Recombinant Idiomarina loihiensis GTP cyclohydrolase folE2 (folE2)

What is Idiomarina loihiensis and why is its GTP cyclohydrolase FolE2 significant for research?

Idiomarina loihiensis is a γ-proteobacterium originally isolated from hydrothermal vents on the Lō'ihi Seamount, Hawaii. This extremophile demonstrates remarkable adaptability, surviving in temperatures ranging from 4°C to 46°C and salinities from 0.5% to 20% NaCl . The organism possesses a relatively compact genome of 2,839,318 bp with a GC content of 47.04%, encoding 2,640 predicted proteins .

The significance of I. loihiensis GTP cyclohydrolase FolE2 lies in its role in the zinc-independent folate biosynthesis pathway. Unlike the human GTP cyclohydrolase (GCYH-IA), bacterial GCYH-IB (encoded by folE2) represents an alternative form of the enzyme that functions under zinc-limiting conditions, making it both metabolically interesting and a potential antibiotic target .

Key characteristics of I. loihiensis:

• Gram-negative, rod-shaped cells (0.35 μm wide, 0.7-1.8 μm long)

• Motile with a single polar flagellum

• Primary carbon metabolism based on amino acid utilization rather than carbohydrates

• Major fatty acid component: iso-C15:0 (32.6%)

How does the structure and function of bacterial FolE2 (GCYH-IB) differ from the human GTP cyclohydrolase (GCYH-IA)?

GCYH-IA and GCYH-IB represent structurally distinct forms of GTP cyclohydrolase despite catalyzing similar reactions in the folate biosynthesis pathway.

Structural differences:

• GCYH-IA (human type): Unimodular, homodecameric, Zn²⁺-dependent enzyme

• GCYH-IB (bacterial type): Bimodular, homotetrameric enzyme that can be activated by various divalent cations

The active site of GCYH-IB is larger and differently shaped compared to GCYH-IA, which has significant implications for inhibitor design. Crystal structures with the 8-oxo-GTP inhibitor bound show that:

• The glycosidic bond angle is anti when bound to GCYH-IA but syn in GCYH-IB

• GCYH-IB has an expanded pocket (approximately 70 ų) near O⁶ of the guanine nucleobase that accommodates two water molecules

• GCYH-IB has another distinct pocket (approximately 100 ų) above the plane of the nucleobase

These structural differences enable the development of selective inhibitors targeting GCYH-IB without affecting human GCYH-IA, making bacterial FolE2 an attractive antibiotic target .

What are the optimal expression and purification protocols for recombinant I. loihiensis FolE2?

Expression System:
The most effective expression system for I. loihiensis FolE2 is E. coli, similar to protocols established for other GCYH-IB enzymes .

Expression Protocol:

• Clone the I. loihiensis folE2 gene into an expression vector with an inducible promoter (e.g., pBAD24)

• Transform into E. coli expression strain (BL21(DE3) or equivalent)

• Culture cells in LB medium with appropriate antibiotic selection

• Induce protein expression with the appropriate inducer when culture reaches OD₆₀₀ of 0.6-0.8

• Continue growth for 4-6 hours at 30°C or overnight at 18°C to minimize inclusion body formation

Purification Method:

• Harvest cells by centrifugation and resuspend in buffer containing protease inhibitors

• Lyse cells using sonication or mechanical disruption

• Clarify lysate by centrifugation (typically 20,000 × g for 30 minutes)

• Purify using affinity chromatography (if tagged) or combination of ion exchange and size exclusion chromatography

• Assess protein purity using SDS-PAGE

• Determine protein concentration spectrophotometrically using the calculated extinction coefficient of 25,590 M⁻¹

Recombinant GCYH-IB enzymes from related organisms have been successfully purified using these methods, yielding protein suitable for enzymatic and structural studies .

What assays are used to measure FolE2 enzymatic activity and how are they optimized?

Researchers utilize two established assays for measuring GCYH-IB activity:

1. Absorbance-based Assay:

• Measures the formation of dihydroneopterin triphosphate (H₂NTP) product by monitoring absorbance at 330 nm

• Useful for initial screening and inhibition studies

• Reaction mixture typically contains buffer (100 mM HEPES, pH 8.0), 100 mM KCl, appropriate divalent metal cofactor, 1 mM DTT, enzyme, and GTP substrate

2. Fluorescence-based Assay:

• Relies on post-reaction oxidation of H₂NTP to fluorescent neopterin

• Monitors neopterin emission at 446 nm with excitation at 365 nm

• More sensitive than the absorbance assay

• Recommended for precise kinetic parameter determination

Optimization Parameters:

• Metal cofactor: Test various divalent cations including Mn²⁺ (optimal for most GCYH-IB enzymes), Mg²⁺, and Zn²⁺ at concentrations ranging from 0.1-5 mM

• Buffer pH: Typically optimized between pH 7.5-8.5

• Temperature: Usually 37°C, but may be adjusted based on the thermal stability of the enzyme

• Substrate concentration: For kinetic studies, use GTP concentrations spanning at least 0.2× to 5× Kₘ

For I. loihiensis FolE2, steady-state kinetic analysis should be performed using substrate concentrations determined through preliminary experiments, similar to the range used for B. subtilis GCYH-IB (3-50 μM GTP) .

How does metal ion dependence affect FolE2 activity, and what are the optimal metal cofactors?

GCYH-IB enzymes, including FolE2 from I. loihiensis, are distinctly different from GCYH-IA in their metal cofactor requirements.

Metal Dependence Characteristics:

• GCYH-IA (folE) is strictly Zn²⁺-dependent

• GCYH-IB (folE2) can utilize various divalent cations and functions during zinc limitation

• For most characterized GCYH-IB enzymes, Mn²⁺ provides optimal activity

Experimental Data for GCYH-IB Metal Preferences:
Studies with B. subtilis GCYH-IB showed that the enzyme exhibited significantly higher activity with Mn²⁺ compared to Zn²⁺, with activity increasing with Mn²⁺ concentration up to approximately 0.5 mM before plateauing .

To determine optimal metal cofactors for I. loihiensis FolE2:

• Conduct enzyme assays with various metal ions (Mn²⁺, Mg²⁺, Fe²⁺, Co²⁺, Ni²⁺, Zn²⁺) at concentrations ranging from 0.1-5 mM

• Include control reactions with EDTA (5 mM) to chelate any metal ions

• Use GTP at a fixed concentration (typically 0.1 mM) to standardize substrate conditions

• Measure enzyme activity using either the absorbance or fluorescence assay methods

Given the genetic relationship between I. loihiensis and other bacteria possessing folE2, it is likely that Mn²⁺ would be the preferred cofactor, but experimental verification is essential for definitive determination.

What is the genomic context of the folE2 gene in I. loihiensis and how is its expression regulated?

The folE2 gene in I. loihiensis exists within a genomic context similar to other bacteria with zinc-responsive regulation systems.

Genomic Organization:
The complete genome sequence of I. loihiensis (2,839,318 bp) provides insights into the organization of essential metabolic genes . While specific operon structure for folE2 in I. loihiensis isn't explicitly detailed in the search results, similar Idiomarina species show that folE2 is often part of zinc-regulated gene clusters.

Expression Regulation:
FolE2 expression is typically controlled by the Zn²⁺-dependent Zur repressor, as demonstrated in B. subtilis and other bacteria . The Zur regulatory system functions as follows:

• Under zinc-replete conditions, Zur binds to specific DNA sequences (Zur boxes) upstream of target genes, repressing their transcription

• During zinc limitation, Zur dissociates from DNA, allowing expression of genes under its control

• This regulatory mechanism ensures folE2 is expressed only when needed for zinc-independent folate biosynthesis

Research in B. subtilis has identified candidate Zur-binding sites upstream of folE2 genes across multiple bacterial genomes, including several in the Gammaproteobacteria class to which I. loihiensis belongs .

Sequence analysis of the region upstream of the I. loihiensis folE2 gene would likely reveal similar Zur box motifs, consistent with the zinc-responsive regulation observed in other bacteria.

How do I design selective inhibitors targeting I. loihiensis FolE2 without affecting human GCYH-IA?

Designing selective inhibitors for bacterial GCYH-IB requires exploiting the structural differences between bacterial and human enzymes.

Strategic Approach:

• Focus on the expanded active site of GCYH-IB compared to human GCYH-IA

• Exploit the different glycosidic bond angles (syn in GCYH-IB vs. anti in GCYH-IA)

• Target the additional pockets present in GCYH-IB but absent in GCYH-IA

Design Principles Based on Structural Data:
Initial work has focused on modifying 8-oxo-GTP, which has shown promising results. Key modifications include:

• Adding ether linkages at O⁶ and O⁸ to displace water molecules from the expanded active site of GCYH-IB

• Designing substituents that project into Pocket 1 (~70 ų) directly outward from O⁶

• Adding groups extending above the nucleobase plane into Pocket 2 (~100 ų)

Example Compound Performance:
A compound designated G3, derived from these principles, demonstrated a three-fold higher potency against bacterial GCYH-IB compared to human GCYH-IA, representing a 31-fold reversal of selectivity compared to the parent compound 8-oxo-GTP .

The next generation of inhibitors should incorporate:

• Phosphate groups or suitable phosphate surrogates to enhance potency through ion pairing with arginine and lysine residues

• Larger substituents targeting the expanded pockets

• Features that maintain selectivity for GCYH-IB over GCYH-IA

What are the key differences between I. loihiensis FolE2 and structurally similar enzymes from other bacterial species?

Comparing I. loihiensis FolE2 with similar enzymes from other bacteria reveals both conservation and diversity in this enzyme family.

Comparative Analysis:
While specific information about I. loihiensis FolE2 structure is limited in the search results, we can infer likely properties based on characterized GCYH-IB enzymes:

• Sequence Conservation:

  • GCYH-IB enzymes belong to the tunneling-fold (T-fold) superfamily, despite having different quaternary structures from GCYH-IA

  • Sequence identity among GCYH-IB enzymes from different bacterial species is typically moderate (40-70%)

• Structural Organization:

  • GCYH-IB enzymes form homotetramers, unlike the homodecameric GCYH-IA

  • The active site architecture includes characteristic pockets that may vary in size and shape among species

• Metal Binding:

  • Crystal structures of GCYH-IB from various bacteria show metal coordination differences

  • In some species, Mn²⁺ is coordinated with an acetate or azide as an equatorial ligand

• Kinetic Parameters:
  Based on data from other GCYH-IB enzymes, typical parameters would include:

  • Kₘ for GTP in the low micromolar range (3-50 μM)

  • Catalytic efficiency influenced by the metal cofactor present

Researchers working with I. loihiensis FolE2 should perform comparative genomic and biochemical analyses to identify any unique features that might distinguish it from other GCYH-IB enzymes.

How can I conduct site-directed mutagenesis to identify key catalytic residues in I. loihiensis FolE2?

Site-directed mutagenesis of I. loihiensis FolE2 requires a systematic approach to identify functionally important residues.

Experimental Design:

• Target Selection:

  • Focus on conserved residues identified through multiple sequence alignment of GCYH-IB enzymes

  • Prioritize residues in proximity to the active site based on homology models or crystal structures

  • Target metal-coordinating residues, substrate-binding residues, and those implicated in catalysis

• Mutagenesis Protocol:

  • Use PCR-based site-directed mutagenesis techniques (QuikChange or similar)

  • Design mutagenic primers with appropriate melting temperatures (Tm ≥ 78°C)

  • Create both conservative and non-conservative mutations to assess the importance of specific chemical properties

• Functional Characterization:

  • Express and purify each mutant protein following the same protocol used for wild-type

  • Determine enzyme activity using the fluorescence-based assay

  • Evaluate metal binding properties for mutations affecting potential cofactor coordination sites

  • Conduct thermal stability analysis to assess structural integrity

• Data Analysis:

  • Calculate kinetic parameters (kcat, Km, kcat/Km) for each mutant

  • Compare with wild-type values to quantify the effect of each mutation

  • Correlate functional changes with structural features to develop a mechanistic model

Expected Outcomes:
Based on studies of related enzymes, mutations likely to affect function would include those involved in:

• Metal coordination (histidine, aspartate, or glutamate residues)

• Substrate binding (basic residues interacting with phosphate groups)

• Catalysis (residues positioned to facilitate ring opening or hydrolysis)

What is the relationship between I. loihiensis FolE2 and bacterial adaptation to extreme environments?

I. loihiensis was isolated from a hydrothermal vent environment, and its FolE2 enzyme likely plays a role in adaptation to this extreme habitat.

Environmental Context and Adaptation:

• Metal Availability in Hydrothermal Vents:

  • Hydrothermal vents often have variable metal compositions, including periods of zinc limitation

  • The ability to use alternative metals (particularly Mn²⁺) for essential enzymes provides a competitive advantage

  • FolE2 enables continued folate biosynthesis during zinc limitation, supporting growth in fluctuating metal conditions

• Genomic Evidence of Adaptation:

  • Comparative genomic analysis of Idiomarina species reveals adaptations to extreme environments

  • Genome reduction is common among Idiomarina species, with selective retention of genes critical for survival

  • Metal tolerance genes and transporters are prevalent, including systems for Fe, Cu, Zn, Pb, and Cd resistance

• Metabolic Specialization:

  • I. loihiensis shows metabolic specialization toward amino acid utilization rather than carbohydrates

  • This specialization is consistent with the protein-rich particulate matter available in their natural habitat

  • FolE2-dependent folate biosynthesis supports this amino acid-centric metabolism by providing essential cofactors for amino acid interconversion

• Stress Response Mechanisms:

  • The use of FolE2 may be part of a broader stress response system in I. loihiensis

  • Similar to how GTP cyclohydrolase II in Candida glabrata provides resistance to nitrosative stress

  • This suggests potential roles beyond folate biosynthesis in extreme environments

Understanding the ecological context of FolE2 function provides insights into bacterial adaptation mechanisms and potential applications in biotechnology related to metal-limited environments.

How do the biochemical properties of recombinant I. loihiensis FolE2 compare with the native enzyme?

When working with recombinant proteins, it's crucial to understand how their properties might differ from the native enzyme.

Key Comparisons:

• Enzyme Activity:

  • Recombinant FolE2 typically exhibits comparable catalytic activity to native enzyme when properly folded

  • Activity measurements using standard assays (absorbance or fluorescence-based) should yield similar kinetic parameters

  • Minor differences may occur due to expression system effects on post-translational modifications

• Metal Content:

  • Native FolE2 from I. loihiensis would contain the physiologically relevant metal cofactor(s)

  • Recombinant enzyme may contain metals present in the expression host or growth media

  • Metal analysis using X-ray emission and fluorescence scans can determine bound metals in both native and recombinant proteins

• Structural Integrity:

  • Native quaternary structure (tetramer for GCYH-IB) should be preserved in recombinant preparations

  • Size exclusion chromatography and analytical ultracentrifugation can confirm oligomeric state

  • Thermal stability profiles may differ slightly between native and recombinant proteins

• Post-translational Modifications:

  • I. loihiensis may employ post-translational modifications absent in E. coli expression systems

  • Mass spectrometry analysis can identify any modifications present in the native enzyme

  • Functional consequences of missing modifications should be evaluated when interpreting recombinant enzyme data

When studying recombinant I. loihiensis FolE2, researchers should include appropriate controls to validate that the recombinant enzyme faithfully represents the native protein's properties.

How can complementation assays be used to verify the function of I. loihiensis FolE2?

Complementation assays provide powerful tools to confirm gene function by testing whether a gene can restore function in a mutant strain.

Complementation Strategy for FolE2:

• Bacterial Strain Selection:

  • Use E. coli folE::KanR strain (lacking GCYH-IA) for complementation

  • Alternative: B. subtilis ΔfolE mutant strain

• Experimental Design:

  • Clone I. loihiensis folE2 into an appropriate expression vector (e.g., pBAD24)

  • Transform the construct into the folE-deficient strain

  • Include positive controls (known folE or folE2) and negative controls (empty vector)

  • Assess growth under conditions requiring folate biosynthesis

• Growth Conditions:

  • Rich medium (LB) with and without thymidine supplementation

  • Minimal medium lacking folate pathway end products

  • Media with metal-limiting conditions (using chelators like EDTA)

• Expected Results:

  • Successful complementation: restoration of growth in folate-dependent conditions

  • Differential complementation: growth only under specific metal conditions

  • Failed complementation: no growth restoration, suggesting functional differences

Previous Complementation Examples:
In similar experiments with other GCYH-IB enzymes:

• B. subtilis ΔfolE strain expressing only GCYH-IB showed thymidine auxotrophy

• In the absence of thymidine, this strain grew with a lag in rich medium

• Growth commenced in stationary phase, reflecting derepression of GCYH-IB under zinc starvation

• Deleting the zur gene eliminated the growth lag due to constitutive GCYH-IB expression

These patterns confirm the functional role of folE2 in zinc-independent folate biosynthesis and provide a framework for testing I. loihiensis FolE2.

What techniques can be used to determine the crystal structure of I. loihiensis FolE2?

Determining the crystal structure of I. loihiensis FolE2 requires a systematic approach to crystallization and structure determination.

Crystal Structure Determination Workflow:

• Protein Preparation:

  • Express and purify recombinant I. loihiensis FolE2 to >95% homogeneity

  • Verify protein quality using SDS-PAGE, mass spectrometry, and dynamic light scattering

  • Concentrate protein to 5-15 mg/mL in a stabilizing buffer

  • Consider addition of substrates, product analogs, or inhibitors for co-crystallization

• Crystallization Screening:

  • Perform initial screening using commercial sparse matrix screens

  • Based on previous GCYH-IB crystallization conditions, focus on:

    • pH range 6.5-8.5

    • PEG-based precipitants

    • Various salts including acetate-containing conditions

  • Optimize promising conditions by varying protein concentration, precipitant concentration, pH, and temperature

• Crystal Optimization:

  • Refine crystallization conditions to produce large, single crystals

  • Consider additives like divalent metal ions (Mn²⁺, Mg²⁺, Zn²⁺)

  • For specific structural questions, co-crystallize with ligands such as GTP or inhibitors

• Data Collection and Processing:

  • Collect X-ray diffraction data at a synchrotron facility

  • Process diffraction data using standard crystallographic software

  • For metal identification, perform X-ray emission and fluorescence scans at appropriate wavelengths

• Structure Determination:

  • Use molecular replacement with known GCYH-IB structures as search models

  • Alternatively, if molecular replacement fails, consider experimental phasing methods

  • Refine the structure iteratively, focusing on the active site and metal coordination

Expected Structural Features:
Based on other GCYH-IB structures, I. loihiensis FolE2 likely exhibits:

• Homotetramer quaternary structure

• T-fold structural motif for each monomer

• Metal binding site with characteristic coordination geometry

• Substrate binding pocket with specific recognition elements for GTP

How does the genomic and proteomic analysis of I. loihiensis inform our understanding of FolE2 evolution?

Genomic and proteomic analyses provide crucial insights into the evolutionary history and functional context of I. loihiensis FolE2.

Evolutionary Insights:

• Phylogenetic Distribution:
  Bacteria possessing FolE2 (GCYH-IB) fall into several distinct groups:

  • Those with only folE (encoding GCYH-IA)

  • Those with only folE2 (encoding GCYH-IB)

  • Those possessing both folE and folE2 (like B. subtilis)

  • Those with multiple copies of both genes

  The presence of folE2 in I. loihiensis likely represents an adaptation to its environment where zinc availability fluctuates.

• Gene Conservation:
  Comparative genomics of Idiomarina species reveals:

  • Core genes related to salinity tolerance and stress response

  • Conservation of metal resistance genes across species

  • Streamlined genomes suggesting selective pressure on retention of essential genes

• Genomic Context:

  • The folE2 gene in related bacteria often occurs in proximity to other zinc-regulated genes

  • Its genomic neighborhood may include genes involved in metal homeostasis

  • Operonic structure can provide clues about co-regulation and functional relationships

• Horizontal Gene Transfer:

  • Some Idiomarina species contain genomic islands suggesting horizontal gene transfer events

  • These events could contribute to the acquisition of metal-independent enzyme variants

  • Analysis of codon bias and GC content can identify potentially transferred genes

Functional Evolution:
The distribution of folE/folE2 genes suggests an evolutionary strategy where bacteria maintain redundant pathways for essential functions like folate biosynthesis, allowing survival in variable environments. The retention of folE2 in I. loihiensis, a bacterium from metal-rich hydrothermal vents, underscores the importance of metal-independent metabolic pathways even in metal-replete environments .

How can I develop an assay to screen for inhibitors of I. loihiensis FolE2 for antimicrobial drug discovery?

Developing a high-throughput screening assay for I. loihiensis FolE2 inhibitors requires a robust and sensitive detection method suitable for large compound libraries.

Assay Development Strategy:

• Primary Screening Assay Design:

  • Adapt the fluorescence-based neopterin detection assay for microplate format

  • Reaction mixture: 100 mM HEPES (pH 8.0), 100 mM KCl, 0.5 mM MnCl₂, 1 mM DTT, purified FolE2 (0.5 μM), and GTP substrate (9-10 μM)

  • Post-reaction oxidation: Convert H₂NTP to fluorescent neopterin

  • Detection: Measure fluorescence at 446 nm (emission) with excitation at 365 nm

• Assay Optimization Parameters:

  • Z'-factor determination to ensure statistical reliability (aim for Z' > 0.5)

  • Signal-to-background ratio optimization

  • DMSO tolerance assessment (typically up to 2-5% final concentration)

  • Miniaturization to 384-well format if needed for higher throughput

• Screening Implementation:

  • Positive controls: Known inhibitors like 8-oxo-GTP derivatives

  • Negative controls: Reaction without inhibitor (DMSO only)

  • Data normalization: Percent inhibition relative to controls

  • Hit criteria: Typically >50% inhibition at screening concentration

• Secondary Assays:

  • Dose-response determination for IC₅₀ values

  • Counter-screen against human GCYH-IA to assess selectivity

  • Alternative assay format (absorbance-based) to eliminate false positives

  • Cell-based assays in bacterial systems dependent on folE2

Selectivity Considerations:
To ensure potential antibiotics target bacterial GCYH-IB without affecting human GCYH-IA:

• Include parallel screening against human GCYH-IA

• Calculate selectivity index (ratio of IC₅₀ values)

• Prioritize compounds with at least 10-fold selectivity for further development

Data Analysis and Validation:
For promising compounds, determine complete inhibition profiles including:

• IC₅₀ values against multiple bacterial GCYH-IB enzymes

• Mechanism of inhibition (competitive, non-competitive, uncompetitive)

• Correlation between enzyme inhibition and antimicrobial activity

• Structure-activity relationships to guide optimization