Recombinant Chromobacterium violaceum Phosphomethylpyrimidine synthase (thiC), partial

What is Phosphomethylpyrimidine synthase (ThiC) and what is its role in C. violaceum?

ThiC (EC 4.1.99.17) is a critical enzyme in the thiamine pyrophosphate (TPP) biosynthesis pathway. In C. violaceum, ThiC catalyzes the complex conversion of aminoimidazole ribotide (AIR) to 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate (HMP-P), which is essential for the pyrimidine moiety formation in thiamine biosynthesis . This enzyme is alternatively known as hydroxymethylpyrimidine phosphate synthase, HMP-P synthase, HMP-phosphate synthase, or HMPP synthase . The gene encoding ThiC (thiC; CV_0235) has been identified in the genome of C. violaceum strain ATCC 12472, highlighting its importance in the metabolic processes of this organism .

How does the thiamine biosynthesis pathway in C. violaceum compare to other bacterial species?

The thiamine biosynthesis pathway in C. violaceum shares similarities with other bacterial species but exhibits some distinct features:

Unlike E. coli where tryptophan biosynthesis genes are organized in an operon, C. violaceum has these genes dispersed throughout the genome . The ThiC enzyme in various bacteria appears to contain iron-sulfur cluster ligands that are required for function in vivo, and this feature is likely conserved in C. violaceum .

What are the key structural and functional domains of ThiC?

The ThiC protein contains several key structural and functional elements:

• Fe-S cluster binding site: Contains potential ligands for an iron-sulfur cluster that are essential for catalytic activity

• Catalytic domain: Responsible for the complex rearrangement reaction that converts AIR to HMP-P

• Substrate binding sites: Regions that interact with AIR and potentially SAM (S-adenosylmethionine)

• Potential regulatory domains: May interact with the TPP riboswitch in some regulatory contexts

Studies suggest that the Fe-S cluster is particularly critical for ThiC function, as mutants defective in iron-sulfur cluster metabolism show reduced efficiency in the synthesis of the pyrimidine moiety of thiamine .

What are the optimal expression systems for recombinant C. violaceum ThiC?

The optimal expression systems for recombinant C. violaceum ThiC production include:

• E. coli-based systems: The most commonly used approach due to high yield and established protocols. The protein can be expressed in E. coli strains like BL21(DE3) with suitable expression vectors .

• Yeast expression systems: For researchers requiring eukaryotic post-translational modifications, expression in Pichia pastoris may provide advantages, though yields are typically lower than E. coli systems .

• Baculovirus expression systems: Used when higher eukaryotic folding machinery is needed, particularly useful if the protein tends to form inclusion bodies in bacterial systems .

The choice of expression system should be guided by specific experimental requirements, including the need for post-translational modifications, protein solubility considerations, and downstream applications.

What are the challenges in expressing and purifying functional ThiC protein?

Expressing and purifying functional ThiC presents several challenges:

• Iron-sulfur cluster integrity: The Fe-S cluster in ThiC is sensitive to oxidative conditions, which can compromise enzyme activity. Purification under anaerobic or reducing conditions may be necessary to maintain full functionality .

• High GC content: The genome of C. violaceum has a very high GC content (64.83%), which can pose difficulties during PCR amplification and cloning. This may require optimized protocols with specialized polymerases and buffer systems .

• Protein solubility: ThiC may form inclusion bodies when overexpressed, necessitating optimization of expression conditions (temperature, induction levels) or refolding protocols.

• Maintaining enzymatic activity: The complex reaction catalyzed by ThiC requires proper folding and cofactor incorporation, which may be lost during purification.

To overcome these challenges, researchers should consider:

• Using specialized E. coli strains engineered for expression of proteins with high GC content

• Including iron and sulfur sources in expression media

• Optimizing induction conditions (temperature, IPTG concentration, duration)

• Adding reducing agents during purification

• Using affinity tags that minimize interference with protein function

What purification strategies yield the highest activity for recombinant ThiC?

The most effective purification strategies for maintaining ThiC activity include:

• Affinity chromatography: Using tags such as His-tag, which can be added to either the N- or C-terminus of the protein. The tag type can be determined during the production process based on specific requirements .

• Anoxic purification: Maintaining reducing conditions throughout purification to preserve Fe-S cluster integrity.

• Buffer optimization: Including compounds like DTT or β-mercaptoethanol to prevent oxidation of the Fe-S cluster.

• Multi-step purification protocol:

  • Initial affinity purification using the engineered tag

  • Ion exchange chromatography for further purification

  • Size exclusion chromatography as a final polishing step

For long-term storage, it is recommended to add 5-50% glycerol (with 50% being the default final concentration) and aliquot for storage at -20°C/-80°C .

What are the established assays for measuring ThiC activity?

Several methods have been developed to assess ThiC enzymatic activity:

• Conversion of AIR to HMP-P: This direct assay monitors the transformation of the substrate (AIR) to product (HMP-P) using techniques such as:

  • HPLC with UV detection

  • LC-MS/MS for more sensitive detection of the product

  • Radiolabeled substrate approaches using 14C-labeled AIR

• Coupled enzyme assays: These indirect methods couple ThiC activity to other enzymatic reactions that generate a measurable signal.

• Complementation assays: Functional ThiC can complement thiamine auxotrophy in appropriate microbial strains, providing an in vivo assessment of activity.

When designing ThiC activity assays, researchers should consider:

• The need for anaerobic conditions to maintain Fe-S cluster integrity

• The requirement for SAM as a potential cofactor

• The complexity of the reaction, which involves extensive molecular rearrangement

How does oxidative stress affect ThiC activity and what methods can mitigate this?

Oxidative stress significantly impacts ThiC activity due to its sensitive Fe-S cluster:

• Effects of oxidative stress:

  • The conversion of AIR to HMP-P is sensitive to oxidative conditions

  • The Fe-S cluster can be damaged or lost under oxidative environments

  • Certain ThiC variants show increased sensitivity to oxidative growth conditions

• Mitigation strategies:

  • Perform all enzymatic assays under anaerobic conditions or in the presence of oxygen scavengers

  • Include reducing agents (DTT, β-mercaptoethanol, or glutathione) in reaction buffers

  • Use sealed reaction vessels with inert gas (N2 or Ar) to minimize oxygen exposure

  • Consider adding Fe2+ and sulfide during protein expression to enhance Fe-S cluster assembly

  • Reconstitute the Fe-S cluster in vitro using established protocols if activity is lost

Research has shown that ThiC variants with altered Fe-S cluster coordination exhibit differential sensitivity to oxidative conditions, suggesting this feature is crucial for the physiological function of the enzyme .

What is the relationship between ThiC activity and the TPP regulatory network?

ThiC activity is integrated into the broader TPP regulatory network through several mechanisms:

• Riboswitch regulation: In many bacteria, ThiC expression is controlled by TPP-sensing riboswitches that respond to intracellular TPP levels. In E. coli, the thiC riboswitch exerts regulation at both the transcriptional and translational levels .

• Feedback inhibition: High levels of TPP can repress the expression of thiamine biosynthesis genes, including thiC, through riboswitch-mediated mechanisms. For example, in Treponema denticola, a TPP-sensing riboswitch (Td thi-box) regulates the expression of TPP transporters via feedback inhibition .

• Coordinated regulation: ThiC activity is coordinated with other enzymes in the thiamine biosynthesis pathway. The expression of these enzymes is often co-regulated to ensure balanced production of the pyrimidine and thiazole moieties of thiamine.

• Iron-sulfur cluster metabolism: There appears to be a connection between iron-sulfur cluster metabolism and ThiC function, suggesting that cellular iron status may influence thiamine biosynthesis capability .

Understanding these regulatory relationships is crucial for interpreting ThiC activity in different physiological contexts and for designing experiments that accurately reflect in vivo conditions.

What are the proven strategies for cloning the thiC gene from C. violaceum?

Successfully cloning the thiC gene from C. violaceum requires addressing several technical challenges:

• High GC content optimization:

  • Use polymerases specifically designed for high-GC templates (e.g., Q5 High-Fidelity DNA Polymerase)

  • Include additives such as DMSO (5-10%) or betaine (1-2 M) in PCR reactions

  • Design primers with balanced GC content at the 3' ends

• Effective amplification strategy:

  • For long sequences, consider dividing the gene into smaller fragments for amplification, similar to the approach used for the violacein biosynthetic cluster

  • Use touchdown PCR protocols to enhance specificity

• Vector selection:

  • For expression in E. coli, vectors like pET303/CT-His have proven successful for other C. violaceum proteins

  • For genomic library construction, vectors like pBBR1MCS-1 have been used successfully with C. violaceum DNA

• Verification methods:

  • Confirm successful cloning by restriction digestion analysis

  • Use Sanger sequencing to verify the entire sequence due to the high GC content that may lead to polymerase errors

In one documented approach with C. violaceum genes, researchers successfully constructed a genomic library by ligating 5-7 kb BamHI fragments into the BamHI site of pBBR1MCS-1, which could be adapted for thiC cloning .

How can I design effective mutagenesis approaches to study ThiC structure-function relationships?

Effective mutagenesis strategies for ThiC structure-function studies include:

• Targeted approaches based on bioinformatic analysis:

  • Focus on putative Fe-S cluster coordination sites, which are critical for function

  • Target conserved residues identified through multiple sequence alignments across species

  • Examine homology models to identify potential catalytic and substrate-binding residues

• Systematic mutagenesis protocols:

  • Alanine-scanning mutagenesis of highly conserved regions

  • Conservative substitutions to examine the importance of specific chemical properties

  • Introduction of mutations that alter sensitivity to oxidative conditions to probe Fe-S cluster function

• Technical considerations:

  • Use site-directed mutagenesis kits optimized for high-GC templates

  • Design mutagenic primers with minimal secondary structure

  • Consider using two-step PCR methods for difficult templates

• Functional validation approaches:

  • Complementation assays in thiamine auxotrophic strains

  • In vitro activity assays comparing wild-type and mutant proteins

  • Structural studies (e.g., circular dichroism) to assess protein folding integrity

When designing mutations, consider their potential impact on protein stability, cofactor binding, and substrate recognition rather than focusing solely on catalytic residues.

What genomic tools are available for studying thiC regulation in C. violaceum?

Several genomic tools can be employed to study thiC regulation in C. violaceum:

• Riboswitch analysis tools:

  • The BLISS database was developed specifically for riboswitch discovery and can identify conserved regulatory elements in 5' UTRs

  • RNA homology search methods can detect potential TPP-sensing riboswitches upstream of thiC

• Promoter analysis approaches:

  • Reporter gene fusions (lacZ, GFP) to analyze promoter activity under different conditions

  • 5' RACE to map transcription start sites and identify regulatory elements

  • ChIP-seq to identify protein factors that might interact with the thiC promoter

• Genome-wide approaches:

  • RNA-seq to examine thiC expression under varying thiamine concentrations

  • Tn-seq to identify genes that affect thiC expression

  • CRISPRi for targeted repression to study regulatory relationships

• Comparative genomics tools:

  • Analysis of the thiC genomic context across Chromobacterium species

  • Identification of conserved non-coding regions that might contain regulatory elements

The genomic context analysis is particularly important as the thiamine biosynthesis genes in C. violaceum are not organized in an operon, unlike in E. coli where tryptophan biosynthesis genes form an operon .

How can recombinant ThiC be used in metabolic engineering applications?

Recombinant ThiC presents several opportunities for metabolic engineering applications:

• Enhanced thiamine production:

  • Overexpression of optimized ThiC can overcome rate-limiting steps in thiamine biosynthesis

  • Co-expression with other pathway enzymes can establish balanced flux through the pathway

  • Integration with hexose monophosphate pathway engineering to increase precursor availability

• Pathway optimization strategies:

  • C. violaceum's incomplete hexose monophosphate pathway (HMP) may enable more efficient conversion of glucose to aromatic amino acids through erythrose-4-phosphate (E4P) generation

  • This feature could be exploited in the production of tryptophan-derived compounds, including violacein

• Engineering considerations:

  • Address iron-sulfur cluster sensitivity by introducing mutations that enhance oxidative stability

  • Optimize codon usage for expression in industrial production hosts

  • Consider fusion protein approaches to enhance stability and activity

• Potential applications:

  • Production of thiamine derivatives for nutritional supplements

  • Creation of biosensors for thiamine pathway intermediates

  • Development of screening platforms for antibiotic discovery targeting thiamine biosynthesis

When engineering ThiC-containing pathways, researchers should consider the connection between E4P availability, tryptophan biosynthesis, and downstream products like violacein, which represents a distinctive feature of C. violaceum metabolism .

What is the relationship between ThiC function and violacein biosynthesis in C. violaceum?

The relationship between ThiC function and violacein biosynthesis involves several metabolic connections:

• Precursor metabolism overlap:

  • ThiC requires aminoimidazole ribotide (AIR), which is derived from the hexose monophosphate pathway (HMP)

  • Violacein biosynthesis requires tryptophan, which shares pathway intermediates with ThiC through the shikimate pathway

  • Both pathways rely on erythrose-4-phosphate (E4P) availability

• Metabolic flux considerations:

  • The incomplete HMP in C. violaceum may redirect more E4P toward aromatic amino acid synthesis, potentially benefiting both tryptophan (violacein precursor) and AIR (ThiC substrate) production

  • Experiments have shown that HMP-defective mutants produce increased amounts of E4P, which is the limiting substrate for tryptophan biosynthesis

• Regulatory interactions:

  • While direct regulatory connections between ThiC and violacein biosynthesis have not been established, both pathways are influenced by cellular metabolic status

  • Quorum sensing systems that regulate violacein production may indirectly affect thiamine biosynthesis through global regulatory networks

Understanding these relationships could inform metabolic engineering strategies that optimize both pathways simultaneously or redirect flux between them based on desired outcomes.

How does ThiC function relate to C. violaceum pathogenicity and virulence?

The relationship between ThiC function and C. violaceum pathogenicity involves several important connections:

• Metabolic requirements during infection:

  • Thiamine pyrophosphate (TPP) is an essential cofactor for central metabolic enzymes

  • Sufficient TPP levels are necessary for bacterial survival and virulence in host environments

  • The ability to synthesize or acquire thiamine may influence pathogen fitness during infection

• Host interaction considerations:

  • While ThiC itself has not been directly implicated as a virulence factor, thiamine metabolism is crucial for pathogen survival

  • C. violaceum infections are rare but often severe, with a high fatality rate

  • The bacterium causes systemic disease with sepsis and multiple organ abscesses, processes requiring robust metabolic function

• Potential virulence connections:

  • Iron acquisition is important for pathogenicity, and the iron requirement for ThiC function (Fe-S cluster) links thiamine metabolism to iron homeostasis

  • TPP-dependent enzymes are involved in central carbon metabolism and stress responses that contribute to pathogen survival in host environments

• Therapeutic implications:

  • ThiC represents a potential antimicrobial target due to its absence in mammals

  • Inhibitors of ThiC might selectively target bacterial pathogens without affecting host metabolism

  • Understanding ThiC function could inform development of novel therapeutics for C. violaceum infections, which often show unexpected antibiotic resistance patterns

While direct evidence linking ThiC to virulence is limited, its essential role in thiamine metabolism suggests it supports the metabolic demands required for successful infection and colonization.

How do Fe-S cluster variations affect ThiC catalytic mechanism and stability?

The Fe-S cluster in ThiC plays a critical role in its function, with important implications for catalytic activity and stability:

• Structural and functional significance:

  • Research indicates that ThiC contains potential ligands for an Fe-S cluster that are required for function in vivo

  • The Fe-S cluster likely participates directly in the complex rearrangement reaction that converts AIR to HMP-P

  • Variants of ThiC with altered Fe-S coordination show increased sensitivity to oxidative growth conditions

• Mechanistic considerations:

  • The Fe-S cluster may facilitate radical-based chemistry similar to other radical SAM enzymes

  • Alterations in Fe-S coordination can change the redox potential of the cluster, affecting its catalytic properties

  • The geometry and oxidation state of the cluster likely influence substrate binding and activation

• Stability determinants:

  • Environmental factors (oxygen, reducing agents, pH) significantly impact Fe-S cluster integrity

  • The protein environment surrounding the cluster contributes to its stability

  • Post-translational modifications may affect Fe-S cluster assembly or maintenance

• Research approaches to investigate Fe-S function:

  • EPR spectroscopy to characterize Fe-S cluster type and oxidation states

  • Site-directed mutagenesis of putative Fe-S ligand residues

  • In vitro Fe-S cluster reconstitution studies

  • Anaerobic enzyme assays comparing native and reconstituted enzyme forms

This area represents an important frontier in ThiC research, as understanding Fe-S cluster dynamics could lead to enhanced enzyme stability for biotechnological applications and provide insights into ThiC evolution.

What are the latest findings on ThiC riboswitch regulation across bacterial species?

Research on ThiC riboswitch regulation has revealed several important insights across bacterial species:

• Regulatory mechanisms:

  • In E. coli, the thiC riboswitch exerts regulation at both transcriptional and translational levels

  • TPP-sensing riboswitches show phylogenetic distribution patterns and preferred expression platform mechanisms in different bacterial groups

  • The binding of TPP to the riboswitch induces conformational changes that affect gene expression

• Structural features:

  • Expanded multiple sequence alignments have revealed new consensus features, structural motifs, and base interactions in several riboswitch classes

  • The thiC riboswitch contains conserved elements that directly interact with TPP

  • Different bacterial groups show variations in riboswitch structure while maintaining the core TPP-binding domain

• Evolution and distribution:

  • ThiC riboswitches are phylogenetically widespread in bacteria

  • The BLISS database has been instrumental in discovering and defining these cis-regulatory RNA motifs

  • Comparative genomics approaches have identified thiamine-related riboswitches (THI-box structures) that recognize TPP

• Functional implications:

  • In Treponema denticola, a TPP-sensing riboswitch (Td thi-box) regulates a TPP ABC transporter operon (tbpABC) via feedback inhibition

  • The regulatory mechanism involves direct binding of TPP to the riboswitch RNA structure

  • This regulation ensures balanced TPP acquisition based on cellular needs

While specific information about ThiC riboswitch regulation in C. violaceum is limited, these findings from other bacterial species provide a framework for investigating similar regulatory mechanisms in Chromobacterium.

What cutting-edge techniques are being developed to study ThiC reaction mechanisms?

Several advanced techniques are being applied or developed to elucidate the complex reaction mechanism of ThiC:

• Time-resolved spectroscopic methods:

  • Stopped-flow spectroscopy to capture transient intermediates in the ThiC reaction

  • Rapid freeze-quench EPR to trap radical intermediates

  • Time-resolved Mössbauer spectroscopy to monitor Fe-S cluster states during catalysis

• Advanced structural biology approaches:

  • Cryo-electron microscopy to visualize ThiC in different conformational states

  • Neutron crystallography to locate hydrogen atoms important for catalysis

  • NMR studies of isotopically labeled ThiC to examine dynamics during catalysis

• Computational methods:

  • Quantum mechanics/molecular mechanics (QM/MM) simulations to model the reaction pathway

  • Machine learning approaches to predict substrate binding modes and reaction coordinates

  • Molecular dynamics simulations to understand protein conformational changes during catalysis

• Novel biochemical approaches:

  • Use of substrate analogs with specific isotopic labels to track atom rearrangements

  • Application of unnatural amino acids to probe specific residue functions

  • Single-molecule enzymology to examine ThiC catalytic heterogeneity

• Systems biology integration:

  • Multi-omics approaches to understand ThiC function in the context of cellular metabolism

  • Synthetic biology frameworks to test hypotheses about ThiC mechanism in reconstituted systems

  • Metabolic flux analysis to quantify the contribution of ThiC to cellular thiamine homeostasis

These cutting-edge approaches are beginning to unravel the extraordinary molecular gymnastics performed by ThiC in converting AIR to HMP-P, a reaction involving complex carbon skeleton rearrangement.

How does ThiC from C. violaceum compare to homologs from other bacterial species?

ThiC from C. violaceum exhibits both conserved features and distinctive characteristics when compared to homologs from other bacterial species:

What evolutionary insights can be gained from studying ThiC across the Chromobacterium genus?

Studying ThiC across the Chromobacterium genus provides valuable evolutionary insights:

• Genomic context evolution:

  • Unlike many bacterial species where thiamine biosynthesis genes are organized in operons, C. violaceum shows a non-operon organization of these genes

  • This genomic arrangement suggests unique evolutionary pressures on thiamine metabolism in Chromobacterium

  • Comparative genomics across Chromobacterium species could reveal the timing and selective pressures driving these genomic rearrangements

• Functional adaptation:

  • The incomplete hexose monophosphate pathway in C. violaceum affects erythrose-4-phosphate (E4P) production, which impacts both thiamine and aromatic amino acid biosynthesis

  • This metabolic configuration may represent an adaptation that enables more efficient channeling of glucose to aromatic amino acids (including tryptophan for violacein production)

  • The connection between primary metabolism (thiamine biosynthesis) and secondary metabolism (violacein production) suggests co-evolution of these pathways

• Environmental adaptation signatures:

  • Chromobacterium species inhabit diverse environments, from soil and water to plant-associated niches

  • Variations in ThiC sequence and regulation across species may reflect adaptation to different thiamine availability in these environments

  • Species-specific features may indicate selective pressures related to thiamine acquisition versus biosynthesis strategies

• Pathogenicity evolution:

  • Some Chromobacterium species are opportunistic pathogens with high virulence

  • ThiC function may support metabolic requirements during host infection

  • Comparative analysis of ThiC from pathogenic versus non-pathogenic Chromobacterium species could reveal adaptations related to virulence

This evolutionary perspective not only informs our understanding of ThiC function but also provides insights into the broader metabolic adaptations that have shaped the Chromobacterium genus.

What is the relationship between ThiC and the broader metabolic network in C. violaceum?

ThiC is integrated into the broader metabolic network of C. violaceum through several key connections:

• Central carbon metabolism integration:

  • ThiC activity depends on aminoimidazole ribotide (AIR), which is derived from the pentose phosphate pathway

  • The incomplete hexose monophosphate pathway (HMP) in C. violaceum affects E4P availability, linking ThiC substrate production to central carbon metabolism

  • This metabolic configuration may allow C. violaceum to convert a larger amount of glucose to aromatic amino acids than other organisms with a complete HMP

• Iron-sulfur cluster metabolism:

  • ThiC requires an Fe-S cluster for activity, connecting it to cellular iron homeostasis

  • Fe-S cluster biogenesis pathways therefore influence ThiC function

  • Studies have shown that mutants defective in iron-sulfur cluster metabolism are less efficient at synthesis of the pyrimidine moiety of thiamine

• Thiamine utilization network:

  • TPP, the product of the thiamine biosynthesis pathway involving ThiC, serves as an essential cofactor for numerous enzymes

  • These TPP-dependent enzymes function in central carbon metabolism, amino acid metabolism, and other pathways

  • The activity of these enzymes creates feedback loops that influence cellular requirements for thiamine

• Secondary metabolism connections:

  • Tryptophan biosynthesis shares precursors with the ThiC reaction through the aromatic amino acid pathway

  • Tryptophan is the precursor for violacein, the distinctive purple pigment produced by C. violaceum

  • Genetic regulation of both pathways may be coordinated to balance primary and secondary metabolism

This metabolic integration highlights the importance of ThiC beyond thiamine biosynthesis and explains why changes in ThiC activity can have broad implications for cellular physiology.