Recombinant Bartonella henselae 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (ispH)

What is Bartonella henselae and what is its clinical significance?

Bartonella henselae is a facultative intracellular bacterium primarily known as the causative agent of cat-scratch disease in humans. Beyond this well-established condition, B. henselae is increasingly associated with several other clinical syndromes, particularly ocular infections and endocarditis . Cats serve as the main reservoir for B. henselae, with the bacteria typically transmitted to cats via cat fleas (Ctenocephalides felis) .

Recent research has identified potential new vectors for B. henselae transmission, notably Ixodes ricinus ticks, which are the most prevalent tick species that bite humans in Western Europe . Experimental studies have demonstrated that I. ricinus can transmit B. henselae through saliva after feeding on infected blood, with the bacteria persisting across developmental stages from larva to nymph to adult . This finding has significant epidemiological implications as it may explain previously unattributed cases of bartonellosis in humans with no history of cat scratches or flea exposure.

What is the role of 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (ispH) in bacterial metabolism?

4-hydroxy-3-methylbut-2-enyl diphosphate reductase (ispH, also known as HDR or LytB) plays a critical role as the terminal enzyme in the methylerythritol phosphate (MEP) pathway for isoprenoid biosynthesis . This enzyme catalyzes the conversion of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate (HMBPP) into the essential isoprenoid precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) .

In photosynthetic organisms, ispH/HDR is considered a light-dependent key regulatory enzyme in the MEP pathway . Research indicates that in the green alga Botryococcus braunii, upregulation of gene expression for HDR results in higher accumulation of carotenoids, suggesting ispH's regulatory role in controlling isoprenoid production . The MEP pathway generates fundamental building blocks for various essential biomolecules including cell membrane components, electron transport chain components, and various secondary metabolites.

The absence of the MEP pathway in mammals (which exclusively use the alternative mevalonate pathway) makes ispH a potential target for developing selective antimicrobial agents against bacterial pathogens like B. henselae.

What methodologies are used to assess the enzymatic activity of recombinant Bartonella henselae ispH?

The enzymatic activity of recombinant B. henselae ispH can be assessed using several complementary methodologies based on established protocols for similar enzymes:

A standard assay mixture for ispH activity measurement typically contains:

• 70 mM potassium phosphate buffer (pH 7.0)

• 1 mM divalent cation (preferably CoCl₂)

• 1 mM DTT

• 20 mM NaF

• 1.5 mM NADH

• 60 μM FAD

• 7 μM 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate (substrate)

• 0.5 mM pamidronate (to inhibit further metabolism of products)

For monitoring product formation, several analytical approaches can be employed:

• Radiochemical assays: Using tritium-labeled substrate ([1-³H]-labeled HMBPP) with detection by:

  • Reversed-phase ion-pair HPLC separation

  • Online liquid scintillation analysis

  • This provides quantitative determination of conversion rates

• NMR-based analysis: Using ¹³C-labeled substrates:

  • [2,2'-¹³C₂], [1,3,4-¹³C₃], or [U-¹³C₅] labeled substrate

  • Analysis of products by NMR spectroscopy after reaction completion

  • Sample preparation in 70% d₄-methanol in ²H₂O

• Chromatographic detection: HPLC separation with specific conditions:

  • Column: Multospher 120 RP 18-AQ-3 (4.6 × 250 mm)

  • Mobile phase: Gradient of methanol in 10 mM tetra-n-butylammonium phosphate (pH 6.0)

  • Flow rate: 0.55 ml/min

  • Retention volumes: 30.5 ml for substrate, 42 ml for products

These methodologies require careful optimization for recombinant B. henselae ispH specifically, as the protocols described were established for homologous enzymes.

What expression systems are most effective for producing functional recombinant Bartonella henselae ispH?

Producing functional recombinant B. henselae ispH presents unique challenges due to its iron-sulfur cluster requirements. Though the search results don't specifically address expression systems for B. henselae ispH, we can infer best practices from studies on similar enzymes:

The most effective expression systems typically include:

• E. coli-based systems:

  • pET expression vectors under T7 promoter control

  • Modified E. coli strains engineered for iron-sulfur protein expression

  • Co-expression with iron-sulfur cluster assembly systems (isc or suf operons)

  • Growth under microaerobic conditions to prevent oxidative damage to the [Fe-S] cluster

  • Induction at lower temperatures (16-20°C) to promote proper folding

  • Supplementation of growth media with iron and sulfur sources

• Key considerations for expression optimization:

  • Codon optimization for E. coli if necessary

  • Addition of a purification tag (His-tag) preferably at the N-terminus to avoid interfering with the C-terminal active site

  • Use of E. coli strains with reduced protease activity

  • Controlled induction conditions to prevent inclusion body formation

The presence of a functional [Fe₄S₄] cluster is critical for activity, as indicated in research on homologous enzymes . Approximately 50% of IspH molecules in crystallographic studies contained the [Fe₄S₄] cluster, highlighting the challenges in producing fully functional enzyme .

What buffer conditions are optimal for maintaining stability of purified recombinant Bartonella henselae ispH?

Maintaining the stability of purified recombinant B. henselae ispH requires careful attention to buffer composition to preserve the integrity of the iron-sulfur cluster. Based on research with similar enzymes, optimal buffer conditions include:

• Buffer components essential for stability:

  • Potassium phosphate buffer (70 mM, pH 7.0) - the pH optimum for enzymatic activity

  • Reducing agents (1-5 mM DTT) to prevent oxidation of the [Fe-S] cluster

  • Glycerol (10-20%) as a stabilizing agent

  • Divalent cations, particularly Co²⁺ (1 mM), which showed the highest efficacy among tested cations

• Additional stabilizing factors:

  • Low concentrations of substrate or substrate analogs

  • Low concentrations of iron and sulfide salts to prevent cluster degradation

  • Protection from oxygen exposure (work under anaerobic conditions when possible)

  • Storage at 4°C for short-term and -80°C (flash-frozen) for long-term preservation

• Compounds to avoid:

  • EDTA, which completely suppresses enzyme activity by chelating essential divalent cations

  • Oxidizing agents

  • High salt concentrations that might destabilize the protein structure

Storage in single-use aliquots is recommended to avoid repeated freeze-thaw cycles, and spectroscopic monitoring of the [Fe-S] cluster integrity (UV-Visible absorption at ~390-420 nm) can help assess stability over time.

How can the incorporation of the iron-sulfur cluster in recombinant Bartonella henselae ispH be verified?

Verification of proper [Fe-S] cluster incorporation into recombinant B. henselae ispH is critical for ensuring enzyme functionality. Several complementary analytical techniques can be employed:

• UV-Visible absorption spectroscopy:

  • [Fe₄S₄] clusters typically exhibit characteristic broad absorption bands at ~390-420 nm

  • The ratio of absorbance at this wavelength compared to protein absorbance at 280 nm provides a measure of cluster incorporation

  • Changes in spectra upon reduction or substrate binding can indicate functional cluster

• Electron Paramagnetic Resonance (EPR) spectroscopy:

  • Detects paramagnetic species, including reduced [Fe₄S₄]⁺ clusters

  • Provides detailed information about the electronic structure and redox state

  • Can detect substrate-induced changes in the electronic properties of the cluster

• Iron and sulfide content analysis:

  • Colorimetric assays for iron (e.g., ferrozine method)

  • Sulfide determination (e.g., methylene blue method)

  • A ratio approaching 4:4 (Fe:S) indicates complete cluster incorporation

• Enzymatic activity assays:

  • Activity correlates with proper cluster incorporation

  • The data from search result shows that enzymatic activity is severely reduced when cofactors required for proper [Fe-S] cluster function are omitted:

• X-ray crystallography:

  • Can directly visualize the [Fe-S] cluster and determine occupancy

  • Search result indicates that in crystallographic studies, the occupancy of the fourth iron (Fe3) was approximately 0.45-0.51, suggesting that about 50% of the enzyme molecules contained a complete [Fe₄S₄] cluster

These techniques provide complementary information and should ideally be used in combination to thoroughly characterize the iron-sulfur cluster status in the recombinant enzyme.

What is the proposed catalytic mechanism of Bartonella henselae ispH and how does it compare to homologs from other species?

The catalytic mechanism of B. henselae ispH likely follows the general pattern established for other bacterial ispH enzymes, though species-specific variations may exist. Based on the search results and known mechanisms:

The core reaction catalyzed by ispH involves the reductive dehydroxylation of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate (HMBPP) to form both isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) . The proposed mechanism includes:

• Binding of substrate to the enzyme:

  • The substrate (HMBPP) likely coordinates to the unique iron site of the [Fe₄S₄] cluster

  • This interaction positions the hydroxyl group for elimination

• Electron transfer to the substrate:

  • Electrons originate from NADH (preferred over NADPH)

  • Transfer occurs via FAD as an intermediate electron carrier

  • The [Fe₄S₄] cluster serves as the immediate electron donor to the substrate

• Formation of a reaction intermediate:

  • A radical or organometallic intermediate forms after initial electron transfer

  • This intermediate undergoes further reaction to eliminate the hydroxyl group

• Product formation:

  • The reaction produces a mixture of IPP and DMAPP

  • The ratio of these products may be species-specific

The mechanism is supported by several experimental observations from search result :

• Omission of NADH reduces activity to 12%

• Omission of FAD reduces activity to 17%

• Co²⁺ is the preferred divalent cation, with omission reducing activity to 20%

• EDTA completely suppresses activity by chelating essential divalent cations

Crystallographic studies have shown that in some ispH enzymes, ligand binding occurs at specific sites, with the occupancy of the fourth iron (Fe3) in the [Fe₄S₄] cluster being approximately 0.45-0.51 .

How do specific cofactors influence the activity of recombinant Bartonella henselae ispH?

Multiple cofactors play critical roles in the catalytic function of ispH, with each contributing distinctly to the enzyme's activity. Based on search result , we can detail their specific influences:

• NADH as primary electron donor:

  • Omission of NADH reduced activity to just 12% of control levels

  • NADH was more effective than NADPH in supporting enzyme activity

  • Serves as the source of electrons needed for the reductive dehydroxylation reaction

• FAD as electron transfer mediator:

  • Removing FAD from the reaction mixture decreased activity to 17%

  • Likely functions as an intermediate carrier, transferring electrons from NADH to the [Fe-S] cluster

  • The concentration of 60 μM used in assays suggests a relatively high affinity interaction

• Divalent cations, particularly Co²⁺:

  • Co²⁺ showed the highest efficacy among tested divalent cations

  • Omission of Co²⁺ reduced activity to 20%

  • May play roles in both enzyme structural stability and catalytic function

  • Addition of EDTA (metal chelator) completely suppressed activity

• The [Fe₄S₄] cluster:

  • Central to catalytic activity as the site of substrate binding and electron transfer

  • Crystallographic studies show approximately 50% occupancy of the fourth iron (Fe3)

  • The integrity of this cluster is essential for function

The relative importance of these cofactors is summarized in the following table derived from search result :

The proposed electron transfer chain is:
NADH → FAD → [Fe₄S₄] cluster → substrate

Each component represents a potential target for inhibitor development or a consideration for optimizing in vitro assay conditions.

What methodologies are most suitable for investigating the interaction between recombinant Bartonella henselae ispH and potential inhibitors?

Investigating interactions between recombinant B. henselae ispH and potential inhibitors requires a multi-faceted approach combining functional, structural, and biophysical methods:

• Enzyme activity assays:

  • Inhibition studies using the established activity assay conditions :

    • 70 mM potassium phosphate, pH 7.0

    • 1 mM CoCl₂

    • 1 mM DTT

    • 20 mM NaF

    • 1.5 mM NADH

    • 60 μM FAD

    • 7 μM substrate (1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate)

  • Determination of IC₅₀ values and inhibition constants (Ki)

  • Analysis of inhibition mechanisms (competitive, non-competitive, uncompetitive)

• Structural studies:

  • X-ray crystallography of enzyme-inhibitor complexes

    • Similar to the approach mentioned in search result where atomic coordinates for IspH bound to ligands were deposited in the Protein Data Bank

  • Computational docking studies using homology models if crystal structures are unavailable

  • Structure-activity relationship (SAR) analysis of inhibitor series

• Biophysical binding assays:

  • Isothermal titration calorimetry (ITC) to determine binding thermodynamics

  • Surface plasmon resonance (SPR) for binding kinetics

  • Thermal shift assays to assess stabilization by inhibitors

  • Microscale thermophoresis for quantifying binding affinities

• Spectroscopic techniques:

  • UV-Visible spectroscopy to monitor changes in [Fe-S] cluster upon inhibitor binding

  • EPR spectroscopy to detect alterations in the electronic structure of the [Fe-S] cluster

  • NMR studies to map binding sites using chemical shift perturbations

• Target-based screening approaches:

  • Fragment-based screening to identify initial binding modules

  • High-throughput screening with focused libraries of compounds likely to interact with [Fe-S] clusters

  • Virtual screening using structural information

Since pyridine-containing compounds have been shown to bind to ispH , compounds with similar structural features could serve as starting points for inhibitor development. The binding position of ligands to ispH may follow patterns similar to those observed in structural studies mentioned in search result .

How can recombinant Bartonella henselae ispH be utilized as a target for antimicrobial development?

Recombinant B. henselae ispH represents a promising target for antimicrobial development due to several advantageous characteristics:

• Pathway exclusivity and selective toxicity:

  • The MEP pathway is absent in humans who exclusively use the mevalonate pathway for isoprenoid biosynthesis

  • This fundamental difference provides a basis for selective toxicity, minimizing potential side effects

  • IspH has no human homolog, reducing the risk of off-target effects

• Essential metabolic function:

  • IspH catalyzes the terminal step in the MEP pathway, producing IPP and DMAPP

  • These compounds are essential precursors for:

    • Cell membrane components

    • Bacterial virulence factors

    • Electron transport chain components

  • Inhibition disrupts multiple critical processes simultaneously

• Unique structural features for drug design:

  • The [Fe₄S₄] cluster at the active site presents distinctive chemical opportunities for inhibitor design

  • The requirement for specific cofactors (NADH, FAD, Co²⁺) offers multiple points for intervention

  • The complex electron transfer pathway necessary for activity provides several vulnerable targets

• Drug development strategies:

  • Structure-based design using crystallographic data of enzyme-inhibitor complexes

  • Development of substrate analogs that interact with both the protein and [Fe-S] center

  • Design of compounds that disrupt the electron transfer chain from NADH through FAD to the [Fe-S] cluster

  • Metal-coordinating inhibitors that interact with the [Fe₄S₄] cluster

• Validation through in vitro studies:

  • Recombinant enzyme provides a platform for high-throughput screening

  • Structure-activity relationship development

  • Mechanism of action studies

  • The established activity assay conditions can be adapted for inhibitor screening

The data from search result demonstrating the dependency of ispH on specific cofactors highlights potential vulnerabilities that could be exploited for inhibitor development. The relative activity reductions when various components are omitted suggest multiple approaches to disrupting enzyme function.

What are the current challenges in expressing and utilizing recombinant Bartonella henselae ispH for structural biology studies?

Expressing and utilizing recombinant B. henselae ispH for structural biology presents several significant challenges:

• [Fe-S] cluster incorporation and stability:

  • Ensuring complete and stable incorporation of the [Fe₄S₄] cluster

  • Crystallographic studies indicate only about 50% occupancy of the fourth iron (Fe3) in the cluster

  • The oxygen-sensitive nature of [Fe-S] clusters complicates expression, purification, and crystallization

• Expression challenges:

  • Achieving sufficient expression levels of soluble, properly folded protein

  • Balancing expression rate with [Fe-S] cluster incorporation

  • Need for specialized expression systems with iron-sulfur cluster assembly machinery

• Purification difficulties:

  • Maintaining reducing conditions throughout purification to preserve [Fe-S] cluster integrity

  • Separating fully assembled holo-enzyme from apo-protein lacking the [Fe-S] cluster

  • Preventing metal-catalyzed oxidation during concentration and crystallization

• Crystallization obstacles:

  • Obtaining homogeneous protein preparations with consistent [Fe-S] cluster occupancy

  • Identifying conditions that maintain protein stability during crystallization

  • Growing crystals of sufficient quality for high-resolution diffraction studies

• Technical requirements for structural analysis:

  • Need for anaerobic or low-oxygen environments during crystal handling

  • Special considerations for synchrotron data collection to minimize radiation damage to the [Fe-S] cluster

  • Challenges in phase determination and model building around the [Fe-S] center

• Enzyme-ligand complex formation:

  • Difficulties in co-crystallizing with substrates due to their reactive nature

  • Need for stable substrate analogs or inhibitors

  • Ensuring consistent ligand occupancy in crystal structures

• Alternative structural approaches:

  • Cryo-EM as an emerging alternative facing challenges with proteins of this size (~40 kDa)

  • NMR studies limited by protein size and paramagnetic effects of the [Fe-S] cluster

  • Small-angle X-ray scattering (SAXS) providing lower resolution but useful conformational information

Despite these challenges, structural studies have been successful with related ispH enzymes, as evidenced by the deposition of atomic coordinates for IspH bound to ligands in the Protein Data Bank .

How can site-directed mutagenesis of recombinant Bartonella henselae ispH contribute to understanding its catalytic mechanism?

Site-directed mutagenesis represents a powerful approach for dissecting the catalytic mechanism of B. henselae ispH and can contribute to our understanding in several key ways:

• Identifying catalytic residues:

  • Mutation of residues near the [Fe-S] cluster can help determine their roles in:

    • Substrate binding

    • Proper positioning of the substrate relative to the [Fe-S] cluster

    • Proton donation/abstraction during catalysis

    • Stabilization of reaction intermediates

  • Systematic alanine scanning of conserved residues around the active site

• Investigating electron transfer pathways:

  • Mutations targeting residues potentially involved in electron transfer from:

    • NADH binding site

    • FAD interaction region

    • Pathways connecting FAD to the [Fe-S] cluster

  • The search results indicate that NADH and FAD are essential for activity (reducing activity to 12% and 17% respectively when omitted)

• Analyzing metal coordination:

  • Mutations affecting residues involved in coordinating the [Fe-S] cluster

  • Alterations to potential secondary coordination sites for Co²⁺, which shows the highest efficacy among divalent cations

  • Modifications to improve cluster stability or alter its redox properties

• Substrate specificity studies:

  • Mutations of residues lining the substrate binding pocket

  • Alterations that might affect the ratio of IPP vs. DMAPP products

  • Engineering variants with modified substrate preferences

• Structure-function correlation:

  • Mutations based on structural information from crystallographic studies

  • Alterations to residues interacting with bound ligands

  • Testing the occupancy effects of the fourth iron (Fe3) by modifying its coordination environment

• Experimental design considerations:

  • Expression and purification of mutants under identical conditions

  • Comprehensive characterization using:

    • Activity assays under standard conditions

    • Spectroscopic analysis of [Fe-S] cluster integrity

    • Determination of kinetic parameters (kcat, KM)

    • Product analysis to assess potential changes in IPP:DMAPP ratio

• Assessment of mutational effects:

  • Relative activity compared to wild-type enzyme

  • Changes in cofactor requirements or preferences

  • Altered sensitivity to inhibitors

  • Modifications to pH optimum (7.0 for the wild-type)

Such mutagenesis studies would complement the information from search result regarding cofactor dependencies and could provide insights into the mechanistic basis for the different activities observed when various components are omitted from the reaction.

How can isotope labeling techniques be applied to study the reaction mechanism of recombinant Bartonella henselae ispH?

Isotope labeling techniques offer powerful approaches to elucidate the reaction mechanism of recombinant B. henselae ispH at a molecular level. Based on search result , several strategic applications can be considered:

• ¹³C-labeled substrate studies:

  • Similar to the approach described in search result , various ¹³C-labeled substrates can be employed:

    • [2,2'-¹³C₂]-labeled 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate

    • [1,3,4-¹³C₃]-labeled substrate

    • [U-¹³C₅]-labeled substrate (uniformly labeled)

  • NMR analysis of products allows tracking of specific carbon atoms through the reaction

  • Sample preparation can follow the described protocol: dissolution in 70% (vol/vol) d₄-methanol in ²H₂O

• Tritium (³H) labeling for sensitive detection:

  • [1-³H]-labeled substrate with detection by liquid scintillation counting

  • Enables highly sensitive quantitative analysis of reaction kinetics

  • HPLC separation can follow conditions described in search result :

    • Column: Multospher 120 RP 18-AQ-3

    • Mobile phase gradient of methanol in tetra-n-butylammonium phosphate

    • Flow rate: 0.55 ml/min

• ²H (deuterium) labeling for mechanism investigation:

  • Deuterated substrates to investigate kinetic isotope effects

  • Can reveal rate-limiting steps involving C-H bond breaking

  • Helps distinguish between concerted and stepwise mechanisms

• ¹⁸O labeling to track oxygen fate:

  • ¹⁸O-labeled at the hydroxyl position of the substrate

  • Determines the fate of the oxygen atom during the reaction

  • Mass spectrometry analysis of products and potential water byproduct

• ¹⁵N labeling of cofactors:

  • ¹⁵N-labeled NADH to trace nitrogen involvement in electron transfer

  • Can provide insights into transient interactions during catalysis

• ⁵⁷Fe enrichment for Mössbauer spectroscopy:

  • Incorporation of ⁵⁷Fe into the [Fe-S] cluster

  • Enables detailed Mössbauer spectroscopic analysis

  • Provides information about oxidation states and electronic environment of iron atoms during catalysis

• Experimental protocols based on search result :

  • Reaction conditions:

    • 70 mM potassium phosphate, pH 7.0

    • 20 mM NaF

    • 1 mM DTT

    • 0.5 mM pamidronate

    • 1.5 mM NADH

    • 60 μM FAD

    • 0.5 mM CoCl₂

    • Labeled substrate (appropriate concentration)

    • Recombinant enzyme preparation

These isotope labeling approaches can provide detailed mechanistic insights that would be impossible to obtain through conventional kinetic or structural studies alone.

What are the potential interactions between recombinant Bartonella henselae ispH and ferredoxin proteins?

The potential interactions between B. henselae ispH and ferredoxin proteins represent an intriguing area of research, particularly considering the parallels with other organisms. Based on search result , we can explore several aspects of this relationship:

• Ferredoxin as potential physiological electron donor:

  • Search result discusses the possible association of HDR (ispH) with ferredoxin in the green alga Botryococcus braunii

  • Docking analysis revealed potential contact between ferredoxin (PETF-like protein) and HDR

  • The distance between the two Fe-S centers in the docked model was 14.7 Å, comparable to the 12.6 Å observed in Plasmodium falciparum

• Structural basis for potential interaction:

  • Ferredoxin might interact with the "backside" of ispH as defined in previous research

  • This interaction would position the Fe-S centers at an appropriate distance for efficient electron transfer

  • B. henselae ispH likely has surface features that could facilitate species-specific ferredoxin interactions

• Functional implications:

  • If B. henselae ispH can accept electrons from ferredoxin, this would represent an alternative electron transfer pathway to the NADH/FAD-dependent mechanism

  • Such flexibility might allow the bacterium to adapt to different metabolic states or environments

  • In photosynthetic organisms, this association enables light-dependent regulation of isoprenoid synthesis

• Experimental approaches to investigate this interaction:

  • Protein-protein docking simulations similar to those described for B. braunii

  • In vitro reconstitution of electron transfer using purified B. henselae ispH and ferredoxins

  • Activity assays comparing NADH/FAD versus ferredoxin-dependent activity

  • Co-immunoprecipitation or pull-down assays to detect physical interaction

  • Site-directed mutagenesis of predicted interface residues

• Evolutionary perspective:

  • Search result notes that "ferredoxin can be involved in two successive key-regulatory enzymatic reactions in MEP pathway of wide range of photosynthetic organisms"

  • While B. henselae is not photosynthetic, it may have retained the ability to interact with ferredoxin-like electron carriers

  • This could represent an evolutionary adaptation or vestige of the ancient origins of the MEP pathway

The potential interaction with ferredoxin represents an additional layer of complexity in understanding B. henselae ispH function and regulation, and could offer new insights into bacterial metabolism and potential drug targets.

How can computational approaches enhance our understanding of substrate binding and catalysis in recombinant Bartonella henselae ispH?

Computational approaches offer powerful tools to complement experimental studies of B. henselae ispH, providing atomic-level insights into substrate binding, catalysis, and inhibitor development:

• Homology modeling and structure prediction:

  • Generation of B. henselae ispH structural models based on crystallographic data from homologs

  • Refinement of models to accurately represent the [Fe₄S₄] cluster environment

  • Assessment of model quality through energy minimization and validation tools

• Molecular docking studies:

  • Similar to the approach in search result where docking analysis was performed between HDR and ferredoxin

  • Docking of substrate (1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate) to predict binding modes

  • Virtual screening of potential inhibitors targeting the active site

  • Analysis of pyridine-containing compounds that bind to ispH as mentioned in search result

• Molecular dynamics simulations:

  • Exploration of protein dynamics and conformational changes during catalysis

  • Investigation of water molecules and proton transfer networks

  • Assessment of the stability of the [Fe₄S₄] cluster under different conditions

  • Special force field parameters to accurately model the [Fe₄S₄] cluster

• Quantum mechanical/molecular mechanical (QM/MM) methods:

  • High-level quantum calculations of the reaction mechanism

  • Accurate modeling of electronic structure changes during catalysis

  • Calculation of energy barriers for different mechanistic proposals

  • Particular focus on the electron transfer steps, given the importance of NADH and FAD

• Network analysis of electron transfer pathways:

  • Identification of potential pathways for electron transfer from NADH through FAD to the [Fe-S] cluster

  • Calculation of electron coupling elements between redox centers

  • Prediction of residues critical for electron transfer that could be targeted by mutagenesis

• Binding free energy calculations:

  • Thermodynamic analysis of substrate and inhibitor binding

  • Comparison of binding affinities for different potential inhibitors

  • Decomposition analysis to identify key interaction residues

• Ligand optimization strategies:

  • Fragment-based design of novel inhibitors

  • Lead optimization guided by calculated structure-activity relationships

  • Scaffold hopping to identify new chemical classes with improved properties

These computational approaches can guide experimental efforts by generating testable hypotheses about substrate binding, catalytic mechanism, and inhibitor design, ultimately accelerating the development of new antimicrobial agents targeting B. henselae ispH.