Recombinant Enterobacter sp. Bifunctional protein aas (aas)

What is the Bifunctional protein aas from Enterobacter sp.?

Bifunctional protein aas from Enterobacter sp. (strain 638) is a dual-function enzyme with the UniProt accession number A4WE11. This protein functions primarily as a 2-acylglycerophosphoethanolamine acyltransferase (EC 2.3.1.40), also known as 2-acyl-GPE acyltransferase. It plays a crucial role in phospholipid metabolism, particularly in membrane biogenesis pathways . The protein consists of 719 amino acids with a complex secondary structure that facilitates its bifunctional catalytic activities.

What are the structural characteristics of Bifunctional protein aas?

The Bifunctional protein aas from Enterobacter sp. is characterized by its full amino acid sequence beginning with mLFGFFRTLFRILFRIRLTGDTQSLQ and continuing through 719 residues . Analysis of recombinant bifunctional proteins typically involves assessment of secondary structure elements using techniques such as circular dichroism, X-ray crystallography, or comparative modeling. While specific structural data for this particular protein is limited in the provided sources, bifunctional proteins generally contain distinct domains that enable their dual catalytic activities, often with flexible linker regions connecting functional domains .

How is recombinant Enterobacter sp. Bifunctional protein aas typically expressed?

Recombinant expression of Enterobacter sp. Bifunctional protein aas typically employs heterologous expression systems similar to those used for other bacterial recombinant proteins. The methodology generally involves:

• Gene cloning into an expression vector (commonly pET-series vectors)

• Transformation into a suitable expression host (typically E. coli strains like BL21(DE3))

• Induction of protein expression (often using IPTG for T7-based systems)

• Cell harvesting and lysis

• Protein purification using affinity chromatography (commonly with His-tag)

For example, in comparable recombinant protein expression studies, proteins are purified from culture media using ion exchange chromatography, followed by SDS-PAGE analysis to confirm purity and correct molecular weight . Recombinant Bifunctional protein aas would likely be expressed with a tag to facilitate purification, as indicated by product listings for the commercially available recombinant protein .

What expression systems are most effective for producing active Recombinant Enterobacter sp. Bifunctional protein aas?

While specific optimization data for Recombinant Enterobacter sp. Bifunctional protein aas is not directly provided in the search results, insights can be drawn from similar recombinant expression systems. Based on comparable bifunctional enzymes, several expression systems might be suitable:

Expression System Comparison for Bifunctional Proteins:

For the Bifunctional protein aas, E. coli expression systems would likely be the first choice given the bacterial origin of the protein, but optimization may be required to ensure proper folding and activity . Researchers should consider factors such as codon optimization, induction temperature, and solubility-enhancing fusion tags when designing expression protocols.

What purification strategies yield the highest purity and activity for Recombinant Enterobacter sp. Bifunctional protein aas?

Effective purification of Recombinant Enterobacter sp. Bifunctional protein aas likely requires a multi-step approach:

• Initial Capture: Affinity chromatography using Ni-Sepharose for His-tagged protein is commonly employed, as seen with similar recombinant proteins .

• Intermediate Purification: Ion exchange chromatography can separate the target protein based on charge properties.

• Polishing Step: Size exclusion chromatography to achieve final purity.

For example, a successful purification protocol for a recombinant bifunctional enzyme involved loading concentrated crude enzyme onto a Ni-Sepharose column equilibrated with 300 mM NaCl and 50 mM phosphate buffer (pH 7.0), washing with 20 mM imidazole, and eluting with 50-500 mM imidazole gradient . This approach typically yields >90% purity when properly optimized.

Activity should be monitored throughout purification steps with appropriate enzyme assays specific to the catalytic functions of the protein. For Bifunctional protein aas, assays measuring 2-acylglycerophosphoethanolamine acyltransferase activity would be essential.

How can Activity-Based Protein Profiling (ABPP) be applied to study Bifunctional protein aas function?

Activity-Based Protein Profiling (ABPP) represents an advanced approach for studying bifunctional enzymes like the Enterobacter sp. Bifunctional protein aas. While not specifically applied to this protein in the provided references, ABPP has been successfully used with other bifunctional enzymes:

ABPP Methodology for Bifunctional Enzymes:

• Probe Selection/Design: Design activity-based probes that selectively target the catalytic residues of the aas protein. For acyltransferases, probes could be designed based on substrate analogs with reactive groups that form covalent bonds with the active site.

• Probe Application: Incubate the recombinant protein or biological samples containing the native protein with the activity-based probe.

• Analysis Strategy: Perform comparative proteomics analysis combining ABPP with mass spectrometry (MS) to identify active sites and catalytic mechanisms .

This approach has been successfully applied for the identification of glycoside hydrolases in extremophilic archaea, where complementary proteomics combined with ABPP and MS-based analysis enabled identification of novel bifunctional enzymes . Applied to Bifunctional protein aas, this methodology could provide insights into substrate specificity, active site architecture, and the coordination between its dual catalytic functions.

What role might Bifunctional protein aas play in antimicrobial resistance mechanisms in Enterobacter species?

While direct evidence linking Bifunctional protein aas to antimicrobial resistance is not explicitly provided in the search results, several findings suggest potential connections that warrant investigation:

• Membrane Remodeling: As a protein involved in phospholipid metabolism, aas may contribute to membrane modifications that affect antibiotic permeability. Studies of Enterobacter isolates have shown that outer membrane proteins play crucial roles in antimicrobial resistance, particularly to β-lactams and cephalosporins .

• Potential Interaction with Resistance Mechanisms: Environmental Enterobacter isolates lacking specific outer membrane proteins (OmpC) showed multidrug resistance profiles . The relationship between membrane composition (potentially influenced by aas activity) and porin expression deserves exploration.

• Metabolic Adaptations: Bifunctional enzymes often operate at metabolic branch points, and altered metabolism has been linked to antibiotic tolerance in many bacteria.

Research approaches to investigate this connection could include:

• Comparative proteomics between resistant and susceptible strains to assess aas expression levels

• Gene knockout/overexpression studies to evaluate the impact on antibiotic MICs

• Lipidomics analysis to correlate aas activity with membrane composition in resistant isolates

How do the dual catalytic activities of Bifunctional protein aas coordinate during cellular metabolic processes?

The coordination between dual catalytic activities in bifunctional enzymes represents a sophisticated aspect of bacterial metabolism. While specific mechanisms for Bifunctional protein aas are not detailed in the provided sources, insights from similar bifunctional systems suggest several coordination mechanisms:

• Substrate Channeling: Intermediates may be transferred directly between active sites without release into the cytosol, increasing efficiency and preventing side reactions.

• Allosteric Regulation: Binding at one active site may induce conformational changes affecting activity at the second site.

• Expression-Level Coordination: As demonstrated with the bifunctional KDC4427/ADH4428 system in Enterobacter sp., the relative expression levels of different domains or partner proteins can regulate metabolic output .

Research by Liu et al. (2023) with another bifunctional enzyme system in Enterobacter showed that KDC4427 exhibits both phenylpyruvate decarboxylase and indolepyruvate decarboxylase activities, with differential kinetic parameters for each substrate . This differential activity, combined with regulated expression, allowed sequential production of secondary metabolites at different growth phases.

To investigate similar coordination in Bifunctional protein aas, researchers could employ:

• Steady-state and pre-steady-state kinetics with various substrates

• Site-directed mutagenesis of individual active sites

• Time-resolved structural studies during catalytic cycles

What are the critical quality control parameters for ensuring the functionality of purified Recombinant Enterobacter sp. Bifunctional protein aas?

Ensuring functionality of purified Recombinant Enterobacter sp. Bifunctional protein aas requires comprehensive quality control measures:

Critical Quality Control Parameters:

For structural model validation, methods similar to those used for the R13 Fae bifunctional enzyme could be applied, including Ramachandran plot analysis (targeting >85% residues in most favored regions) and ERRAT evaluation (quality factor >80%) . These methods verify proper folding and stability of the recombinant protein.

How can researchers accurately measure the distinct catalytic activities of Bifunctional protein aas in isolation and in complex cellular environments?

Measuring the distinct catalytic activities of Bifunctional protein aas presents methodological challenges that require specialized approaches:

For Isolated Protein:

• Coupled Enzyme Assays: Design assays that specifically measure 2-acylglycerophosphoethanolamine acyltransferase activity by coupling product formation to detectable reactions.

• Direct Product Analysis: Use chromatographic separation (HPLC/LC-MS) to directly quantify substrates and products of the reaction.

• Active Site-Specific Inhibitors: Employ selective inhibitors to distinguish between different catalytic activities of the bifunctional protein.

For Complex Cellular Environments:

• Activity-Based Protein Profiling: Apply activity-based probes selective for acyltransferase activity in cellular lysates, followed by detection via mass spectrometry .

• Metabolic Labeling: Use isotope-labeled substrates to track specific activity within cellular metabolism.

• Domain-Specific Mutations: Generate variants with mutations in one active site but not the other to differentiate activities in cellular contexts.

Correlation Between Activities:
Based on studies of other bifunctional proteins, researchers might investigate potential coordinated regulation between the dual activities. For example, Liu et al. (2023) demonstrated that the bifunctional enzyme KDC4427 in Enterobacter sp. exhibits differential activity toward different substrates (higher kcat for phenylpyruvate but higher affinity for indolepyruvate), which contributes to sequential metabolite production during different growth phases .

What protein-protein interactions have been identified for Bifunctional protein aas that influence its cellular functions?

While specific protein-protein interactions for Bifunctional protein aas are not directly reported in the provided search results, research on similar bifunctional proteins suggests several potential interaction mechanisms worthy of investigation:

• Regulatory Protein Interactions: Similar to how TyrR and RpoS interact with the promoter region of KDC4427-ADH4428 to regulate expression in Enterobacter sp. , transcriptional regulators likely influence aas expression in response to metabolic needs.

• Membrane Protein Complexes: As an enzyme involved in membrane phospholipid metabolism, aas may interact with membrane proteins including porins like OmpA, OmpX, and OmpF, which have been associated with antibiotic resistance in Enterobacter species .

• Metabolic Enzyme Networks: Bifunctional enzymes often participate in metabolic complexes that facilitate substrate channeling and coordinated pathway regulation.

Research approaches to identify these interactions could include:

• Affinity purification coupled with mass spectrometry (AP-MS)

• Bacterial two-hybrid screening

• Co-immunoprecipitation followed by proteomic analysis

• Proximity labeling techniques such as BioID

Understanding these interactions would provide insights into how Bifunctional protein aas is integrated into broader cellular metabolic networks and regulatory systems.

How does the expression of Bifunctional protein aas vary under different environmental conditions and stress responses in Enterobacter species?

• Growth Phase-Dependent Regulation: Similar to the bifunctional KDC4427 enzyme in Enterobacter sp., which shows differential expression patterns during different growth phases (upregulated during exponential growth) , aas expression may vary throughout the bacterial growth cycle.

• Stress Response Integration: Environmental stressors likely influence aas expression. For example, in Enterobacter species, outer membrane protein expression changes in response to antibiotic exposure, with consequences for resistance profiles .

• Nutrient Availability: Phospholipid metabolism enzymes often respond to changes in available carbon and phosphate sources.

Research Methodology to Investigate Expression Patterns:

Understanding these expression patterns would provide insights into the physiological roles of aas in bacterial adaptation and potentially identify conditions where it becomes particularly important for cellular function or survival.

How conserved is the Bifunctional protein aas across different Enterobacter species and related bacterial genera?

Although comprehensive conservation data specifically for Bifunctional protein aas isn't provided in the search results, approaches to assess evolutionary conservation can be derived from comparative genomic studies of Enterobacter species and related bacteria:

The Bifunctional protein aas appears to be present across multiple bacterial genera within Enterobacteriaceae. Commercial availability of recombinant variants from diverse species including Escherichia coli, Enterobacter sp., Erwinia tasmaniensis, Cronobacter sakazakii, and Citrobacter koseri suggests conservation of this protein across these related genera .

A comparative genomic approach similar to that used for Enterobacter cloacae complex (ECC) analysis would be appropriate to systematically assess aas conservation:

• Sequence Homology Analysis: Identify homologs across bacterial genomes using BLAST searches and calculate sequence identity percentages.

• Functional Domain Conservation: Examine conservation patterns of specific functional domains versus linker regions.

• Phylogenetic Analysis: Construct phylogenetic trees of aas proteins to trace evolutionary relationships.

• Synteny Analysis: Evaluate conservation of genomic context surrounding the aas gene to identify potentially co-evolving genes.

What insights can structural modeling provide regarding the evolution of bifunctionality in the aas protein?

Structural modeling approaches can provide valuable insights into the evolution of bifunctionality in proteins like aas, though specific structural data is not provided in the search results. Drawing from methodologies applied to other bifunctional enzymes, the following approach could be used:

• Homology Modeling: Generate structural models based on related proteins with known structures, similar to the approach used for R13 Fae of Rahnella sp. .

• Domain Interface Analysis: Examine interactions between catalytic domains to understand how they evolved to function together.

• Ancestral Sequence Reconstruction: Infer ancestral sequences and model their structures to trace the evolution of bifunctionality.

• Binding Site Comparison: Analyze conservation of binding sites across homologs, as done with R13 Fae, which showed binding sites for amylose (residues LYS355, GLU359, ILE363, LYS364, PHE365, TYR367, HIS368, and GLU369) and amylopectin (residues PHE187, GLU243, PHE244, TYR245, ASN248, ALA250, ALA251, ASP253, and PRO315) .

Quality assessment metrics for structural models should include Ramachandran plot analysis (targeting >85% residues in most favored regions) and ERRAT evaluation (quality factor >80%), as demonstrated with other bifunctional enzymes .

Such structural analysis would help determine whether Bifunctional protein aas evolved through domain fusion of initially separate proteins or through diversification of binding sites within a single ancestral protein.

Could Bifunctional protein aas serve as a potential antimicrobial target for treating Enterobacter infections?

While direct evidence for Bifunctional protein aas as an antimicrobial target is not provided in the search results, several factors suggest it could be a promising candidate for investigation:

• Essential Metabolic Function: As an enzyme involved in phospholipid metabolism, aas likely plays an important role in membrane biogenesis, which is essential for bacterial viability.

• Structure-Based Drug Design Potential: The bifunctional nature provides multiple potential binding sites for inhibitor development. Similar to approaches used with other bifunctional proteins, structural modeling and docking analysis could identify potential inhibitor binding sites .

• Context in Antimicrobial Resistance: The correlation between membrane composition, outer membrane proteins, and antibiotic resistance in Enterobacter species suggests that targeting phospholipid metabolism could potentially sensitize resistant strains to existing antibiotics.

Research approaches to explore this potential:

• Gene essentiality studies using conditional knockout systems

• High-throughput screening for selective inhibitors

• In silico docking studies against modeled structures

• Combination studies with existing antibiotics to identify potential synergies

The development of selective inhibitors would require careful consideration of homologous proteins in human cells to avoid toxicity issues.

What biotechnological applications could leverage the dual catalytic activities of Recombinant Enterobacter sp. Bifunctional protein aas?

The dual catalytic capabilities of Bifunctional protein aas offer several potential biotechnological applications, though specific applications are not directly described in the search results. Based on the enzyme's functions and examples from other bifunctional enzymes, several promising applications can be proposed:

• Biocatalysis for Phospholipid Modification: The 2-acylglycerophosphoethanolamine acyltransferase activity could be harnessed for enzymatic synthesis of modified phospholipids with applications in liposome technology, drug delivery systems, or membrane protein research.

• Biosensor Development: Similar to how other bifunctional enzymes have been applied, aas could potentially be engineered as part of biosensor systems for detecting specific lipids or metabolites.

• Metabolic Engineering: The ability to perform multiple catalytic steps within a single enzyme could improve efficiency in engineered metabolic pathways, particularly those involving lipid metabolism.

• Protein Engineering Platform: The bifunctional nature provides an interesting scaffold for protein engineering efforts aimed at creating novel catalytic functions or improving existing ones.

For example, the bifunctional KDC4427 enzyme from Enterobacter sp. demonstrates how such dual-function enzymes can mediate sequential production of different metabolites (2-PE and IAA) that influence interspecies interactions . This suggests potential applications in agricultural or environmental biotechnology where controlled production of multiple compounds could be beneficial.

What are the major technical challenges in characterizing the kinetic properties of both catalytic functions in Bifunctional protein aas?

Characterizing the kinetic properties of bifunctional enzymes like Enterobacter sp. Bifunctional protein aas presents several technical challenges:

• Interdependent Catalytic Activities: If the two catalytic functions influence each other, standard Michaelis-Menten kinetics may not adequately describe the system. This requires more complex kinetic models and experimental designs.

• Domain-Specific Activity Measurement: Developing assays that can independently measure each catalytic function without interference from the other activity can be technically challenging, especially if they share substrates or cofactors.

• Structural Dynamics During Catalysis: Capturing conformational changes that may occur during catalysis often requires specialized biophysical techniques like hydrogen-deuterium exchange mass spectrometry (HDX-MS) or single-molecule FRET.

• Membrane Association Challenges: As an enzyme involved in phospholipid metabolism, aas likely interacts with membranes, complicating in vitro assays that may not properly replicate the membrane environment.

Future methodological approaches could include:

• Development of domain-specific mutations that selectively inactivate one function while preserving the other

• Time-resolved structural studies to capture intermediate states

• Reconstitution in nanodiscs or liposomes to better mimic the native membrane environment

• Advanced kinetic modeling approaches that account for potential allosteric effects between domains

How might systems biology approaches enhance our understanding of Bifunctional protein aas in the context of Enterobacter metabolism and pathogenesis?

Systems biology approaches offer powerful frameworks to understand multifunctional proteins like Bifunctional protein aas in their broader biological context:

• Multi-omics Integration: Combining transcriptomics, proteomics, and metabolomics data could reveal how aas expression correlates with global metabolic states and virulence traits. Similar approaches have been used to study outer membrane proteins and virulence in Enterobacter isolates .

• Network Analysis: Placing aas within protein-protein interaction and metabolic networks would illuminate its connections to other cellular processes. For example, research on Enterobacter has shown correlations between outer membrane proteins like OmpA, OmpX, and virulence factors .

• Genome-Scale Metabolic Modeling: Incorporating aas functions into genome-scale metabolic models could predict the system-wide effects of altered aas activity and identify potential synthetic lethal interactions.

• Host-Pathogen Interaction Modeling: Similar to studies examining ROS generation by neutrophils in response to Enterobacter infection , systems approaches could reveal how aas activity influences host immune responses.

• Comparative Systems Biology: Comparing system-level behaviors across different Enterobacter species and strains with varying aas properties could reveal evolutionary adaptations and niche-specific functions.

These approaches would help transition from understanding aas as an isolated protein to comprehending its role in the complex network of interactions that govern bacterial physiology and pathogenesis.

What is the recommended protocol for expressing and purifying Recombinant Enterobacter sp. Bifunctional protein aas with optimal yield and activity?

While a specific optimized protocol for Recombinant Enterobacter sp. Bifunctional protein aas is not provided in the search results, a recommended protocol can be derived from successful approaches used with similar bifunctional bacterial proteins:

Expression and Purification Protocol:

1. Cloning and Vector Construction:

• Clone the aas gene (UNIPROT: A4WE11) into pET-38b(+) expression vector with a C-terminal His6-tag

• Confirm sequence integrity by DNA sequencing

2. Expression Conditions:

• Transform expression construct into E. coli BL21(DE3)

• Culture in LB medium supplemented with appropriate antibiotic at 37°C until OD600 reaches 0.6-0.8

• Induce with 0.5 mM IPTG

• Reduce temperature to 18°C and continue expression for 16-18 hours

• Harvest cells by centrifugation at 5,000 × g for 15 minutes at 4°C

3. Cell Lysis and Initial Clarification:

• Resuspend cell pellet in lysis buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 1 mM PMSF, 1 mg/mL lysozyme)

• Incubate on ice for 30 minutes

• Disrupt cells by sonication (6 cycles of 30 seconds on/30 seconds off)

• Clarify lysate by centrifugation at 15,000 × g for 30 minutes at 4°C

4. Purification Steps:

• Load clarified lysate onto Ni-Sepharose column equilibrated with binding buffer (50 mM phosphate buffer pH 7.0, 300 mM NaCl)

• Wash with binding buffer containing 20 mM imidazole

• Elute protein with linear gradient of imidazole (50-500 mM) in binding buffer

• Pool active fractions and concentrate using 30 kDa MWCO centrifugal filter

• Perform buffer exchange to storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol)

• Confirm purity by SDS-PAGE (expected MW ~80 kDa)

5. Activity Verification:

• Develop specific assay for 2-acylglycerophosphoethanolamine acyltransferase activity

• Store purified protein in aliquots at -80°C to maintain activity

This protocol incorporates elements from successful purifications of other bifunctional enzymes, such as the approach used for recombinant bifunctional alginate lyase AlyS02 , adapted to the specific characteristics of the aas protein.

How can researchers effectively analyze the structure-function relationship of Recombinant Enterobacter sp. Bifunctional protein aas?

Effective analysis of structure-function relationships for Bifunctional protein aas requires an integrated approach combining computational, biochemical, and biophysical methods:

Computational Approaches:

• Homology Modeling: Generate a structural model using related proteins with known structures as templates.

• Molecular Dynamics Simulations: Explore conformational dynamics and potential interactions between domains.

• Binding Site Prediction: Identify potential substrate binding sites and catalytic residues.

• Structural Validation: Apply evaluation methods such as Ramachandran plot analysis and ERRAT quality factor assessment, targeting >85% residues in most favored regions and quality scores >80%, respectively .

Biochemical Approaches:

• Site-Directed Mutagenesis: Create variants with mutations at predicted catalytic residues or domain interfaces.

• Domain Truncation/Fusion: Generate constructs expressing individual domains to assess their independent function.

• Activity Assays: Measure enzyme kinetics for different substrates and compare wild-type with mutant variants.

Biophysical Approaches:

Integration and Analysis:
Correlate structural features with functional properties by analyzing how specific mutations affect:

• Substrate binding affinity (Km)

• Catalytic efficiency (kcat/Km)

• Protein stability (ΔTm)

• Domain interactions and communication