence of cofactors or substrates

  • Redox state (disulfide bond formation)

• Engineering Approaches:

  • Introduce mutations at interfaces to disrupt unwanted interactions

  • Modify surface charges to prevent non-specific aggregation

  • Add or remove cysteine residues to control disulfide formation

  • Design stabilizing interactions for desired oligomeric states

• Validation Methods:
  Research on CPSase from M. smithii used chemical cross-linking and size exclusion chromatography to confirm a homodimeric/tetrameric structure consistent with dimer-based CPSase activity . Similar approaches can validate the correct oligomeric state of recombinant carA.

What are the best approaches for scaling up recombinant carA production for structural studies?

Scaling up recombinant carA production for structural studies requires careful consideration of several factors:

• Fermentation Strategies:

  • Batch cultivation: Simple but limited biomass yield

  • Fed-batch cultivation: Higher cell densities and protein yields

  • Continuous cultivation: Steady-state production for consistent quality

  • High-density fermentation: Maximizes volumetric productivity

• Expression System Selection:

  • E. coli: Easiest to scale, but may have limitations for complex proteins

  • Baculovirus/insect cells: Successfully used for human CPS1 expression leading to crystallization

  • Yeast systems: Combine ease of scaling with some eukaryotic processing benefits

  • Mammalian cells: Most complex processing but challenging to scale

• Purification Scale-Up Considerations:

  • Transition from gravity columns to automated systems (ÄKTA or similar)

  • Invest in larger chromatography columns and increased resin volumes

  • Develop efficient capture steps to handle large volumes

  • Implement tangential flow filtration for buffer exchange and concentration

• Quality Control Metrics:

  • Establish robust activity assays for batch validation

  • Implement analytical techniques to confirm structural integrity

  • Monitor batch-to-batch variation in yield and specific activity

  • Validate oligomeric state consistency across batches

For structural biology applications, recombinant human CPS1 was successfully produced in a baculovirus/insect cell expression system, yielding enzyme in an active and highly purified form suitable for crystallization and X-ray diffraction studies .