Recombinant Acinetobacter sp. GTP cyclohydrolase folE2 (folE2)

What is the fundamental biochemical function of Acinetobacter sp. folE2?

Acinetobacter sp. folE2 encodes GTP cyclohydrolase IB (GCYH-IB), which catalyzes the conversion of GTP to dihydroneopterin triphosphate, the first committed step in the de novo folate biosynthesis pathway. Unlike the canonical GCYH-IA (encoded by folE), GCYH-IB is a metal-promiscuous enzyme that can function with various divalent cations beyond zinc, including manganese, iron, and other metals . This characteristic allows the enzyme to maintain folate biosynthesis under zinc-limiting conditions, providing metabolic resilience in environments where zinc availability is restricted . The reaction catalyzed involves a complex ring-opening of the guanine moiety of GTP, followed by rearrangement to form the pterin ring system characteristic of folates.

What expression systems are recommended for producing recombinant Acinetobacter sp. folE2?

For laboratory-scale production of recombinant Acinetobacter sp. folE2, Escherichia coli remains the preferred heterologous expression system due to its well-established genetics, rapid growth, and high protein yields. Based on established protocols for other bacterial GCYH-IB enzymes, the following approach is recommended:

Expression System Protocol:

• Clone the folE2 gene into a pET-series vector (pET-28a or pET-21a) with an N-terminal His6-tag for purification

• Transform into E. coli BL21(DE3) or Rosetta(DE3) cells for expression

• Grow cultures at 37°C until OD600 reaches 0.6-0.8

• Induce with 0.5-1.0 mM IPTG and shift to 18-25°C for 16-18 hours

• Harvest cells by centrifugation and lyse using sonication in buffer containing:

  • 50 mM HEPES, pH 8.0

  • 300 mM NaCl

  • 10% glycerol

  • 1 mM DTT

  • Protease inhibitor cocktail

This approach has been successful for producing biochemically active GCYH-IB enzymes from Bacillus subtilis and Neisseria gonorrhoeae , and should be adaptable for Acinetobacter sp. folE2 with minimal modifications.

How can I verify the enzymatic activity of recombinant Acinetobacter sp. folE2?

The enzymatic activity of recombinant Acinetobacter sp. folE2 can be verified using a fluorescence-based assay that detects the formation of neopterin, which is derived from the enzymatic product. The methodology is as follows:

Fluorescence-Based Activity Assay Protocol:

• Prepare reaction mixture containing:

  • 100 mM HEPES buffer (pH 8.0)

  • 100 mM KCl

  • 0.5-1.0 mM divalent metal ion (MnCl₂, ZnCl₂, FeCl₂, or MgCl₂)

  • 1 mM DTT

  • 0.1-0.5 μM purified enzyme

  • 0.1 mM GTP (substrate)

• Incubate at 37°C for 30-60 minutes

• Add 100 μL of acidic iodine solution (1% I₂/2% KI in 1 M HCl)

• Incubate at room temperature for 15 minutes in the dark

• Add 5 μL of 1 M NaOH and 100 μL of 1 M HCl

• Measure fluorescence using excitation at 365 nm and emission at 446 nm

This assay has been successfully used for characterizing GCYH-IB enzymes from other bacterial species and should work equivalently for the Acinetobacter sp. enzyme.

How does metal dependency differ between folE and folE2 enzymes?

The metal dependency profiles of folE (GCYH-IA) and folE2 (GCYH-IB) enzymes represent a fundamental biochemical distinction with significant physiological implications:

To experimentally characterize the metal dependency of Acinetobacter sp. folE2, prepare the recombinant enzyme in metal-free form by extensive dialysis against buffer containing 5-10 mM EDTA, followed by dialysis against metal-free buffer. Then conduct activity assays in the presence of various concentrations (0.01-1.0 mM) of different divalent metal ions to determine activation profiles .

What is the recommended protocol for determining metal ion preference of Acinetobacter sp. folE2?

To rigorously determine the metal ion preference of Acinetobacter sp. folE2, follow this comprehensive protocol:

Metal Preference Determination Protocol:

• Metal Removal:

  • Dialyze purified protein against 50 mM HEPES (pH 8.0), 100 mM KCl, 5 mM EDTA for 12 hours at 4°C

  • Perform two subsequent dialysis steps against metal-free buffer to remove EDTA

• Activity Screening:

  • Prepare reaction mixtures containing apo-enzyme (0.5 μM) and 0.5 mM of different divalent metals (Mn²⁺, Fe²⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Mg²⁺)

  • Measure activity using the fluorescence assay described previously

  • Calculate relative activity compared to the most active metal condition

• Metal Titration:

  • For metals showing significant activation, perform titrations using concentrations ranging from 0.001 to 2.0 mM

  • Plot activity versus metal concentration to determine EC₅₀ values

• Competition Experiments:

  • Prepare reaction mixtures with optimal concentration of the preferred metal

  • Add increasing concentrations of competing metals

  • Determine whether other metals enhance or inhibit activity

This approach will yield a comprehensive metal activation profile and provide insights into the physiological metal preference of the Acinetobacter sp. folE2 enzyme under various environmental conditions .

What are the key structural features of GCYH-IB enzymes and how can they be investigated?

GCYH-IB enzymes, including Acinetobacter sp. folE2, possess distinctive structural features that differentiate them from the canonical GCYH-IA enzymes despite catalyzing the same reaction. Key structural characteristics include:

• A bimodular architecture, consisting of:

  • An N-terminal domain with a tunneling-fold (T-fold) motif that forms the core of the active site

  • A C-terminal domain that contributes to oligomerization and substrate binding

• Homotetrameric quaternary structure (compared to the homodecameric structure of GCYH-IA)

• A distinct metal-binding site architecture that accommodates various divalent cations

To investigate these structural features in Acinetobacter sp. folE2, employ the following approaches:

Structural Characterization Workflow:

• Crystallization Screening:

  • Purify protein to >95% homogeneity and concentrate to 10-15 mg/mL

  • Screen crystallization conditions using commercial sparse matrix screens

  • Optimize promising conditions varying precipitant concentration, pH, and additives

  • Co-crystallize with substrate analogs (8-oxo-GTP) and different metal ions

• X-ray Crystallography:

  • Collect diffraction data at synchrotron radiation facility

  • Process data using XDS or MOSFLM

  • Solve structure by molecular replacement using known GCYH-IB structures (e.g., N. gonorrhoeae, PDB: 3D1T)

• Alternative Structural Approaches:

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

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to probe conformational dynamics

  • Negative-stain electron microscopy to visualize quaternary organization

These approaches will provide insights into the structural basis for the metal promiscuity and catalytic mechanism of Acinetobacter sp. folE2 .

How do I design site-directed mutagenesis experiments to probe the active site of Acinetobacter sp. folE2?

Site-directed mutagenesis is a powerful approach to investigate structure-function relationships in folE2 enzymes. To effectively design such experiments for Acinetobacter sp. folE2:

Site-Directed Mutagenesis Strategy:

• Target Residue Selection:

  • Identify putative metal-coordinating residues (typically His, Glu, Asp) based on sequence alignment with characterized GCYH-IB enzymes

  • Target conserved residues in the active site that may participate in substrate binding or catalysis

  • Consider residues that might confer metal specificity

• Mutation Design:

  • For metal-coordinating residues: Substitute with Ala to eliminate coordination or with alternative coordinating residues (His→Glu) to alter metal preference

  • For substrate-binding residues: Create conservative substitutions that maintain charge but alter geometry (e.g., Asp→Glu)

  • For catalytic residues: Replace with isosteric but catalytically inactive residues

• Experimental Evaluation:

  • Express and purify mutant proteins using the same protocol as wild-type

  • Determine steady-state kinetic parameters (kcat, KM) for each mutant with various metal cofactors

  • Analyze metal binding affinity using isothermal titration calorimetry (ITC)

  • Assess structural integrity through circular dichroism (CD) spectroscopy

Expected outcomes from these experiments include identification of residues critical for catalysis versus metal coordination, and insights into the structural basis for metal promiscuity in Acinetobacter sp. folE2 .

How is folE2 expression regulated in Acinetobacter sp. in response to zinc availability?

In bacteria possessing both folE and folE2 genes, such as Bacillus subtilis, folE2 expression is typically regulated by zinc-responsive transcription factors. For Acinetobacter sp., the following regulatory mechanism is likely operational:

Regulatory Mechanism:

• Under zinc-replete conditions, the Zur (zinc uptake regulator) protein, bound to Zn²⁺, acts as a transcriptional repressor by binding to a specific sequence (Zur box) in the promoter region of folE2

• During zinc limitation, Zur releases from DNA, allowing RNA polymerase access to the promoter and initiating folE2 transcription

• This results in increased GCYH-IB production, enabling folate biosynthesis despite zinc limitation

To experimentally investigate this regulation in Acinetobacter sp.:

Experimental Approach for Studying Regulation:

• Promoter Analysis:

  • Identify putative Zur-binding sites upstream of the folE2 gene using bioinformatics tools

  • Create transcriptional fusions between the folE2 promoter and a reporter gene (lacZ or gfp)

  • Measure reporter activity under varying zinc concentrations

• Transcriptional Analysis:

  • Grow Acinetobacter sp. in defined media with varying zinc concentrations

  • Extract RNA and perform RT-qPCR to quantify folE2 expression levels

  • Perform RNA-seq to identify co-regulated genes in the zinc depletion response

• Protein Binding Studies:

  • Express and purify the Acinetobacter sp. Zur protein

  • Perform electrophoretic mobility shift assays (EMSA) with labeled folE2 promoter fragments

  • Use DNase I footprinting to precisely map the Zur binding site

This approach will elucidate the molecular mechanisms controlling folE2 expression and provide insights into how Acinetobacter sp. adapts to zinc limitation .

What experimental design would best demonstrate the physiological role of folE2 in Acinetobacter sp. during zinc limitation?

To definitively establish the physiological role of folE2 in Acinetobacter sp. during zinc limitation, a comprehensive experimental design incorporating genetics, biochemistry, and physiological measurements is required:

Experimental Design for Physiological Role Determination:

• Genetic Manipulation:

  • Generate deletion mutants: ΔfolE, ΔfolE2, and ΔfolE/ΔfolE2 double mutant

  • Create complementation strains expressing folE or folE2 from controlled promoters

  • Construct folE2 variants with altered metal specificity through site-directed mutagenesis

• Growth Phenotype Analysis:

  • Compare growth curves of wild-type and mutant strains in media with:

    • Defined zinc concentrations (1 nM to 10 μM ZnSO₄)

    • Metal chelators (EDTA, TPEN)

    • Competing metals (excess Mn²⁺, Fe²⁺)

  • Perform growth competition assays between wild-type and ΔfolE2 strains under zinc limitation

• Metabolic Analysis:

  • Measure intracellular folate levels using LC-MS/MS

  • Quantify GTP and other nucleotide levels

  • Analyze expression of folate-dependent metabolic pathways

• Metal Homeostasis Evaluation:

  • Determine intracellular zinc and manganese concentrations using ICP-MS

  • Analyze expression of zinc transport systems in response to folE2 deletion

  • Measure metal distribution using metalloproteomics approaches

Expected outcomes include growth defects in the ΔfolE2 strain specifically under zinc limitation, rescue by folE2 complementation, and metabolic signatures of folate deficiency in the absence of folE2 during zinc starvation .

How can I design experiments to investigate potential moonlighting functions of Acinetobacter sp. folE2?

Beyond its canonical role in folate biosynthesis, emerging evidence suggests GCYH-IB enzymes may have additional functions. To investigate potential moonlighting roles of Acinetobacter sp. folE2:

Experimental Strategy for Moonlighting Function Discovery:

• Interactome Analysis:

  • Perform bacterial two-hybrid screening to identify protein interaction partners

  • Use pull-down assays with tagged folE2 followed by mass spectrometry

  • Validate interactions using biolayer interferometry or surface plasmon resonance

• Metabolic Impact Assessment:

  • Conduct untargeted metabolomics comparing wild-type and ΔfolE2 strains

  • Focus on pathways beyond folate biosynthesis showing significant alterations

  • Perform 13C-labeling studies to track metabolic flux changes

• Alternative Substrate Screening:

  • Test activity of purified folE2 with GTP analogs and other nucleotides

  • Analyze reaction products using HPLC and mass spectrometry

  • Investigate potential roles in modified nucleotide formation for tRNA modification

• Stress Response Studies:

  • Expose wild-type and ΔfolE2 strains to various stressors (oxidative, nitrosative, acid stress)

  • Measure survival rates and stress response gene expression

  • Investigate potential protein-nucleic acid interactions under stress conditions

This comprehensive approach may reveal unexpected functions of folE2, particularly in connection with the 7-deazapurine biosynthesis pathway for tRNA modification or other cellular processes beyond folate metabolism .

What approaches can be used to investigate the evolution of metal specificity in the folE2 enzyme family?

The metal promiscuity of folE2 enzymes presents a fascinating case for studying metalloenzyme evolution. To investigate evolutionary aspects of metal specificity in the folE2 family:

Evolutionary Analysis Protocol:

• Phylogenetic Analysis:

  • Collect folE2 sequences from diverse bacterial phyla, focusing on Acinetobacter and related genera

  • Construct maximum-likelihood phylogenetic trees

  • Map metal preferences (where known) onto the phylogeny

  • Identify clades with apparent shifts in metal preference

• Ancestral Sequence Reconstruction:

  • Infer ancestral folE2 sequences at key nodes in the phylogeny

  • Express and characterize reconstructed ancestral enzymes

  • Determine metal preference profiles of ancestral enzymes

• Comparative Structural Analysis:

  • Align structures of folE2 enzymes with different metal preferences

  • Identify structural elements correlating with specific metal utilization

  • Design chimeric enzymes combining domains from different folE2 variants

• Horizontal Gene Transfer Analysis:

  • Examine genomic context of folE2 genes across species

  • Compare folE2 phylogeny with species phylogeny to detect horizontal transfer events

  • Correlate gene transfer events with habitat transitions or metal availability

This approach will provide insights into how metal utilization evolved in folE2 enzymes and may reveal adaptations specific to Acinetobacter sp. habitats and lifestyle .

How can computational approaches be used to predict metal binding sites and substrate specificity in Acinetobacter sp. folE2?

Computational methods offer powerful tools for predicting functional properties of Acinetobacter sp. folE2 before experimental validation:

Computational Prediction Workflow:

• Homology Modeling:

  • Generate structural models using SWISS-MODEL or I-TASSER

  • Use known GCYH-IB structures (e.g., N. gonorrhoeae, PDB: 3D1T) as templates

  • Refine models focusing on active site geometry and metal-binding sites

• Molecular Docking:

  • Dock GTP and analogs into the active site using AutoDock Vina or GOLD

  • Evaluate binding poses and interaction energies

  • Identify key residues for substrate recognition

• Molecular Dynamics Simulations:

  • Prepare systems with different metal ions in the active site

  • Run extended (>100 ns) simulations to analyze metal coordination dynamics

  • Calculate free energy of metal binding using enhanced sampling methods

• Quantum Mechanics/Molecular Mechanics (QM/MM):

  • Investigate the reaction mechanism with different metals at the active site

  • Calculate energy barriers for the rate-limiting steps

  • Compare catalytic efficiency with different metals

• Machine Learning Approaches:

  • Train models on known metalloenzyme structures to predict metal-binding sites

  • Use sequence-based features to predict metal preference from primary structure

  • Apply graph neural networks to predict protein-ligand interactions

These computational approaches will generate testable hypotheses about metal coordination, substrate binding, and catalytic mechanism in Acinetobacter sp. folE2, guiding subsequent experimental work .

How can Acinetobacter sp. folE2 be utilized as a tool for studying bacterial adaptation to metal limitation?

Acinetobacter sp. folE2 provides an excellent model system for investigating bacterial strategies for coping with metal limitation, a critical aspect of host-pathogen interactions and environmental adaptation:

Research Applications in Metal Homeostasis:

• Metal Limitation Reporter System:

  • Develop biosensors using folE2 promoter fusions to fluorescent proteins

  • Monitor zinc limitation in real-time during infection or environmental transitions

  • Screen for compounds that disrupt bacterial metal acquisition systems

• Competitive Index Studies:

  • Create tagged wild-type and ΔfolE2 strains for competition experiments

  • Track population dynamics in environments with fluctuating metal availability

  • Identify environmental niches where folE2 provides a competitive advantage

• Host-Pathogen Interaction Models:

  • Investigate the role of folE2 in Acinetobacter pathogenesis

  • Determine whether host nutritional immunity (metal sequestration) induces folE2 expression

  • Assess whether folE2-dependent metal flexibility contributes to virulence

• Evolutionary Adaptation Experiments:

  • Subject Acinetobacter sp. to long-term metal limitation

  • Monitor genetic changes in folE2 sequence and regulation

  • Analyze shifts in metal utilization strategies

These approaches leverage folE2 as a model for understanding fundamental aspects of bacterial metal physiology with potential applications in developing new antimicrobial strategies targeting metal homeostasis .

What is the recommended protocol for purifying recombinant Acinetobacter sp. folE2 while maintaining its native metal content?

Preserving the native metal content of recombinant folE2 is critical for accurate biochemical characterization. The following protocol is designed specifically for this purpose:

Metal-Preserving Purification Protocol:

• Expression Optimization:

  • Supplement E. coli growth medium with 0.1-0.5 mM of the metal of interest (Mn²⁺, Fe²⁺, or Zn²⁺)

  • Use low IPTG concentration (0.1-0.2 mM) and lower growth temperature (18°C) to promote proper folding

• Lysis and Buffer Composition:

  • Prepare all buffers using high-purity water treated with Chelex-100 resin

  • Include 0.1 mM of the metal of interest in all buffers

  • Avoid metal chelators like EDTA unless specifically needed

  • Use the following lysis buffer:

    • 50 mM HEPES, pH 8.0

    • 300 mM NaCl

    • 10% glycerol

    • 0.1 mM of appropriate metal salt

    • 1 mM DTT

    • Protease inhibitor cocktail (EDTA-free)

• Chromatography Strategy:

  • Use immobilized metal affinity chromatography (IMAC) with Co²⁺ or Ni²⁺ resin

  • Include 0.05 mM of the metal of interest in all chromatography buffers

  • Elute with imidazole gradient (20-250 mM)

  • Follow with size exclusion chromatography using metal-supplemented buffer

• Metal Content Analysis:

  • Determine protein concentration by Bradford assay or UV absorption

  • Analyze metal content using inductively coupled plasma mass spectrometry (ICP-MS)

  • Calculate metal-to-protein stoichiometry

  • Verify enzymatic activity correlates with metal content

This protocol will yield recombinant Acinetobacter sp. folE2 with well-defined metal content for subsequent biochemical and structural studies .

How should I interpret contradictory results in metal activation studies of Acinetobacter sp. folE2?

Contradictory results in metal activation studies are common with metal-promiscuous enzymes like folE2. Consider these interpretations and troubleshooting approaches:

Contradiction Analysis Framework:

• Buffer Components Influence:

  Buffer Component	Potential Effect	Troubleshooting Approach
  Phosphate	May precipitate certain metals	Use HEPES or Tris buffers
  Tris	Can weakly coordinate metals	Compare with HEPES results
  Reducing agents	May alter metal oxidation states	Try different reducers (DTT vs. TCEP)
  pH	Affects metal solubility and coordination	Test pH range 7.0-8.5
  Salt concentration	Competes with protein-metal interactions	Vary NaCl from 50-300 mM

• Protein Preparation Factors:

  • Residual metals from expression host may influence results

  • Storage conditions may lead to oxidation or metal exchange

  • Freeze-thaw cycles can affect metal retention

  • Tag position (N- vs. C-terminal) may influence metal binding

• Experimental Design Considerations:

  • Order of addition (metal first vs. substrate first) may yield different results

  • Pre-incubation time with metal affects activation profiles

  • Assay temperature influences metal binding equilibria

  • Metal contamination in commercial reagents can be significant

• Resolution Strategies:

  • Systematically vary all buffer components individually

  • Test multiple protein preparations from independent expressions

  • Use metal chelation resin to remove trace metal contaminants

  • Perform isothermal titration calorimetry to directly measure metal binding

By systematically addressing these factors, you can resolve contradictions and obtain a more accurate understanding of the metal activation properties of Acinetobacter sp. folE2 .

What are common pitfalls in studying recombinant Acinetobacter sp. folE2 and how can they be avoided?

Research with recombinant Acinetobacter sp. folE2 presents several challenges that can compromise experimental outcomes. Here are key pitfalls and solutions:

Common Pitfalls and Solutions:

• Expression and Solubility Issues:

  • Pitfall: Inclusion body formation

  • Solution: Lower induction temperature to 18°C, reduce IPTG concentration, co-express with molecular chaperones, or use solubility-enhancing fusion tags (SUMO, MBP)

• Enzyme Stability Challenges:

  • Pitfall: Activity loss during purification or storage

  • Solution: Include glycerol (10-20%) in buffers, add reducing agents, avoid freeze-thaw cycles, and store at higher protein concentration (>1 mg/mL)

• Metal Contamination:

  • Pitfall: Trace metals in buffers confounding metal specificity studies

  • Solution: Treat all buffers with Chelex-100 resin, use high-purity reagents, include negative controls with EDTA, and verify metal content by ICP-MS

• Kinetic Assay Limitations:

  • Pitfall: Non-linear enzyme kinetics or substrate inhibition at high GTP concentrations

  • Solution: Use lower enzyme concentrations, optimize GTP concentration range, and consider potential product inhibition

• Structural Analysis Challenges:

  • Pitfall: Conformational heterogeneity hindering crystallization

  • Solution: Perform limited proteolysis to identify stable domains, screen with substrate analogs or product, and consider surface entropy reduction mutations

• Functional Redundancy:

  • Pitfall: Difficulty observing phenotypes in genetic studies due to redundant folE

  • Solution: Create double knockout strains, use zinc-limiting conditions to focus on folE2 function, and monitor expression of both enzymes

By anticipating these challenges and implementing the suggested solutions, researchers can significantly improve the quality and reliability of experiments with recombinant Acinetobacter sp. folE2 .