Recombinant Acinetobacter sp. Orotate phosphoribosyltransferase (pyrE)

What is Orotate phosphoribosyltransferase (pyrE) and what is its function in Acinetobacter species?

Orotate phosphoribosyltransferase (pyrE), also known as OPRT or OPRTase (EC 2.4.2.10), is an essential enzyme in the de novo pyrimidine biosynthesis pathway. In Acinetobacter species, pyrE catalyzes the conversion of orotate to orotidine-5'-monophosphate (OMP) using phosphoribosyl pyrophosphate (PRPP) as a co-substrate. This reaction is a critical step in nucleotide biosynthesis, making pyrE essential for cellular growth and replication in these organisms. The enzyme plays a vital role in the metabolic pathways that support bacterial survival and potentially contributes to virulence and antimicrobial resistance mechanisms .

What are the optimal storage conditions for recombinant pyrE preparations?

The stability and activity of recombinant pyrE are highly dependent on proper storage conditions. Based on empirical data from commercial preparations, the following storage protocols are recommended:

• Lyophilized form: Store at -20°C/-80°C for up to 12 months

• Liquid form: Store at -20°C/-80°C for up to 6 months

• Working aliquots: Store at 4°C for up to one week

For long-term storage, it is advisable to add glycerol (5-50% final concentration, with 50% being optimal) to prevent freeze-thaw damage to the protein structure. Repeated freeze-thaw cycles should be strictly avoided as they significantly reduce enzyme activity through protein denaturation and aggregation .

What is the recommended reconstitution protocol for lyophilized recombinant pyrE?

For optimal reconstitution of lyophilized recombinant pyrE:

• Briefly centrifuge the vial containing lyophilized protein to bring all content to the bottom

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (optimally 50%)

• Prepare small working aliquots to minimize freeze-thaw cycles

• Store reconstituted aliquots at -20°C/-80°C for long-term storage

This protocol maintains protein stability and enzymatic activity while minimizing degradation. The addition of glycerol is particularly important as it prevents ice crystal formation that can damage protein structure during freezing .

How can recombinant pyrE be used in antimicrobial resistance studies?

Recombinant pyrE can serve as a valuable tool in antimicrobial resistance studies through several experimental approaches:

• Metabolic pathway analysis: As a critical enzyme in nucleotide biosynthesis, pyrE functionality can be studied in relation to growth rates of resistant vs. susceptible strains to understand metabolic adaptations.

• Target validation studies: Because pyrE is essential for Acinetobacter survival, it can be evaluated as a potential drug target. Researchers can design inhibitors specific to pyrE and assess their efficacy against resistant strains.

• Evolutionary studies: Comparing pyrE sequences from susceptible and resistant isolates can reveal evolutionary adaptations. Studies on Acinetobacter baumannii have shown a concerning progression from multidrug resistance (MDR) to extensive drug resistance (XDR) and ultimately to pandrug resistance, particularly evident in isolates from 2019-2020 .

• Metabolic reprogramming investigation: The relationship between pyrimidine metabolism (via pyrE) and antimicrobial resistance mechanisms can be investigated using recombinant pyrE in enzymatic assays to compare activity levels between resistant and susceptible strains.

These approaches are particularly relevant given that A. baumannii is on the WHO list of priority pathogens requiring urgent development of new antimicrobial agents due to its high resistance to current antibiotics .

What expression systems are most effective for producing recombinant pyrE from Acinetobacter species?

Based on commercial production data and research protocols, E. coli expression systems have proven most effective for recombinant pyrE production from Acinetobacter species. The following factors contribute to successful expression:

• Codon optimization: Adapting the Acinetobacter pyrE sequence for optimal codon usage in E. coli

• Affinity tags: Including purification tags that can be determined during manufacturing

• Expression vector selection: Vectors with strong promoters for high yield production

• Induction conditions: Optimized temperature, IPTG concentration, and induction time

Both commercial products (CSB-EP738833AWW and CSB-EP481807AWN-B) utilize E. coli expression systems, achieving >85% purity as verified by SDS-PAGE, demonstrating the effectiveness of this approach for both Acinetobacter sp. and Acinetobacter baumannii pyrE production .

How does pyrE contribute to Acinetobacter pathogenicity and survival in clinical settings?

The pyrE enzyme contributes to Acinetobacter pathogenicity through multiple mechanisms that support bacterial adaptation and survival in clinical environments:

• Essential metabolic function: As a key enzyme in pyrimidine biosynthesis, pyrE is crucial for DNA and RNA synthesis, supporting the rapid growth and adaptation of Acinetobacter species in hospital settings.

• Stress response: Under antibiotic pressure, nucleotide metabolism pathways involving pyrE may be modulated to support cellular stress responses, potentially contributing to antimicrobial resistance development.

• Biofilm formation: Nucleotide metabolism plays a role in biofilm development, which is a significant virulence factor in Acinetobacter infections. pyrE's role in providing nucleotide building blocks may indirectly support biofilm establishment.

• Persistence in clinical environments: Studies have shown that A. baumannii isolates have developed increasing resistance over time, from MDR to XDR and ultimately to pandrug resistance. This evolution suggests metabolic adaptations in which pyrE function may be preserved despite other cellular changes .

Clinical data from 2013-2020 demonstrates this concerning progression, with pandrug-resistant isolates appearing only in the 2019-2020 period, highlighting the remarkable adaptability of these pathogens in which fundamental metabolic enzymes like pyrE continue to function despite significant antimicrobial pressure .

What research approaches can be used to explore pyrE as a potential therapeutic target?

Given the essential nature of pyrE in bacterial metabolism, several research approaches can be employed to evaluate its potential as a therapeutic target:

• Structure-based drug design: Using the known sequence and predicted structure of Acinetobacter pyrE (as provided in product specifications), researchers can design small molecule inhibitors that specifically target this enzyme. Comparative analysis of the sequences from different strains (e.g., CSB-EP738833AWW and CSB-EP481807AWN-B) can inform the design of broad-spectrum inhibitors .

• High-throughput screening: Establishing enzyme activity assays using recombinant pyrE to screen compound libraries for potential inhibitors.

• Genetic knockdown/knockout studies: Using RNA interference or CRISPR-Cas9 techniques to modulate pyrE expression and assess the impact on bacterial viability and virulence.

• Resistance bypass strategies: Investigating synthetic lethal interactions with pyrE to identify combination therapies that might overcome resistance mechanisms.

• Cross-species comparative analysis: Similar to the approach used with BamA protein (another potential target), researchers could explore the immunogenic potential and cross-reactivity of pyrE-based therapeutics. The BamA study demonstrated that immunization provided 40% increased survival in challenged mice, suggesting immunological approaches may be valuable for pyrE as well .

When developing such approaches, considerations of specificity are crucial. As seen with BamA research, cross-reactivity with other bacteria (including potential impacts on the human microbiome) must be carefully evaluated .

What are the regulatory and safety considerations for working with recombinant pyrE from Acinetobacter species?

Research involving recombinant pyrE from Acinetobacter species must comply with institutional biosafety guidelines and national regulations governing recombinant DNA work. Key considerations include:

• NIH Guidelines compliance: Institutions receiving NIH funding must follow the NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules (April 2019 or latest edition). This applies to all researchers regardless of funding source .

• Risk assessment and classification: Principal Investigators must determine if they are conducting recombinant or synthetic nucleic acid (r/sNA) research and correctly classify their experiments according to NIH Guidelines .

• Biosafety measures: Appropriate combination of work practices, protective equipment (PPE), and engineering controls must be implemented. For Acinetobacter work, this is particularly important given the pathogen's clinical significance and resistance profile .

• Institutional Biosafety Committee (IBC) approval: Both exempt and non-exempt r/sNA research must be registered and authorized by the IBC before research begins .

• Handling of MDR/XDR strains: Special considerations apply when working with multidrug-resistant or extensively drug-resistant Acinetobacter isolates, which have shown increasing prevalence in clinical settings (93-99% resistance to carbapenems, fluoroquinolones, and aztreonam) .

The trend toward pandrug resistance in recent isolates (2019-2020) emphasizes the need for stringent biosafety protocols when handling Acinetobacter samples or recombinant proteins derived from these organisms .

What are the most reliable assays for measuring pyrE enzymatic activity in experimental settings?

For accurate assessment of recombinant pyrE activity, the following methodological approaches are recommended:

• Spectrophotometric assays: Measure the conversion of orotate to OMP by monitoring absorbance changes at 295-300 nm.

• Coupled enzyme assays: Link pyrE activity to subsequent enzymatic reactions that produce easily detectable products.

• Radiometric assays: Use radioactively labeled substrates (14C-orotate) to measure product formation with high sensitivity.

• HPLC-based methods: Separate and quantify substrate and product to determine reaction kinetics and inhibition parameters.

For all assay types, the following considerations are critical:

• Buffer optimization: Activity is optimal at pH 7.0-7.5 with Mg2+ as a cofactor

• Temperature control: Standard assays are conducted at 37°C to mimic physiological conditions

• Substrate concentration: Saturating concentrations of both orotate and PRPP must be established

• Purity verification: Protein preparations should be >85% pure as verified by SDS-PAGE

When evaluating pyrE from clinical isolates, particularly those with antimicrobial resistance, comparative enzymatic profiling against reference strains can provide insights into potential metabolic adaptations associated with resistance phenotypes.

How might pyrE research contribute to addressing the growing Acinetobacter antimicrobial resistance crisis?

The global health challenge posed by antimicrobial-resistant Acinetobacter species necessitates novel research approaches, with pyrE offering several promising avenues:

The research on phenotypic profile changes in A. baumannii from 2013-2020 demonstrates a clear progression from MDR to XDR to pandrug resistance, highlighting the urgent need for alternative approaches like pyrE-targeted interventions .

What methodological advances are needed to enhance pyrE research outcomes?

To advance pyrE research and its applications in addressing Acinetobacter infections, several methodological improvements are needed:

• Structural biology approaches: High-resolution crystal structures of Acinetobacter pyrE (currently unavailable) would facilitate structure-based drug design and comparative analysis with pyrE from other species.

• In vivo models: Development of improved animal models that better recapitulate human Acinetobacter infections would enhance the translational potential of pyrE research.

• Single-cell techniques: Methods to study pyrE activity at the single-cell level would help understand heterogeneity in bacterial populations, particularly regarding metabolic states associated with persistence.

• Systems biology integration: Incorporating pyrE research into broader metabolic network models would provide context for understanding the system-wide impacts of targeting this enzyme.

• Rapid screening technologies: Development of high-throughput methods to assess pyrE enzymatic activity and inhibition would accelerate drug discovery efforts.

• Clinical isolate repositories: Establishing comprehensive collections of temporally and geographically diverse Acinetobacter clinical isolates would support comparative studies of pyrE sequence, structure, and function across the resistance spectrum.

With current treatment options becoming increasingly limited (colistin, tigecycline, and minocycline remain the most effective with resistance rates of only 18-23%), advancing these methodological approaches for pyrE research represents a promising direction for combating the growing threat of antimicrobial-resistant Acinetobacter infections .