Recombinant Rhodopirellula baltica Thiamine-monophosphate kinase (thiL)

What is Thiamine-monophosphate kinase (thiL) and what role does it play in R. baltica?

Thiamine-monophosphate kinase (thiL) is an essential enzyme that catalyzes the final step in thiamine pyrophosphate (TPP) biosynthesis. The enzyme specifically phosphorylates thiamine monophosphate (TMP) to produce TPP, which serves as a critical cofactor for various pivotal cellular processes in all living organisms, including bacteria . In Rhodopirellula baltica, a marine member of the phylum Planctomycetes, thiL plays a fundamental role in both de novo TPP synthesis and potentially in thiamine salvage pathways, similar to its function in other bacterial species .

TPP is physiologically essential as it participates as a cofactor in key metabolic processes including carbohydrate, lipid, and amino acid metabolism. As microorganisms such as R. baltica can synthesize TPP via de novo biosynthetic pathways that mammals lack, thiL represents an interesting target for comparative biochemistry studies .

What expression systems are available for producing recombinant R. baltica thiL?

Recombinant R. baltica thiL can be produced using multiple expression systems, each offering distinct advantages depending on research requirements. The following expression platforms are documented for thiL production:

The biotinylated variant utilizes AviTag-BirA technology, where E. coli biotin ligase (BirA) specifically attaches biotin to the AviTag peptide, creating a covalent amide linkage between biotin and a specific lysine residue .

How does R. baltica thiL compare structurally and functionally with thiL from other bacterial species?

R. baltica thiL shows both similarities and distinct differences when compared to thiL enzymes from other bacterial species. While sharing the fundamental catalytic function of phosphorylating TMP to TPP, comparative analysis reveals important distinctions.

Unlike the extensively characterized thiL from Pseudomonas aeruginosa (PaThiL), detailed kinetic parameters for R. baltica thiL have not been comprehensively documented in the available literature. For context, PaThiL demonstrates a Vmax value of 4.0±0.2 nmol·min^-1 with KM values of 111±8 μM for ATP and 8.0±3.5 μM for TMP in a random Bi-Bi mechanism .

R. baltica belongs to a deeply branching bacterial phylum (Planctomycetes), suggesting its thiL may exhibit unique evolutionary adaptations compared to those from proteobacteria like P. aeruginosa. The structural and functional implications of these evolutionary differences remain an area for further investigation.

What are the optimal conditions for measuring R. baltica thiL enzymatic activity in vitro?

The optimal conditions for measuring R. baltica thiL enzymatic activity require careful consideration of buffer composition, pH, temperature, and substrate concentrations based on the marine environment from which R. baltica originates.

While specific optimized conditions for R. baltica thiL have not been extensively documented, insights can be drawn from related thiL enzymes. For reference, P. aeruginosa thiL activity has been successfully measured in a reaction buffer containing 50 mM Tris-HCl (pH 7.5), 5 mM MgCl2, and 350 mM KCl . Given R. baltica's marine origin, slightly higher salt concentrations may be beneficial for maintaining optimal activity.

A recommended starting protocol for enzymatic assays would include:

• Reaction buffer: 50 mM Tris-HCl (pH 7.5-8.0), 5-10 mM MgCl2, 350-500 mM KCl

• Substrate concentrations: 100-200 μM ATP and 10-50 μM TMP

• Temperature: 25-30°C (reflecting R. baltica's mesophilic nature)

• Detection methods: Either a coupled enzyme assay monitoring ADP formation or direct measurement of TPP production via HPLC

Researchers should systematically optimize these parameters to establish enzyme-specific conditions, particularly since R. baltica's deep-branching phylogenetic position may confer unique biochemical properties to its thiL enzyme.

How can substrate specificity of R. baltica thiL be determined experimentally?

Determining the substrate specificity of R. baltica thiL requires a systematic approach that examines both phosphoryl donors and acceptors. Based on methodologies applied to other thiL enzymes, the following experimental approach is recommended:

Experimental Design for Substrate Specificity Analysis:

• Phosphoryl donor specificity:

  • Test various nucleoside triphosphates (ATP, GTP, CTP, UTP) at equimolar concentrations

  • Measure initial reaction rates using a luminescent kinase assay that detects nucleoside diphosphate formation

  • Calculate relative activity compared to the preferred substrate (likely ATP)

• Phosphoryl acceptor specificity:

  • Test TMP analogs including oxythiamine monophosphate, pyrithiamine monophosphate, and other thiamine derivatives

  • Include controls: thiamine, TPP, and other non-phosphorylated thiamine analogs

  • Analyze products via HPLC or LC-MS for confirmation of phosphoryl transfer

For reference, PaThiL has demonstrated the ability to phosphorylate oxythiamine monophosphate (a TMP analog) with a KM value of 15.2±2.0 μM, while thiamine and other thiamine analogs without a monophosphate group were not phosphorylated, indicating that prior acquisition of monophosphate is a requirement for PaThiL substrates . Similar substrate constraint patterns may exist for R. baltica thiL.

What strategies can be employed to improve the solubility and stability of recombinant R. baltica thiL?

Improving solubility and stability of recombinant R. baltica thiL requires addressing multiple factors from expression to storage. The following strategies are recommended based on general principles for challenging recombinant enzymes and specific considerations for thiL proteins:

Expression Optimization:

• Lower induction temperature (16-20°C) to reduce inclusion body formation

• Reduce inducer concentration (IPTG 0.1-0.5 mM for E. coli systems)

• Co-express with molecular chaperones (GroEL/GroES, DnaK/DnaJ/GrpE)

• Consider fusion partners (MBP, SUMO, or Thioredoxin) to enhance solubility

Buffer Optimization:

• Test various buffering agents (HEPES, Tris, Sodium phosphate) at pH 7.0-8.0

• Include 5-10% glycerol as a stabilizing agent

• Add reducing agents (1-5 mM DTT or 2-10 mM β-mercaptoethanol) to prevent oxidation

• For R. baltica specifically, include higher salt concentrations (300-500 mM NaCl or KCl) to reflect its marine origin

Storage Conditions:

• Store purified enzyme at high concentration (>1 mg/ml) to prevent surface denaturation

• Add stabilizers such as trehalose or sucrose (5-10%)

• Flash-freeze in liquid nitrogen in small aliquots to prevent freeze-thaw cycles

• Consider lyophilization with appropriate cryoprotectants for long-term storage

These recommendations should be systematically tested and optimized for R. baltica thiL specifically, as the enzyme from this deep-branching marine bacterium may have unique stability requirements compared to more commonly studied bacterial enzymes.

How can inhibitor screening assays be designed for R. baltica thiL, and what are potential applications?

Designing effective inhibitor screening assays for R. baltica thiL requires consideration of assay sensitivity, throughput, and physiological relevance. While thiL from R. baltica has not been specifically targeted in published inhibitor screens, approaches used for related thiL enzymes can be adapted.

Recommended Screening Methodology:

• Primary Screening Assay:

  • A luminescent kinase assay monitoring ATP consumption during a 10-minute reaction

  • Reaction mixture: purified thiL (~5 μg), 10 μM TMP, 100 μM test compound

  • Positive control: known inhibitors of related thiL enzymes (such as WAY213613)

  • Z-factor determination to ensure assay robustness

• Secondary Confirmation Assays:

  • Direct measurement of TPP formation via HPLC

  • Orthogonal assays measuring ADP production

  • Thermal shift assays to confirm direct binding to thiL

• Inhibition Mechanism Characterization:

  • Kinetic studies varying both ATP and TMP concentrations in the presence of inhibitors

  • Determination of Ki values and inhibition types (competitive, non-competitive, uncompetitive)

For reference, inhibitor screening of P. aeruginosa thiL identified WAY213613 as a noncompetitive inhibitor with respect to TMP with a Ki value of 13.4±2.3 μM, while 5-hydroxyindolacetic acid was identified as an uncompetitive inhibitor with a Ki value of 114±27 μM .

The potential applications of R. baltica thiL inhibitors include:

• Comparative biochemistry tools to study evolutionary divergence in thiL enzymes

• Selective growth inhibitors for ecological studies of marine planctomycetes

• Starting points for developing antimicrobials against related pathogenic species

What are the critical considerations when designing expression constructs for R. baltica thiL?

Designing optimal expression constructs for R. baltica thiL requires careful consideration of several key factors to ensure proper enzyme production and activity:

Critical Design Elements:

• Codon Optimization:

  • R. baltica has a GC content of approximately 55%, which differs from common expression hosts

  • Optimize codons for the target expression system (E. coli, yeast, etc.)

  • Avoid rare codons near the 5' end of the transcript

• Start Codon Verification:

  • Verify the correct start codon through sequence alignment with known functional thiL enzymes

  • Be cautious of annotation errors as illustrated by other ThiL studies where incorrect start codons led to inactive proteins

  • For example, in P. aeruginosa thiL studies, a corrected shorter 398-amino acid enzyme had the expected activity, whereas the initial 478-amino acid sequence with 80 additional N-terminal amino acids was inactive

• Tag Selection and Placement:

  • Consider the impact of tags on enzyme activity and substrate binding

  • N-terminal tags are generally preferred as the C-terminus may be involved in substrate binding

  • Include TEV or PreScission protease cleavage sites for tag removal if necessary

  • For structural studies, consider His6, GST, or MBP fusion tags

• Promoter Selection:

  • For E. coli: T7 promoter for high expression or araBAD for tighter regulation

  • For yeast: GAL1 promoter for inducible expression

  • For mammalian systems: CMV or EF1α promoters

• Secretion Signals:

  • R. baltica thiL is naturally cytoplasmic, so secretion signals are generally unnecessary

  • For certain applications, consider testing SEC or PelB signal sequences for periplasmic expression in E. coli

These considerations are essential for avoiding the expression of non-functional protein variants and ensuring high yields of active enzyme.

How can isothermal titration calorimetry (ITC) be optimized for studying substrate binding kinetics of R. baltica thiL?

Isothermal Titration Calorimetry (ITC) provides direct thermodynamic information about substrate binding and can be optimized for R. baltica thiL using the following methodology:

ITC Experimental Design for R. baltica thiL:

• Sample Preparation:

  • Purify thiL to >95% homogeneity via size exclusion chromatography

  • Dialyze protein and substrates extensively against identical buffer to minimize buffer mismatch effects

  • Recommended buffer: 50 mM HEPES pH 7.5, 350 mM KCl, 5 mM MgCl2

  • Degas all solutions prior to experiments to prevent bubble formation

• Experimental Parameters:

  • Cell concentration: 10-20 μM thiL

  • Syringe concentration: 200-400 μM substrate (TMP or ATP)

  • Temperature: 25°C (standard for mesophilic enzymes)

  • Reference power: 10 μcal/sec

  • Injection schedule: 25-30 injections of 1.5-2 μL each

  • Spacing between injections: 180-240 seconds

• Experimental Strategy:

  • For the two-substrate system (ATP and TMP), perform separate titrations for each substrate

  • For ATP binding studies, pre-incubate thiL with saturating TMP (100 μM)

  • For TMP binding studies, pre-incubate thiL with AMP-PNP (non-hydrolyzable ATP analog)

  • Run control experiments: substrate into buffer and buffer into protein

• Data Analysis:

  • Fit the integrated heat data to appropriate binding models (one-set, two-set, or sequential binding sites)

  • Determine binding stoichiometry (n), association constant (Ka), enthalpy (ΔH), and entropy (ΔS)

  • Calculate Gibbs free energy (ΔG) using the equation: ΔG = ΔH - TΔS

• Expected Results Interpretation:

  • Based on similar enzymes, TMP binding is likely to have higher affinity (lower KD) than ATP

  • The binding process may be enthalpically driven (negative ΔH) due to hydrogen bonding and electrostatic interactions

  • Conformational changes upon substrate binding may contribute to entropy changes

This optimized ITC approach should provide valuable insights into the thermodynamics of substrate binding for R. baltica thiL, which can complement kinetic studies and inform structural biology efforts.

What approaches can be used to investigate potential protein-protein interactions involving R. baltica thiL in its native cellular context?

Investigating protein-protein interactions (PPIs) involving R. baltica thiL requires a multi-faceted approach that considers the unique challenges of working with this marine planctomycete. The following methodologies are recommended:

In Vivo Approaches:

• Bacterial Two-Hybrid System:

  • Adapt the bacterial adenylate cyclase two-hybrid system for R. baltica

  • Clone thiL into bait vectors and create a prey library from R. baltica genomic DNA

  • Screen for interactions on selective media containing X-gal

  • Validate positive interactions through reciprocal testing

• Proximity-Dependent Biotinylation:

  • Express thiL fused to BioID2 or TurboID in R. baltica

  • Induce biotinylation of proximal proteins

  • Purify biotinylated proteins via streptavidin and identify by mass spectrometry

  • This approach is particularly useful for detecting transient interactions

• Co-Immunoprecipitation with Native Antibodies:

  • Generate specific antibodies against R. baltica thiL

  • Perform immunoprecipitation from R. baltica cell lysates

  • Identify co-precipitating proteins via LC-MS/MS

  • Validate interactions using reciprocal co-IP

In Vitro Validation Approaches:

• Surface Plasmon Resonance (SPR):

  • Immobilize purified thiL on a sensor chip using the biotinylated version

  • Flow candidate interacting proteins and measure binding kinetics

  • Determine KD values for specific interactions

• Microscale Thermophoresis (MST):

  • Label thiL with a fluorescent dye

  • Titrate potential interacting partners

  • Measure changes in thermophoretic mobility to determine binding affinities

• Size Exclusion Chromatography with Multi-Angle Light Scattering (SEC-MALS):

  • Analyze thiL alone and in combination with potential binding partners

  • Determine absolute molecular weights to confirm complex formation

  • This method is particularly useful for stable complexes

Bioinformatic Prediction:

• Genomic Context Analysis:

  • Examine gene neighborhoods around thiL in R. baltica

  • Identify conserved gene clusters across related species

  • Based on search results, thiL may be functionally related to metabolic enzymes involved in thiamine metabolism

• Co-expression Network Analysis:

  • Analyze transcriptomic data to identify genes co-regulated with thiL

  • Construct correlation networks to predict functional associations

This comprehensive approach would provide valuable insights into the functional context of thiL within the unique cellular environment of R. baltica.

How can structural studies of R. baltica thiL contribute to understanding the evolution of thiamine metabolism in deep-branching bacteria?

Structural studies of R. baltica thiL can provide significant insights into the evolution of thiamine metabolism, especially given the deep-branching phylogenetic position of Planctomycetes. The following approach would maximize these evolutionary insights:

Structural Biology Approach:

• Comparative Structural Analysis:

  • Determine the crystal structure of R. baltica thiL at high resolution (<2.0 Å)

  • Compare with existing thiL structures from diverse bacterial phyla

  • Identify conserved catalytic residues versus lineage-specific structural elements

  • Map sequence conservation onto structural elements to distinguish functional constraints from evolutionary drift

• Evolutionary Structure-Function Relationships:

  • Analyze the active site architecture across thiL orthologs

  • Identify structural adaptations that might reflect R. baltica's marine environment

  • Compare substrate binding pockets to understand specificity differences

  • Examine oligomerization interfaces for evolutionary conservation

• Ancestral Sequence Reconstruction:

  • Use thiL sequences from diverse bacteria to reconstruct ancestral sequences

  • Express and characterize reconstructed ancestral thiL enzymes

  • Compare kinetic parameters of ancestral and modern thiL variants

  • This approach can reveal the evolutionary trajectory of thiL function

Expected outcomes would include identification of:

• Core structural elements conserved across all thiL enzymes, representing ancestral features

• Planctomycete-specific structural adaptations that may reflect their unique cellular organization

• Potential co-evolution patterns with other enzymes in the thiamine biosynthesis pathway

• Structural basis for substrate specificity differences between R. baltica thiL and other bacterial thiL enzymes

These structural insights would contribute significantly to understanding how essential metabolic pathways have evolved in deep-branching bacterial lineages, potentially revealing ancient features of thiamine metabolism.

What is the potential for using R. baltica thiL as a model system for studying enzyme adaptation to marine environments?

R. baltica thiL offers a valuable model system for studying enzyme adaptation to marine environments, particularly given the unique ecological niche of Planctomycetes. Several research avenues can exploit this potential:

Marine Adaptation Research Framework:

The results from these studies would contribute to our understanding of how essential metabolic enzymes adapt to marine environments while maintaining their critical cellular functions. The insights gained could have broader implications for understanding marine microbial ecology and evolution.