Recombinant Leptospira interrogans serogroup Icterohaemorrhagiae serovar copenhageni Phosphomethylpyrimidine synthase (thiC)

What is the biological significance of phosphomethylpyrimidine synthase (thiC) in Leptospira interrogans metabolism?

Phosphomethylpyrimidine synthase (thiC) catalyzes a critical early step in thiamin (vitamin B1) biosynthesis in Leptospira interrogans. This enzyme specifically converts 5-aminoimidazole ribotide (AIR) to 4-amino-2-methyl-5-phosphomethylpyrimidine (HMP-P), producing formate and carbon monoxide as byproducts . The thiamin pyrophosphate eventually produced serves as an essential cofactor for enzymes involved in carbohydrate metabolism and energy production.

The reaction catalyzed by thiC is remarkable for its complexity, representing "the most complex enzyme-catalyzed radical cascade identified to date" . The enzyme contains a [Fe₄S₄] cluster that participates in the generation of radical intermediates during the complex rearrangement reaction. Since mammals lack this biosynthetic pathway and must obtain thiamin through dietary sources, thiC represents a potential antimicrobial target for treating leptospirosis .

How does Leptospira interrogans pathogenesis relate to thiamin biosynthesis?

Leptospira interrogans is the causative agent of leptospirosis, a zoonotic disease that affects over one million humans annually and causes approximately 60,000 deaths worldwide . The bacteria can survive for weeks to months in soil and water environments , requiring robust metabolic pathways that support persistence in diverse conditions.

The thiamin biosynthesis pathway, including the thiC-catalyzed step, is crucial for bacterial survival under conditions where exogenous thiamin is limited. While the search results don't directly link thiC activity to virulence, several connections can be drawn:

• Environmental persistence: The ability to synthesize essential cofactors like thiamin likely contributes to L. interrogans' remarkable environmental persistence .

• Metabolic adaptation: During infection, bacteria face nutrient-limited conditions where de novo thiamin synthesis could provide a competitive advantage.

• Stress response: L. interrogans upregulates various metabolic pathways in response to environmental stresses, similar to what might occur with thiamin biosynthesis enzymes .

What is the current understanding of the thiC enzyme reaction mechanism?

The thiC reaction mechanism has been recently elucidated through the trapping of five intermediates that provide "snapshots of the reaction coordinate" . The current mechanistic understanding involves:

• Initial stage: The 5'-dA radical formed from S-adenosylmethionine (SAM) abstracts a hydrogen atom from C5' of AIR (compound 1) to generate radical species (compound 13) .

• Middle stages: Acid-catalyzed ring opening followed by electron transfer from the electron-rich aminoimidazole to the alkene radical cation produces compound 33, which undergoes β-scission to give compounds 34 and 35. These intermediates then recombine .

• Final stages: A series of additional rearrangements, including a decarbonylation step that releases carbon monoxide, ultimately produces HMP-P .

Key catalytic residues include a glutamate (Glu413 in C. crescentus ThiC) that forms a hydrogen bond with the C2' hydroxyl of AIR and a cysteine (Cys474) that participates in hydrogen atom transfer later in the reaction .

What strategies have proven effective for capturing the transient radical intermediates in the thiC-catalyzed reaction?

Trapping transient radical intermediates in the thiC reaction requires sophisticated experimental approaches:

• Chemical trapping agents: O-(2,3,4,5,6-pentafluorobenzyl) hydroxylamine (PFBHA) has been successfully employed to capture aldehyde-containing fragments generated during the reaction. This derivatization strategy allows detection of intermediates by LC-MS as E/Z isomer pairs .

• Site-directed mutagenesis: Strategic mutations of catalytic residues can disrupt the normal reaction pathway, causing accumulation of intermediates. For example:

  • E413Q mutation in C. crescentus ThiC prevents HMP-P formation and leads to accumulation of intermediates like compound P24 .

  • C474S mutation blocks a key hydrogen atom transfer step, resulting in accumulation of different intermediates .

• Substrate analogues: Synthetic AIR analogues with stabilized chemical bonds have been developed. For instance, compound 43 replaces a labile C-N bond with a stable C-C bond, allowing detection of formyl imidazole intermediate 39 .

• Advanced analytical techniques: LC-MS analysis of reaction mixtures after derivatization has been crucial for detecting and characterizing intermediates. MS-MS fragmentation patterns help confirm intermediate structures .

These approaches collectively contributed to a comprehensive understanding of the complex radical cascade catalyzed by thiC, leading to the identification of key intermediates that would otherwise be too unstable for characterization.

How can protein engineering approaches be applied to modify the catalytic properties of Leptospira interrogans thiC?

Based on structural and mechanistic insights from thiC enzymes in other organisms, several protein engineering approaches could be applied to L. interrogans thiC:

• Active site modifications: Targeted mutations of residues coordinating the substrate or involved in catalysis could alter reaction specificity or rate. For example:

  • Mutations equivalent to E413Q in C. crescentus ThiC could potentially alter the acid-base catalysis properties .

  • Modifications to the cysteine residue equivalent to C474 would likely affect hydrogen atom transfer steps .

• Iron-sulfur cluster coordination: Engineering the residues that coordinate the [Fe4S4] cluster could alter its redox properties and therefore the radical generation capability of the enzyme.

• Substrate binding pocket modifications: Altering residues that form hydrogen bonds with AIR could potentially modify substrate specificity or binding affinity.

• Directed evolution approaches: Developing a functional selection or screening system for thiC could enable the evolution of variants with enhanced stability, altered substrate specificity, or improved catalytic efficiency.

The challenge in applying these approaches lies in developing appropriate assays for thiC activity, especially given the complex nature of the reaction and the instability of the intermediates and products.

What are the methodological considerations for studying thiC activity in cell-free extracts versus purified enzyme systems?

Studying thiC activity presents unique challenges due to its complex radical chemistry and cofactor requirements:

Cell-free extract considerations:

• Advantages:

  • Native environment with potential stabilizing factors

  • Presence of physiological reducing systems that maintain the iron-sulfur cluster in the active state

  • Availability of partner proteins that might assist in cofactor incorporation

• Challenges:

  • Background activities that might interfere with assays

  • Difficulty in quantifying enzyme concentration

  • Potential for product degradation by other enzymes

Purified enzyme considerations:

• Advantages:

  • Defined system allowing precise kinetic measurements

  • Absence of competing reactions

  • Ability to control all reaction components

• Challenges:

  • Maintaining iron-sulfur cluster integrity during purification

  • Reconstitution of enzyme activity after purification

  • Establishing appropriate reducing conditions

Methodological recommendations:

• Perform purification under anaerobic conditions to preserve the iron-sulfur cluster

• Include reducing agents (DTT, β-mercaptoethanol) and iron chelators in buffers

• Consider adding glycerol or other stabilizing agents

• Use LC-MS for product detection rather than spectrophotometric assays

• Include controls with heat-inactivated enzyme and without SAM to account for non-enzymatic reactions

For studies focusing on the Leptospira interrogans thiC specifically, the protocols developed for other ThiC enzymes (like those from A. thaliana or C. crescentus ) could serve as starting points, with optimization for the specific properties of the L. interrogans enzyme.

What expression systems are optimal for producing active recombinant Leptospira interrogans thiC?

Based on successful expression of other Leptospira proteins and related iron-sulfur enzymes, the following expression systems and conditions are recommended for L. interrogans thiC:

Expression system recommendations:

• E. coli expression strains:

  • BL21(DE3) derivatives: Particularly those optimized for iron-sulfur proteins

  • Rosetta or CodonPlus strains: To address potential rare codon usage in Leptospira genes

  • SHuffle strains: For potentially improved disulfide bond formation if relevant

• Expression vectors:

  • pET system vectors (particularly pET28a): Successfully used for other Leptospira proteins

  • Gateway system vectors (pDEST17): Demonstrated success with some Leptospira proteins

• Expression conditions:

  • Low temperature induction (15-18°C): To maximize proper protein folding

  • Extended expression times (16-24 hours): To allow sufficient time for iron-sulfur cluster incorporation

  • Supplementation with iron (ferric ammonium citrate or ferrous sulfate) and cysteine: To enhance iron-sulfur cluster formation

What purification strategy maintains the catalytic activity of thiC?

Purifying catalytically active ThiC requires special considerations due to its oxygen-sensitive iron-sulfur cluster:

Recommended purification protocol:

• Cell lysis:

  • Perform under anaerobic conditions or with degassed buffers

  • Include protease inhibitors to prevent degradation

  • Use mild sonication (3 × 20 s) to prevent protein denaturation

• Buffer composition:

  • Base buffer: 50 mM Tris-HCl or HEPES, pH 7.5-8.0

  • Salt: 300-500 mM NaCl to enhance stability

  • Reducing agents: 1-5 mM DTT or β-mercaptoethanol

  • Glycerol: 10-20% to enhance protein stability

  • Iron chelator: 0.1-0.5 mM EDTA to prevent non-specific iron binding

• Purification steps:

  • Initial purification: Ni-NTA affinity chromatography under native conditions

  • Secondary purification: Size exclusion chromatography to remove aggregates

  • Consider anaerobic columns or adding sodium dithionite to buffers

• Iron-sulfur cluster reconstitution:

  • If activity is low, consider in vitro reconstitution of the iron-sulfur cluster

  • Typical procedure includes incubation with ferrous ammonium sulfate, sodium sulfide, and DTT under anaerobic conditions

• Activity verification:

  • Measure UV-Vis spectrum to confirm iron-sulfur cluster presence (typical absorption peaks around 320 and 420 nm)

  • Verify enzymatic activity through LC-MS detection of HMP-P formation from AIR

How can researchers assess the integrity of the iron-sulfur cluster in recombinant thiC preparations?

Evaluating the integrity of the [Fe4S4] cluster in thiC is critical for ensuring enzyme activity. The following analytical approaches are recommended:

• UV-Visible spectroscopy:

  • [Fe4S4] clusters typically exhibit absorption features at ~320 nm and ~420 nm

  • The ratio of A420/A280 provides an estimate of cluster occupancy

  • Monitor spectral changes during anaerobic-to-aerobic transitions to assess oxygen sensitivity

• Electron Paramagnetic Resonance (EPR) spectroscopy:

  • Can directly detect paramagnetic [Fe4S4]+ cluster (reduced state)

  • Requires reduction with dithionite or photoreduction

  • Characteristic g-values can confirm cluster type and environment

• Iron and sulfide quantification:

  • Iron content: Determined by colorimetric assays (e.g., ferene method)

  • Acid-labile sulfide: Measured using methylene blue formation

  • Theoretical [Fe4S4]:protein ratio should be 1:1 for fully loaded enzyme

• Circular Dichroism (CD) spectroscopy:

  • Provides information on secondary structure and integrity of the iron-sulfur center

  • Can detect structural changes upon cluster oxidation or degradation

• Activity correlation:

  • Measure enzymatic activity (AIR to HMP-P conversion) and correlate with spectroscopic features

  • Activity loss with corresponding spectral changes would indicate cluster degradation

These complementary approaches provide a comprehensive assessment of iron-sulfur cluster integrity, essential for interpreting structure-function relationships and ensuring reproducible enzymatic assays.

What structural features of thiC are essential for its catalytic function?

ThiC possesses several critical structural features that enable its complex radical rearrangement chemistry:

• Iron-sulfur cluster binding site: The enzyme contains a non-canonical [Fe4S4] cluster coordinated by three cysteine residues in a CX2CX4C motif . This cluster is essential for SAM binding and the generation of the 5'-deoxyadenosyl radical that initiates the reaction.

• SAM binding site: ThiC contains a novel mononuclear iron site involved in SAM binding , positioning it optimally for radical generation.

• Substrate binding pocket: Key residues position AIR for the initial hydrogen atom abstraction from C5', including:

  • A glutamate residue (Glu422 in A. thaliana ThiC; Glu413 in C. crescentus ThiC) that hydrogen bonds with the C2' hydroxyl of AIR

  • Residues that coordinate the aminoimidazole portion of AIR

• Catalytic residues:

  • Cys474 (in C. crescentus ThiC) plays a critical role in hydrogen atom transfer during the reaction

  • Acidic residues likely facilitate proton transfers during the complex rearrangement

• Protein fold: ThiC belongs to the radical SAM enzyme superfamily, with a partial (β/α)8 TIM barrel fold that provides the structural framework for positioning all the catalytic components .

The complex interplay between these structural elements creates the precise geometric arrangement necessary for controlling the radical chemistry and directing the multistep rearrangement toward the correct product.

What insights do recent intermediate trapping experiments provide about the thiC reaction mechanism?

Recent intermediate trapping experiments have provided breakthrough insights into the complex thiC reaction mechanism:

• Resolution of reaction pathway controversy: The identification of compound 30 (a ribose fragment) and formyl aminoimidazole 39 has resolved conflicting mechanistic proposals by demonstrating that the C1'-C2' bond cleavage occurs prior to the attachment of ribose to the aminoimidazole .

• Identification of five new intermediates: The trapped intermediates provide "snapshots of the ThiC reaction coordinate" that have enabled a revised mechanism for this complex radical rearrangement:

  • Compound P24: An aldehyde-containing intermediate trapped using PFBHA derivatization

  • Compound 30: A fragment revealing early C-C bond cleavage

  • Formyl aminoimidazole 39: Confirming fragmentation pathway

  • Compounds 58, 67, and 68: Later-stage intermediates trapped with the C474S mutant

• Revised mechanistic proposal: The trapping experiments support a mechanism where:

  • Initial hydrogen abstraction from C5' of AIR forms radical 13

  • Acid-catalyzed ring opening followed by electron transfer produces species 33

  • β-scission produces fragments 34 and 35, which recombine to form 36

  • Subsequent transformations eventually lead to pyrimidine formation

• Role of key residues: The experiments with E413Q and C474S mutants revealed:

  • Glu413 is critical for acid-catalyzed steps early in the mechanism

  • Cys474 participates in hydrogen atom transfer during later stages of the reaction

These findings represent a significant advance in understanding "the most complex enzyme-catalyzed radical cascade identified to date" and provide a framework for further studies on thiC enzymes from various organisms, including Leptospira interrogans.

How do mutations in key residues affect the reaction pathway of thiC?

Mutations in key thiC residues dramatically alter the reaction pathway, providing valuable mechanistic insights:

1. Glutamate mutant (E413Q in C. crescentus ThiC):

• Observed effects:

  • Complete loss of HMP-P production

  • Accumulation of intermediates P24 and compound 30

  • No detectable carbon monoxide formation

• Mechanistic implications:

  • The glutamate residue is essential for acid-catalyzed ring opening early in the reaction

  • Without proper ring opening, the reaction stalls at early intermediates

  • The side products formed indicate premature fragmentation of the reaction pathway

2. Cysteine mutant (C474S in C. crescentus ThiC):

• Observed effects:

  • Loss of HMP-P production

  • Accumulation of compound 58, which reacts with bisulfite and thiosulfate to form compounds 67 and 68

  • Absence of carbon monoxide formation

• Mechanistic implications:

  • Cys474 is involved in hydrogen atom transfer during later stages of the reaction

  • The C474S mutation blocks progression from intermediate 60 to 61

  • Deprotonation and electron transfer in the absence of hydrogen atom transfer leads to alternative product 66, which tautomerizes to the observed trapped product 58

3. Effect on reaction energetics and specificity:

• Mutations alter the energy landscape of the reaction

• Loss of specific acid-base catalysis or hydrogen atom transfer capabilities creates alternative reaction pathways

• The accumulation of specific intermediates with different mutations provides a map of where each residue functions in the reaction coordinate

These findings demonstrate how critical residues control the precise choreography of radical transfer, electron movement, and proton abstraction/donation in this complex reaction. Similar studies on L. interrogans thiC would likely reveal comparable effects, given the high conservation of key catalytic residues in this enzyme family.

What evidence supports thiC as a potential antimicrobial target against Leptospira interrogans?

Multiple lines of evidence support phosphomethylpyrimidine synthase (thiC) as a promising antimicrobial target against Leptospira interrogans:

• Target validation considerations:

  • Pathway essentiality: Thiamin is an essential cofactor for central metabolic enzymes, and its biosynthesis is likely crucial under conditions where exogenous thiamin is limited .

  • Absence in humans: The thiamin biosynthetic pathway is absent in vertebrates, who obtain thiamin through dietary sources, providing inherent selectivity .

  • Uniqueness of the enzyme: ThiC catalyzes an unusual radical rearrangement reaction with no mechanistic counterpart in human metabolism .

• Structural and biochemical evidence:

  • Structure characterization: The crystal structure of related phosphomethylpyrimidine synthases has been determined, facilitating structure-based drug design .

  • Active site accessibility: The SAM and substrate binding sites present accessible targets for small molecule inhibitors.

  • Mechanistic understanding: Recent elucidation of the reaction mechanism provides insights for designing mechanism-based inhibitors .

• Precedent from related pathways:

  • The related enzyme phosphomethylpyrimidine kinase has been explicitly proposed as "a potential chemotherapeutic target for antileptospiral treatment" .

  • The structure of this enzyme from L. interrogans has been modeled and deposited in the Protein Data Bank (PDB ID: 2G53) .

• Environmental and infectious context:

  • L. interrogans can survive for months in environmental water sources , suggesting robust metabolic pathways like thiamin biosynthesis contribute to this persistence.

  • During infection, bacteria encounter nutrient-limited conditions where de novo thiamin synthesis would be advantageous.

While direct genetic evidence (e.g., gene knockout studies demonstrating essentiality in L. interrogans) is not presented in the search results, the biochemical logic strongly supports thiC as a promising antimicrobial target that warrants further investigation.

What approaches could be used to design selective inhibitors of Leptospira interrogans thiC?

Designing selective inhibitors of L. interrogans thiC could employ several complementary approaches:

• Structure-based design strategies:

  • SAM binding site targeting: Develop analogues of SAM that specifically inhibit the radical generation step

  • Substrate competitive inhibitors: Design AIR analogues that bind but cannot undergo the radical rearrangement

  • Intermediate-inspired inhibitors: Utilize the recently trapped reaction intermediates as templates for inhibitor design

  • Allosteric inhibitors: Target unique surface features or conformational states of the L. interrogans enzyme

• Mechanism-based inhibitor approaches:

  • Radical traps: Incorporate functional groups that quench the 5'-deoxyadenosyl radical

  • Covalent modifiers: Target key catalytic residues such as the active site cysteine equivalent to C474 in C. crescentus ThiC

  • Iron chelators: Design compounds that disrupt the iron-sulfur cluster

• Fragment-based approaches:

  • Screen small molecular fragments that bind to different regions of thiC

  • Link promising fragments to develop high-affinity inhibitors

  • Utilize NMR or X-ray crystallography to guide fragment elaboration

• Computational methods:

  • Virtual screening: Use docking to identify potential inhibitors from compound libraries

  • Molecular dynamics: Study protein flexibility to identify transient binding pockets

  • Quantum mechanical calculations: Model the radical reaction to identify vulnerable steps for inhibition

The recently reported trapped intermediates in the thiC reaction provide valuable templates for inhibitor design, particularly compounds that mimic the transition states or unstable intermediates in the reaction pathway.

What methodologies are most appropriate for evaluating potential thiC inhibitors against Leptospira interrogans?

A comprehensive evaluation of thiC inhibitors requires a multi-level testing strategy:

Biochemical Assays

• Primary enzymatic assays:

  • Endpoint assays: Measure HMP-P formation using LC-MS/MS detection

  • Coupled enzyme assays: Connect HMP-P formation to a detectable signal

  • SAM cleavage assays: Monitor 5'-deoxyadenosine formation as a proxy for enzyme activity

• Binding assays:

  • Thermal shift assays: Measure changes in protein thermal stability upon inhibitor binding

  • Surface plasmon resonance: Determine binding kinetics and affinity

  • Isothermal titration calorimetry: Characterize thermodynamics of inhibitor binding

Structural Biology Approaches

• X-ray crystallography: Determine co-crystal structures of thiC with inhibitors

• HDX-MS: Identify conformational changes induced by inhibitor binding

• NMR studies: Characterize inhibitor binding sites and protein dynamics

Cellular Assays

• Growth inhibition assays: Determine minimum inhibitory concentration (MIC) against L. interrogans

• Thiamin rescue experiments: Confirm inhibition is due to thiamin pathway disruption

• Cell-based target engagement: Develop assays to confirm on-target activity in cells

Mechanistic and Specificity Studies

• Selectivity panels: Test against human enzymes to ensure safety

• Mode of inhibition: Determine competitive, non-competitive, or uncompetitive mechanisms

• Resistance development: Assess frequency of resistance and characterize resistant mutants

Advanced Validation Models

• Ex vivo kidney tubule models: Test efficacy in environments mimicking renal colonization

• Animal models of leptospirosis: Evaluate efficacy in standard hamster models

• Biofilm inhibition assays: Assess activity against L. interrogans biofilms, which contribute to environmental persistence