Recombinant Acinetobacter sp. Non-canonical purine NTP pyrophosphatase (ACIAD0526)

What is ACIAD0526 and what is its primary function?

ACIAD0526 is a non-canonical purine NTP pyrophosphatase from Acinetobacter species that belongs to the Maf/ham1-like pyrophosphatase family. Its primary function is to hydrolyze non-canonical nucleoside triphosphates, preventing their incorporation into nucleic acids during replication and transcription. This enzyme plays a crucial role in maintaining nucleotide pool quality by removing potentially mutagenic modified nucleotides .

The enzyme catalyzes the following reaction:
Non-canonical nucleoside triphosphate + H₂O → Non-canonical nucleoside monophosphate + Pyrophosphate

This activity is similar to that observed in other pyrophosphatases, such as the inorganic pyrophosphatase from E. coli, which hydrolyzes inorganic pyrophosphate in the cell .

How does ACIAD0526 differ from canonical pyrophosphatases?

ACIAD0526 differs from canonical pyrophosphatases in several key aspects:

Unlike inorganic pyrophosphatases that primarily serve housekeeping functions in energy metabolism, ACIAD0526 has a specialized role in preventing incorporation of damaged or modified nucleotides into genomic material .

What are the expression patterns of ACIAD0526 in Acinetobacter species?

The expression of ACIAD0526 in Acinetobacter species appears to be regulated in response to nucleotide stress and DNA damage. While specific expression data for ACIAD0526 is limited in the provided search results, related research on Acinetobacter baumannii gene regulation suggests that:

• Expression may be upregulated during oxidative stress conditions, which increase the levels of damaged nucleotides

• The gene may be co-regulated with other DNA repair and maintenance systems

• Expression patterns may vary between different growth phases and environmental conditions

Similar to other bacterial pyrophosphatases, ACIAD0526 expression is likely maintained at basal levels under normal conditions, with increased expression during stress responses that generate non-canonical nucleotides .

What are the optimal expression systems for producing recombinant ACIAD0526?

Based on successful strategies for similar recombinant proteins, the following expression systems are recommended for ACIAD0526:

The choice of vector system should include a fusion tag (His₆, GST, or MBP) to facilitate purification. Based on experience with similar pyrophosphatases, expression in E. coli BL21(DE3) using a pET-based vector with an N-terminal His₆-tag has shown good results for recombinant production of pyrophosphatases in the milligram scale .

What purification strategy yields the highest purity and activity of ACIAD0526?

A multi-step purification approach is recommended to obtain highly pure and active ACIAD0526:

• Initial capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin for His-tagged protein

  • Buffer: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole

  • Elution with imidazole gradient (50-300 mM)

• Intermediate purification: Ion exchange chromatography

  • Buffer: 20 mM Tris-HCl pH 8.0, 50 mM NaCl

  • Linear gradient elution with increasing NaCl (50-500 mM)

• Polishing step: Size exclusion chromatography

  • Buffer: 50 mM Tris-HCl pH 8.0, 150 mM NaCl

  • Separates monomeric from potential aggregated forms

• Optional tag removal: If needed, remove fusion tags using appropriate proteases (TEV or thrombin)

This approach has been successfully applied to similar enzymes such as inorganic pyrophosphatase from E. coli, resulting in homogeneous enzyme preparations suitable for crystallographic studies .

How can I assess the proper folding and activity of recombinant ACIAD0526?

Multiple complementary techniques should be employed to evaluate proper folding and activity:

For activity assays, a coupled enzymatic assay can be used where the release of pyrophosphate is coupled to the oxidation of NADH, which can be monitored spectrophotometrically. Alternatively, direct detection of non-canonical nucleoside monophosphate products using HPLC or mass spectrometry provides a more definitive assessment of substrate specificity .

What are the known substrates of ACIAD0526 and how can substrate specificity be determined?

As a non-canonical purine NTP pyrophosphatase, ACIAD0526 likely hydrolyzes a range of modified nucleotides. Based on related Maf/ham1-like pyrophosphatases, potential substrates include:

To determine substrate specificity:

• Enzyme kinetics approach:

  • Measure initial reaction rates with various substrates

  • Determine Km and kcat values for each substrate

  • Calculate specificity constants (kcat/Km) to rank preferences

• Competition assays:

  • Use a mixture of potential substrates

  • Analyze reaction products by LC-MS to determine preferential hydrolysis

• Structural biology approach:

  • Obtain crystal structures with bound substrate analogs

  • Identify key interactions in the active site

Similar approaches have been used successfully for studying pyrophosphatases from E. coli and other organisms .

How does ACIAD0526 interact with viral RNA-dependent RNA polymerases?

Recent research suggests that Maf/ham1-like pyrophosphatases, including potential homologs of ACIAD0526, can interact with viral RNA-dependent RNA polymerases (RdRps). These interactions appear to be host-specific and may play important roles in viral replication .

Key aspects of these interactions include:

• Functional roles:

  • The pyrophosphatase may prevent incorporation of non-canonical nucleotides into viral RNA

  • The interaction may enhance fidelity of viral RNA synthesis

  • Some viral RdRps may recruit host pyrophosphatases to sanitize nucleotide pools

• Experimental approaches to study interactions:

  • Co-immunoprecipitation to verify physical interaction

  • Pull-down assays with subsequent mass spectrometry (similar to methods used for AamA in A. baumannii)

  • Blue native gel electrophoresis to detect stable complexes

  • Bimolecular fluorescence complementation for in vivo interaction studies

• Interaction dynamics:

  • Some interactions may be transient rather than forming stable complexes

  • Interactions might be regulated by cellular conditions or viral infection stage

Understanding these interactions could provide insights into host-virus relationships and potentially identify new targets for antiviral interventions .

What structural features contribute to ACIAD0526 substrate recognition?

While specific structural data for ACIAD0526 is not provided in the search results, insights can be extrapolated from related pyrophosphatases. The following structural features likely contribute to substrate recognition:

• Active site architecture:

  • Conserved catalytic residues for metal coordination (typically Mg²⁺)

  • Positively charged binding pocket for triphosphate moiety

  • Specificity-determining residues that interact with the non-canonical base

• Key domains:

  • Nucleotide binding pocket with conserved motifs

  • Substrate discrimination loop/region

  • Potential oligomerization interfaces that may influence activity

• Structural elements determining specificity:

Small-angle X-ray scattering (SAXS) could be applied to study solution structures of ACIAD0526 alone and in combination with potential interaction partners, similar to approaches used for AamA studies .

How can ACIAD0526 be engineered for enhanced activity or altered specificity?

Engineering ACIAD0526 for modified properties requires strategic approaches based on structure-function relationships:

Specific strategies might include:

• Altering substrate specificity:

  • Mutate residues in the base recognition pocket

  • Target the design approach based on principles similar to those used for engineering PylRS variants for different substrates

  • Create a library of variants with systematically altered binding pocket residues

• Enhancing catalytic activity:

  • Optimize metal coordination sites

  • Modify flexible loops that might limit substrate access

  • Engineer improved protein stability to maintain activity under challenging conditions

• Developing application-specific variants:

  • Create variants with improved activity against specific harmful non-canonical nucleotides

  • Engineer constructs with additional domains for specific cellular targeting

The methodological approach can be inspired by the engineering of Pyrrolysyl-tRNA Synthetase variants described in search result , adapting similar principles to ACIAD0526.

What role does ACIAD0526 play in Acinetobacter pathogenicity and antibiotic resistance?

While direct evidence linking ACIAD0526 to pathogenicity is limited in the search results, potential roles can be inferred based on understanding of nucleotide metabolism and bacterial stress responses:

• Stress response and adaptation:

  • ACIAD0526 may help Acinetobacter species survive oxidative stress during host infection by preventing mutagenic nucleotide incorporation

  • This function could contribute to the bacterium's ability to persist in hospital environments

• Genomic integrity maintenance:

  • By sanitizing nucleotide pools, ACIAD0526 might contribute to genomic stability

  • This could affect mutation rates and evolution of antibiotic resistance

• Potential interaction with host immune responses:

  • Non-canonical nucleotides can trigger immune sensing pathways

  • ACIAD0526 might help evade such detection by removing immunostimulatory nucleotides

• Research approaches to investigate these roles:

  • Generate knockout strains and assess virulence in infection models

  • Compare expression levels between antibiotic-resistant and susceptible strains

  • Analyze nucleotide pools in wild-type vs. ACIAD0526-deficient strains under antibiotic stress

These investigations would contribute to understanding how nucleotide metabolism enzymes like ACIAD0526 may influence the challenging nosocomial pathogen behavior observed with Acinetobacter baumannii .

How can ACIAD0526 be utilized in developing novel antimicrobial strategies?

Given the role of pyrophosphatases in maintaining nucleotide pool quality, ACIAD0526 presents several opportunities for antimicrobial development:

• Direct targeting of ACIAD0526:

  • Develop small molecule inhibitors that specifically target the enzyme

  • Structure-based drug design utilizing crystal structures (similar to approaches used for E. coli inorganic pyrophosphatase)

  • High-throughput screening of compound libraries against purified enzyme

• Nucleotide analog approach:

  • Design nucleotide analogs that are resistant to ACIAD0526 hydrolysis

  • These analogs could accumulate and interfere with bacterial replication

  • Requires detailed understanding of substrate recognition mechanisms

• Immunological targeting:

  • Develop ACIAD0526-based vaccine components

  • Similar to approaches using recombinant PKF protein from A. baumannii

  • Test different adjuvants and immunization routes for optimal protection

For vaccine applications, intramuscular immunization with AS03 adjuvant or intranasal immunization with LTK63 could be explored, as these approaches have shown promise with other recombinant proteins from Acinetobacter species .

How can I resolve common challenges in recombinant expression of ACIAD0526?

Recombinant expression of pyrophosphatases can encounter several challenges. Here are solutions to common issues:

Additionally:

• If expression yields are consistently low, consider cell-free expression systems

• For proteins forming inclusion bodies, refolding protocols can be optimized using gradual dialysis

• If activity is lower than expected, ensure proper metal cofactors (typically Mg²⁺) are present in activity assays

• For aggregation issues, perform buffer screening using thermal shift assays to identify stabilizing conditions

These approaches have been successfully applied to the recombinant production of similar proteins including inorganic pyrophosphatases from E. coli .

What are the critical parameters for designing kinetic assays for ACIAD0526?

Designing robust kinetic assays for ACIAD0526 requires careful consideration of several parameters:

• Assay conditions optimization:

• Detection method selection:

  • Malachite green assay for released phosphate (sensitive but end-point)

  • Coupled enzymatic assay for real-time monitoring

  • Direct HPLC analysis of substrate consumption and product formation

  • Mass spectrometry for detailed product characterization

• Substrate considerations:

  • Pre-test substrate stability under assay conditions

  • Ensure sufficient substrate purity (>95%)

  • Use appropriate substrate concentration range (typically 0.1-10× Km)

  • Include control reactions without enzyme

• Data analysis guidelines:

  • Apply appropriate enzyme kinetic models (Michaelis-Menten, substrate inhibition)

  • Use initial velocity conditions (<10% substrate conversion)

  • Perform reactions in at least triplicate

  • Include proper statistical analysis

Following these parameters will help ensure reliable and reproducible kinetic characterization of ACIAD0526, similar to approaches used for other pyrophosphatases .

How can structural studies of ACIAD0526 be optimized for success?

Structural studies of pyrophosphatases require careful preparation and optimization:

• Protein preparation for structural studies:

  • Ultra-high purity (>95% by SDS-PAGE)

  • Verify monodispersity by dynamic light scattering

  • Concentrate to 5-15 mg/ml depending on technique

  • Ensure long-term stability at 4°C

• X-ray crystallography optimization:

• Alternative structural approaches:

  • Small-angle X-ray scattering (SAXS) for solution structure

  • Cryo-electron microscopy for larger complexes with interaction partners

  • NMR for dynamic studies of smaller domains

• Analysis of protein-ligand interactions:

  • Co-crystallization with substrate analogs or product molecules

  • Isothermal titration calorimetry for binding thermodynamics

  • Surface plasmon resonance for interaction kinetics

These approaches have been successfully applied to inorganic pyrophosphatase from E. coli (crystal structure determined to 2.5 Å resolution) and could be adapted for ACIAD0526 structural studies.

What are the future research directions for ACIAD0526?

Future research on ACIAD0526 should focus on several promising directions:

• Comprehensive structural and functional characterization:

  • Determination of high-resolution crystal structures

  • Detailed substrate specificity profiling

  • Investigation of catalytic mechanism and role of metal cofactors

• Biological significance in Acinetobacter:

  • Gene knockout studies to determine physiological role

  • Transcriptomic analysis under various stress conditions

  • Investigation of potential roles in pathogenicity and antibiotic resistance

• Interaction studies:

  • Identification of protein interaction partners in Acinetobacter

  • Exploration of interactions with viral components

  • Characterization of potential regulatory mechanisms

• Translational applications:

  • Development of inhibitors as potential antimicrobials

  • Exploration as a potential vaccine component

  • Use in biotechnological applications for nucleotide pool sanitization

These research directions will contribute to a deeper understanding of ACIAD0526 and may lead to novel applications in both fundamental research and applied biotechnology .

How can researchers integrate ACIAD0526 studies with broader research on nucleotide metabolism?

ACIAD0526 research can be integrated with broader nucleotide metabolism studies through:

• Systems biology approaches:

  • Metabolomics to analyze changes in nucleotide pools

  • Integration with transcriptomic and proteomic data

  • Computational modeling of nucleotide metabolism networks

• Comparative studies across species:

  • Evolutionary analysis of Maf/ham1-like pyrophosphatases

  • Functional comparison with homologs from other bacteria and eukaryotes

  • Investigation of species-specific adaptations

• Connection to stress responses:

  • Study of ACIAD0526 regulation under various stress conditions

  • Integration with DNA damage response pathways

  • Analysis of cross-talk between nucleotide metabolism and other cellular processes

• Methodological integration:

  • Development of unified protocols for studying pyrophosphatases

  • Creation of assay systems applicable across different organisms

  • Standardization of data reporting for comparative analyses