Recombinant Vibrio sp. Aspartate carbamoyltransferase catalytic chain (pyrB)

What is the basic structure of the Vibrio sp. ATCase pyrB gene and its relationship to the pyrBI operon?

The Vibrio sp. aspartate carbamoyltransferase catalytic chain is encoded by the pyrB gene, which forms part of a pyrBI operon structure homologous to that found in Escherichia coli. In this arrangement, pyrB encodes the catalytic chain while pyrI encodes the regulatory chain of the enzyme complex. The genes constitute a bicistronic operon with a single transcriptional control region preceding the structural gene for the catalytic polypeptide . The pyrBI operon in Vibrio strain 2693 has been successfully cloned by complementation in E. coli, demonstrating the functional conservation of this genetic arrangement across bacterial species .

How does the amino acid sequence of Vibrio sp. ATCase pyrB compare to that of E. coli?

The amino acid sequences of Vibrio and E. coli PyrB polypeptides exhibit approximately 74% identity, indicating a high degree of conservation . This substantial sequence similarity suggests that the catalytic (c) chains of both enzymes likely present closely comparable tertiary structures. Notably, most of the differences between the sequences (52 of 81 changes, with 53 of them being conservative) are localized in the equatorial (or aspartate-binding) domain. Multiple amino acid sequence alignments reveal that active site residues that contact carbamoyl phosphate (CP) or aspartate in E. coli ATCase are integrally conserved in the Vibrio enzyme .

What are the key structural elements involved in subunit interactions in Vibrio sp. ATCase?

The strong sequence similarities between Vibrio and E. coli ATCases include extensive conservation of residues involved in interactions between subunits, suggesting that the two enzymes have very similar tertiary and quaternary structures . Most of the residues which, in E. coli, are involved in interactions at the c1-c2 interface (between subunits of the same catalytic trimer) are conserved in Vibrio sp. This conservation of interface residues indicates that the quaternary structure and the mechanism of communication between subunits are likely preserved in the Vibrio enzyme, despite its adaptation to lower temperatures .

How does temperature affect the activity and stability of Vibrio sp. ATCase?

Vibrio sp. ATCase demonstrates characteristic psychrophilic (cold-loving) adaptations. The enzyme remains considerably active at low temperatures, suitable for its psychrophilic host, which has an optimal growth temperature of 6°C. Temperature-dependent activity studies show that the enzyme maintains significant catalytic function at temperatures well below the optimal growth temperature of E. coli . Thermal stability measurements indicate that Vibrio ATCase is much more thermolabile than its E. coli counterpart, with a marked decrease in stability at temperatures above approximately 30°C .

What is the kinetic behavior of Vibrio sp. ATCase with respect to its substrates?

Vibrio sp. ATCase displays distinctive kinetic properties:

The enzyme shows weak homotropic interactions with respect to aspartate (n₁ = 1.3 ± 0.05), with an S₀.₅ value of 40 ± 4 mM. For carbamoyl phosphate, the saturation curves are hyperbolic with an apparent Km of 0.3 ± 0.02 mM . These kinetic parameters suggest a different regulatory behavior compared to E. coli ATCase, possibly reflecting adaptation to the cold environment of its host organism.

How do nucleotides regulate Vibrio sp. ATCase activity compared to E. coli ATCase?

Unlike E. coli ATCase, Vibrio sp. ATCase exhibits distinct regulatory responses to nucleotides:

The Vibrio enzyme is inhibited by CTP but is not activated by ATP and does not show the synergistic inhibition by CTP and UTP that is characteristic of E. coli ATCase . These differences in allosteric regulation may represent adaptations to the specific metabolic requirements of the psychrophilic lifestyle.

What are the optimal conditions for cloning and expressing recombinant Vibrio sp. pyrB in E. coli?

For successful cloning and expression of Vibrio sp. pyrB in E. coli, researchers should consider using complementation strategies similar to those employed in the original characterization of the enzyme. The cloning can be performed using standard molecular biology techniques with appropriate selection markers to identify successful transformants. Expression should be optimized considering the temperature sensitivity of the enzyme, with lower induction temperatures (15-25°C) potentially yielding better results than standard E. coli expression conditions .

When designing expression constructs, it's important to consider whether to express the catalytic chain alone or in conjunction with the regulatory chain (pyrI). For studying the complete enzyme complex and its regulatory properties, co-expression of both chains is recommended to ensure proper assembly of the holoenzyme.

What methods are most effective for purifying recombinant Vibrio sp. ATCase?

Purification of recombinant Vibrio sp. ATCase should take into account its thermolability. A multi-step purification strategy might include:

• Cell lysis under gentle conditions at low temperature (0-4°C)

• Ammonium sulfate precipitation

• Ion-exchange chromatography

• Gel filtration chromatography

Throughout the purification process, it's critical to maintain low temperatures to preserve enzyme activity. Additionally, including stabilizing agents such as glycerol (10-20%) in the buffers may help maintain enzyme integrity. The purification buffers should ideally be maintained at pH 9.0, as this has been used successfully in previous work with this enzyme .

How can researchers accurately measure the enzymatic activity of recombinant Vibrio sp. ATCase?

The standard assay for measuring ATCase activity involves quantifying the production of carbamoyl-aspartate. The specific activity can be expressed as units per mg protein, where one unit represents the amount of enzyme that synthesizes 1 μmol carbamoyl-aspartate per hour. For Vibrio sp. ATCase, assays should be performed at temperatures relevant to its psychrophilic nature (typically 6-30°C) to obtain physiologically meaningful results .

For kinetic studies, researchers should use a range of substrate concentrations:

• Aspartate: 0-100 mM

• Carbamoyl phosphate: 0-5 mM

When studying the effects of nucleotides on enzyme activity, concentrations of approximately 5 mM of each nucleotide should be used, with measurements taken at both low (6-15°C) and moderate (30°C) temperatures to fully characterize the regulatory behavior .

What structural adaptations distinguish psychrophilic Vibrio sp. ATCase from mesophilic and thermophilic homologs?

The psychrophilic Vibrio sp. ATCase exhibits specific structural adaptations that distinguish it from mesophilic (e.g., E. coli) and thermophilic homologs. These adaptations typically include:

• Increased structural flexibility, particularly in regions not directly involved in catalysis

• Reduced number of stabilizing interactions (such as salt bridges, hydrogen bonds, or hydrophobic interactions)

• Modifications in amino acid composition that favor flexibility over rigidity

With respect to Pyrococcus abyssi (thermophilic) and E. coli (mesophilic) ATCases, Vibrio ATCase presents marked differences in composition which have been related to its psychrophilic character . These adaptations allow the enzyme to maintain sufficient flexibility for catalysis at low temperatures where mesophilic or thermophilic enzymes would be too rigid to function efficiently.

How does the transcriptional regulation of the pyrBI operon in Vibrio sp. compare to that in E. coli?

The transcriptional regulation of the pyrBI operon in Vibrio sp. shares some similarities with E. coli but also shows distinctive features. In Vibrio strain 2693, the operon is expressed from a promoter that has been experimentally characterized, with a predominant transcription start at a G residue preceded by a putative -10 TAAAAT sequence element . Unlike the typical bacterial promoter structure, no corresponding -35 motif could be identified at a canonical distance in the Vibrio promoter.

What evolutionary insights can be gained from comparing pyrB genes across different bacterial species?

Comparative analysis of pyrB genes across bacterial species provides valuable insights into the evolution of ATCase and its adaptation to different environmental niches. The high degree of sequence similarity (74% identity) between Vibrio and E. coli PyrB polypeptides suggests a common ancestral origin with subsequent divergence as these organisms adapted to different ecological niches .

The conservation of catalytic residues across species highlights the functional constraints on enzyme active sites, while variations in regulatory properties (such as the response to nucleotides) reflect adaptations to different metabolic needs. The psychrophilic adaptations observed in Vibrio sp. ATCase demonstrate how protein structures can be fine-tuned to function optimally under specific environmental conditions while maintaining the core catalytic function.

How can structural studies of Vibrio sp. ATCase contribute to understanding cold adaptation in enzymes?

Vibrio sp. ATCase serves as an excellent model system for investigating cold adaptation mechanisms in enzymes. Detailed structural studies using X-ray crystallography or cryo-electron microscopy could reveal the specific molecular features that contribute to its psychrophilic character. Comparing these structures with those of mesophilic and thermophilic homologs would provide direct evidence of adaptive changes in protein architecture .

Particularly valuable insights might come from analyzing:

• The flexibility of loop regions, especially those in the equatorial domain where most sequence variations are located

• The nature and distribution of electrostatic interactions across the protein surface

• The hydration patterns around the protein

• The dynamics of conformational changes associated with catalysis and regulation

Such structural information would not only enhance our understanding of Vibrio sp. ATCase but would also contribute to broader principles of protein adaptation to extreme environments.

What experimental approaches can be used to investigate the allosteric regulation differences between Vibrio sp. and E. coli ATCase?

To investigate the distinct allosteric regulation patterns of Vibrio sp. ATCase compared to E. coli ATCase, researchers could employ several complementary approaches:

• Site-directed mutagenesis - Introducing specific mutations at positions known to be involved in nucleotide binding in E. coli ATCase to identify the residues responsible for the differential response to ATP and CTP/UTP

• Chimeric enzyme construction - Creating hybrid enzymes with domains from both Vibrio and E. coli ATCases to map which regions determine the specific regulatory properties

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS) - To compare the dynamics and conformational changes induced by nucleotide binding in both enzymes

• Isothermal titration calorimetry (ITC) - To precisely characterize the thermodynamics of nucleotide binding to the regulatory sites

These approaches would help elucidate the molecular basis for the observed differences in allosteric behavior and potentially reveal new principles of allostery in enzymes adapted to different thermal environments.

How might directed evolution be applied to modify the catalytic or regulatory properties of Vibrio sp. ATCase?

Directed evolution represents a powerful approach for engineering modified versions of Vibrio sp. ATCase with altered catalytic or regulatory properties. This experimental strategy could be implemented through:

• Random mutagenesis - Using error-prone PCR to generate libraries of pyrB variants with point mutations throughout the gene

• DNA shuffling - Recombining pyrB sequences from Vibrio sp. with homologous genes from other species to create chimeric enzymes with novel properties

• Focused mutagenesis - Targeting specific regions known to be involved in catalysis or regulation for higher mutation rates

Selection or screening strategies could be designed to identify variants with desired properties such as:

• Enhanced catalytic efficiency at specific temperatures

• Altered substrate specificity

• Modified regulatory responses to nucleotides

• Increased thermostability while maintaining cold activity

Such engineered variants would not only have potential biotechnological applications but would also provide insights into structure-function relationships in this important enzyme.

What are common challenges in maintaining the stability of recombinant Vibrio sp. ATCase during purification and storage?

The psychrophilic nature of Vibrio sp. ATCase presents specific challenges for maintaining stability during purification and storage. Common issues include:

• Thermal denaturation - The enzyme is considerably more thermolabile than its E. coli counterpart, with significant loss of activity above 30°C

• Proteolytic degradation - Increased flexibility may make the enzyme more susceptible to proteases

• Oxidative damage - Sulfhydryl groups may be more exposed and reactive in the flexible structure

To address these challenges, researchers should consider:

• Maintaining strictly controlled low temperatures (0-4°C) throughout all handling procedures

• Including protease inhibitors in all buffers

• Adding reducing agents (e.g., DTT or β-mercaptoethanol) to prevent oxidation

• Incorporating stabilizing agents such as glycerol (10-20%) or appropriate osmolytes

• Storing the purified enzyme in small aliquots at -80°C to minimize freeze-thaw cycles

How can researchers address discrepancies in kinetic measurements between native and recombinant forms of Vibrio sp. ATCase?

When comparing kinetic measurements between native and recombinant forms of Vibrio sp. ATCase, researchers might encounter discrepancies that could stem from several factors:

• Post-translational modifications - The E. coli expression system might not reproduce all modifications present in the native enzyme

• Quaternary structure differences - Improper assembly of the holoenzyme in the recombinant system, especially if expressing only the catalytic chain

• Buffer composition effects - Different ionic conditions might affect enzyme behavior differently between preparations

To address these issues, researchers should:

• Compare enzyme preparations using multiple complementary activity assays

• Characterize the quaternary structure using analytical techniques like size exclusion chromatography or native PAGE

• Test enzyme activity under various buffer conditions to identify optimal measurement parameters

• Consider co-expression of both pyrB and pyrI to ensure proper assembly of the holoenzyme

• Validate findings using site-directed mutagenesis to confirm the roles of specific residues

Research indicates that when properly expressed, the recombinant Vibrio ATCase in E. coli extracts shows similar temperature dependence of activity and stability to the native enzyme in Vibrio extracts .

What experimental controls are essential when comparing the properties of Vibrio sp. ATCase with those of other species?

When conducting comparative studies between Vibrio sp. ATCase and homologs from other species, several essential controls should be implemented:

• Standardized enzyme preparations - Ensure consistent purification protocols that yield comparable enzyme purity and integrity

• Temperature controls - Perform comparative measurements at multiple temperatures relevant to each enzyme's native environment (e.g., 6°C for Vibrio, 37°C for E. coli)

• Assay validation - Verify that assay conditions (pH, ionic strength, substrate concentrations) are appropriate for detecting the full range of activity for each enzyme

• Structural integrity confirmation - Use methods like circular dichroism or fluorescence spectroscopy to confirm that each enzyme preparation maintains its native fold

• Statistical rigor - Perform multiple independent experiments with appropriate replicates and statistical analysis

These controls help ensure that observed differences reflect genuine biological adaptations rather than artifacts of experimental conditions or preparation methods.