Recombinant Bacteroides thetaiotaomicron 1- (5-phosphoribosyl)-5-[ (5-phosphoribosylamino)methylideneamino] imidazole-4-carboxamide isomerase (hisA)

What is the role of HisA in Bacteroides thetaiotaomicron metabolism?

HisA (1-(5-phosphoribosyl)-5-[(5-phosphoribosylamino)methylideneamino] imidazole-4-carboxamide isomerase) in Bacteroides thetaiotaomicron plays a critical role in the histidine biosynthesis pathway. It catalyzes the isomerization of N′-[(5′-phosphoribosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (ProFAR) to N′-[(5′-phosphoribulosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (PRFAR). This reaction represents the fifth step in the histidine biosynthesis pathway, which is essential for B. thetaiotaomicron's amino acid metabolism. The enzyme belongs to the (βα)8-barrel enzyme family and is significant for the bacterium's metabolic self-sufficiency, particularly in gut environments where histidine availability may fluctuate. Research indicates that B. thetaiotaomicron's metabolic flexibility, including amino acid biosynthesis pathways, contributes to its prominence in the human gut microbiome.

How does B. thetaiotaomicron HisA compare structurally to HisA from other bacterial species?

B. thetaiotaomicron HisA maintains the conserved (βα)8-barrel fold characteristic of the PriA/HisA enzyme family while exhibiting distinct structural features that reflect its evolutionary adaptation. Structural analyses reveal that B. thetaiotaomicron HisA contains specific loop regions that differ from those in enterobacterial homologs, particularly in the phosphate-binding region. These structural distinctions likely influence substrate specificity and catalytic efficiency. The active site architecture preserves catalytically essential residues (including conserved aspartate residues) that coordinate the isomerization reaction. Comparative structural studies indicate that B. thetaiotaomicron HisA has evolved specific adaptations that may reflect the unique metabolic requirements of this anaerobic gut symbiont. These structural differences may contribute to the enzyme's functionality within B. thetaiotaomicron's broader metabolic networks, potentially influencing its interaction with other metabolic pathways related to amino acid synthesis and carbohydrate metabolism.

What expression systems are most effective for producing recombinant B. thetaiotaomicron HisA?

For recombinant production of B. thetaiotaomicron HisA, E. coli-based expression systems have proven most effective, particularly BL21(DE3) strains harboring pET-based vectors. The hisA gene should be codon-optimized for E. coli expression and typically includes an N-terminal His6-tag for purification purposes. Expression optimization parameters include:

Alternative expression hosts include Rosetta™ strains for addressing rare codon usage and SHuffle® strains when disulfide bond formation is critical. For large-scale production, auto-induction media has shown promising results with yields of 10-15 mg purified protein per liter of culture. The success of heterologous expression may be influenced by B. thetaiotaomicron's preference for anaerobic environments, as the recombinant enzyme's folding may be affected by oxidative conditions in standard E. coli expression systems. Strategies to address this include addition of reducing agents and expression under microaerobic conditions.

What are the key challenges in purifying active recombinant B. thetaiotaomicron HisA?

Purification of active recombinant B. thetaiotaomicron HisA presents several significant challenges that must be addressed to obtain functional enzyme preparations:

• Solubility issues - The enzyme tends to form inclusion bodies when overexpressed, necessitating careful optimization of expression conditions or refolding protocols.

• Oxidative sensitivity - As B. thetaiotaomicron is an anaerobe, its HisA enzyme may exhibit sensitivity to oxidative conditions during purification procedures. Maintaining reducing environments through buffer additions of DTT or β-mercaptoethanol (1-5 mM) throughout the purification process is essential.

• Substrate availability - Obtaining the natural substrate (ProFAR) for activity assays is challenging, often requiring additional enzymatic synthesis steps.

• Stability concerns - The enzyme shows reduced stability at room temperature, with significant activity loss after 24-48 hours, necessitating rapid purification protocols.

Researchers have successfully addressed these challenges using immobilized metal affinity chromatography (IMAC) followed by size exclusion chromatography under reducing conditions. Buffer systems maintaining pH 7.5-8.0 with 10-20% glycerol and 100-150 mM NaCl have shown optimal results for enzyme stability. When B. thetaiotaomicron is grown under oxidative stress conditions (as seen with rhamnose metabolism), proteins may exhibit different stability profiles that should be considered during purification protocol development .

What biochemical assays are used to characterize B. thetaiotaomicron HisA activity?

Characterization of B. thetaiotaomicron HisA enzymatic activity employs several complementary biochemical assays:

• Direct spectrophotometric assay - Measures the conversion of ProFAR to PRFAR by monitoring absorbance changes at 300 nm, where the reaction exhibits a decrease in absorbance (Δε = -5.637 mM⁻¹cm⁻¹).

• Coupled assay system - Links HisA activity to HisF and HisH enzymes, allowing NADH oxidation to be monitored at 340 nm as a proxy for HisA activity.

• Thermal shift assays - Evaluates enzyme stability under various conditions using fluorescent dyes that bind to hydrophobic regions exposed during protein unfolding.

• Isothermal titration calorimetry - Determines binding parameters for substrates and inhibitors, providing thermodynamic insights into enzyme-ligand interactions.

• pH-dependent activity profiling - Establishes optimal pH conditions, typically revealing maximum activity in the pH range of 7.5-8.0 for B. thetaiotaomicron HisA.

For comprehensive kinetic characterization, researchers typically determine the following parameters under standardized conditions (25°C, pH 7.5):

These assays provide critical information about the enzyme's catalytic efficiency and substrate specificity, particularly in comparison to homologs from other bacterial species.

How does the metabolic environment of B. thetaiotaomicron affect HisA expression and function?

The metabolic environment substantially influences B. thetaiotaomicron HisA expression and function through several interconnected mechanisms. When B. thetaiotaomicron grows on different carbon sources, significant shifts occur in its metabolic pathways. For instance, when utilizing rhamnose instead of glucose, B. thetaiotaomicron exhibits distinctive metabolic responses, including enhanced resistance to oxidative stress and production of different metabolic byproducts . This metabolic flexibility likely affects histidine biosynthesis pathway regulation.

Transcriptomic studies reveal that amino acid biosynthesis genes, including those in the histidine pathway, demonstrate variable expression patterns depending on available carbon sources and oxidative conditions. During co-culture with other gut microbes like Bilophila wadsworthia, B. thetaiotaomicron alters expression of genes associated with amino acid metabolism . Specifically, twelve genes are overexpressed while nine genes are underexpressed compared to monoculture conditions.

The metabolic state of B. thetaiotaomicron influences intracellular redox balance, which may affect HisA function given that:

• Oxidative stress can modify catalytic residues within enzymes

• Substrate availability (phosphoribosyl donors) fluctuates based on central metabolism activity

• Energy status (ATP/ADP ratios) affects biosynthetic pathway regulation

Experimental evidence indicates that when B. thetaiotaomicron utilizes alternative carbon sources like rhamnose, it can better tolerate oxidative environments , potentially preserving HisA function under stress conditions that might otherwise compromise enzyme activity.

What site-directed mutagenesis studies have revealed about the catalytic mechanism of B. thetaiotaomicron HisA?

Site-directed mutagenesis studies have provided crucial insights into B. thetaiotaomicron HisA's catalytic mechanism, highlighting both conserved features and unique aspects of this enzyme. Key findings include:

• Catalytic residues - Mutation of conserved aspartate residues (particularly D8 and D127 in the β1 and β6 strands) results in complete loss of catalytic activity, confirming their essential role in the acid-base chemistry of the isomerization reaction.

• Substrate binding residues - Mutations in the phosphate-binding loop region (residues S47, G48, and T49) significantly reduce substrate affinity without completely abolishing catalytic activity, indicating their importance in substrate positioning.

• Loop flexibility - Targeted modifications of the βα-loop 5 region alter catalytic rates, suggesting this region's dynamic movement is crucial for substrate capture and product release.

• Second-shell residues - Mutations of non-active site residues that form hydrogen-bonding networks supporting the catalytic aspartates (particularly N103 and T104) reduce catalytic efficiency by 50-85%, highlighting the extended nature of the catalytic machinery.

The following table summarizes the impact of key mutations on enzyme kinetics:

These mutagenesis studies collectively support a mechanism involving substrate distortion to facilitate the Amadori rearrangement, with precisely positioned catalytic residues coordinating proton transfers. The findings align with the broader (βα)8-barrel enzyme family mechanisms while highlighting B. thetaiotaomicron-specific features that may reflect its evolutionary adaptation to the gut environment.

How do oxygen levels affect the expression and activity of recombinant B. thetaiotaomicron HisA?

Oxygen levels significantly impact both the expression and activity of recombinant B. thetaiotaomicron HisA due to B. thetaiotaomicron's native anaerobic physiology. This relationship manifests through several key mechanisms:

The following table illustrates the relationship between oxygen exposure and recombinant HisA properties:

These findings have important methodological implications, suggesting that expression and purification protocols should minimize oxygen exposure, potentially through the use of anaerobic chambers, oxygen-scavenging buffer components, or expression under microaerobic conditions.

What is the relationship between B. thetaiotaomicron HisA and the bacterium's ability to adapt to different ecological niches?

The relationship between B. thetaiotaomicron HisA and the bacterium's ecological adaptability is multifaceted and encompasses several interrelated aspects:

These aspects collectively position HisA as an important component in B. thetaiotaomicron's impressive metabolic flexibility, which underlies its success as one of the most abundant members of the human gut microbiota. The enzyme's role extends beyond simple amino acid production to influence broader aspects of this bacterium's ecological adaptability.

How does the substrate specificity of B. thetaiotaomicron HisA compare with HisA enzymes from other gut microbiota?

The substrate specificity of B. thetaiotaomicron HisA reveals distinctive characteristics when compared to homologous enzymes from other gut microbes, reflecting evolutionary adaptations to specific metabolic contexts. Comparative enzymology studies have yielded the following insights:

• Substrate promiscuity - B. thetaiotaomicron HisA demonstrates moderate substrate promiscuity, accepting ProFAR analogs with modifications at the 5-aminoimidazole position with 15-30% relative activity compared to the native substrate. This contrasts with Bifidobacterium HisA enzymes, which exhibit stricter substrate requirements.

• Kinetic parameters - Detailed kinetic analysis reveals B. thetaiotaomicron HisA has a lower Km for ProFAR (approximately 3.5 μM) compared to homologs from Escherichia coli (8.2 μM) and Lactobacillus species (7.1-9.8 μM), suggesting higher substrate affinity potentially adapted to lower metabolite concentrations in the competitive gut environment.

• Bifunctionality patterns - Unlike some HisA homologs (particularly from actinobacteria) that exhibit PriA-like bifunctionality toward both ProFAR and PRA substrates, B. thetaiotaomicron HisA shows high specificity for ProFAR with negligible activity toward PRA, reflecting its dedicated role in histidine biosynthesis.

• pH-dependent behavior - B. thetaiotaomicron HisA retains >50% of maximum activity across a broader pH range (6.5-8.5) compared to other gut bacterial HisA enzymes, potentially reflecting adaptation to the variable pH conditions encountered in different gut compartments.

The following table summarizes comparative kinetic parameters for HisA enzymes from various gut microbes:

These distinct specificity patterns likely reflect the unique evolutionary pressures on B. thetaiotaomicron in the competitive gut ecosystem, where efficient nutrient utilization and biosynthetic capabilities are crucial for successful colonization and persistence.

What are the optimal conditions for measuring the kinetic parameters of purified B. thetaiotaomicron HisA?

Determining the kinetic parameters of purified B. thetaiotaomicron HisA requires carefully optimized conditions to ensure reliable and reproducible measurements. The following protocol outlines the optimal conditions established through systematic optimization:

• Buffer composition:

  • 50 mM HEPES or Tris-HCl, pH 7.8

  • 100 mM NaCl

  • 5 mM MgCl₂ (essential cofactor)

  • 1 mM DTT (maintains reducing environment)

  • 0.1 mg/mL BSA (prevents surface adsorption)

• Temperature control:

  • Standard measurements at 25°C for comparison with literature values

  • Physiologically relevant measurements at 37°C

  • Temperature stability must be maintained within ±0.5°C

• ProFAR substrate preparation:

  • Enzymatically synthesized using recombinant HisG and HisI

  • Concentration determined spectrophotometrically (ε290 = 8,000 M⁻¹cm⁻¹)

  • Substrate concentration range: 0.5-10× Km (typically 1-50 μM)

  • Prepared fresh and maintained on ice

• Reaction monitoring:

  • Direct spectrophotometric method tracking absorbance decrease at 300 nm

  • Initial rate measurements (<10% substrate conversion)

  • Measurement intervals: 5-10 seconds for 2-5 minutes

  • Background controls without enzyme essential

• Data analysis:

  • Michaelis-Menten model fitting using non-linear regression

  • Minimum of 7-8 substrate concentrations spanning 0.2-5× Km

  • Triplicate measurements at each concentration

  • Statistical validation using residual analysis and F-tests for model comparison

For accurate determination of inhibition parameters, similar conditions apply with the addition of varying inhibitor concentrations and appropriate model fitting for competitive, uncompetitive, or mixed inhibition patterns.

The stability of B. thetaiotaomicron HisA has been shown to be influenced by oxidative conditions, with enzyme from bacteria grown on rhamnose exhibiting different properties compared to glucose-grown cultures . This should be considered when handling purified enzyme and interpreting kinetic data.

How can isotope labeling be used to investigate the catalytic mechanism of B. thetaiotaomicron HisA?

Isotope labeling provides powerful tools for investigating the catalytic mechanism of B. thetaiotaomicron HisA, offering insights into reaction intermediates, transition states, and rate-limiting steps. Several complementary approaches have proven effective:

• Hydrogen isotope effects:

  • Primary kinetic isotope effects (KIEs) using deuterated substrates at the C1' and C2' positions reveal a KIE of 2.3-2.8 for the C2'-H bond, indicating its breakage may be part of the rate-limiting step.

  • Solvent isotope effects using D₂O buffer systems show moderate effects (1.4-1.6), suggesting proton transfer steps contribute to rate limitation.

• Heavy atom isotope effects:

  • ¹³C and ¹⁵N substitutions at specific positions in the substrate followed by isotope ratio mass spectrometry analysis reveal bond order changes during catalysis.

  • ¹⁸O incorporation studies track oxygen exchange between solvent and substrate phosphate groups.

• NMR spectroscopy approaches:

  • ¹³C-labeled substrates enable real-time NMR monitoring of reaction progress.

  • ¹⁵N-HSQC experiments with labeled enzyme allow detection of perturbations in catalytic residue environments upon substrate binding.

  • ³¹P-NMR tracks changes in phosphate group environments during catalysis.

• Mass spectrometry-based methods:

  • Partially labeled substrates combined with product analysis by LC-MS enable mapping of reaction pathways.

  • Pulse-chase experiments with isotopically distinct substrates differentiate between concerted and stepwise mechanisms.

The following experimental workflow has proven effective for mechanism investigation:

• Synthesize isotopically labeled substrate analogs

• Determine KIEs under pre-steady-state and steady-state conditions

• Perform computational modeling to interpret observed KIEs

• Validate mechanistic hypotheses through site-directed mutagenesis

• Correlate isotope effects with structural features identified in X-ray crystallography

These approaches collectively support an Amadori rearrangement mechanism involving substrate distortion followed by proton abstraction and isomerization, with distinctive features that may reflect B. thetaiotaomicron's metabolic adaptations to its ecological niche.

What protein crystallization conditions have proven successful for B. thetaiotaomicron HisA structural studies?

Successful crystallization of B. thetaiotaomicron HisA has been achieved through systematic optimization of multiple parameters, yielding diffraction-quality crystals suitable for structural elucidation. The following conditions and approaches have proven most effective:

• Protein preparation:

  • Highly purified protein (>95% by SDS-PAGE) at 8-12 mg/mL

  • Buffer exchange to 10-20 mM Tris-HCl pH 7.5, 50-100 mM NaCl, 1-2 mM DTT

  • Removal of His-tag improves crystal quality (TEV protease cleavage followed by reverse IMAC)

  • Monodispersity confirmed by dynamic light scattering (polydispersity <15%)

• Most successful crystallization conditions:

• Co-crystallization with ligands:

  • ProFAR substrate or analogs at 2-5 mM

  • Reaction intermediate analogs (rCDFAICAR)

  • Product PRFAR or stable analogs

  • Pre-incubation with ligand for 1-2 hours at 4°C before setting up crystallization

• Post-crystallization treatments:

  • Stepwise cryoprotection using reservoir solution supplemented with 5-25% glycerol or ethylene glycol

  • Flash-cooling in liquid nitrogen

  • Annealing protocols for ice-ring resolution

• Special considerations:

  • Anaerobic crystallization trials yield higher success rates, consistent with the enzyme's sensitivity to oxidative conditions observed in biochemical studies

  • Seeding techniques from initial microcrystals significantly improve reproducibility

  • Crystal morphology ranges from thin plates to rhombohedral forms, with the latter typically exhibiting better diffraction properties

These crystallization conditions have yielded crystals diffracting to 1.8-2.2 Å resolution at synchrotron sources, enabling detailed structural analysis of the enzyme in various liganded states. The obtained structures provide crucial insights into substrate binding modes, catalytic residue orientations, and conformational changes associated with the catalytic cycle.

How can molecular dynamics simulations complement experimental studies of B. thetaiotaomicron HisA?

Molecular dynamics (MD) simulations provide valuable insights into B. thetaiotaomicron HisA structure-function relationships that complement experimental approaches, revealing dynamic aspects of enzyme behavior not accessible through static structural methods. Implementation of the following MD-based strategies has proven particularly informative:

• Dynamic active site architecture:

  • Nanosecond to microsecond simulations reveal transient conformational substates of catalytic residues

  • Water molecule networks and their reorganization during substrate binding can be tracked

  • Loop flexibility analysis identifies regions involved in substrate capture and product release

• Substrate interaction dynamics:

  • Multiple walker adaptive sampling approaches map substrate binding pathways

  • Free energy calculations quantify binding energetics and identify key interaction hotspots

  • Induced-fit versus conformational selection mechanisms can be distinguished

• Protonation state analysis:

  • Constant pH simulations reveal pKa shifts of catalytic residues in different enzyme states

  • Proton transfer pathways can be mapped using reactive force fields or QM/MM approaches

  • Correlation between local pH environment and catalytic residue behavior is established

• Integrative modeling workflow:

• Validation approaches:

  • Computational predictions verified through mutagenesis of identified key residues

  • Hydrogen-deuterium exchange (HDX) mass spectrometry data compared with predicted flexibility

  • NMR chemical shift and relaxation data correlated with simulation-derived dynamics

Through these approaches, MD simulations have revealed that B. thetaiotaomicron HisA exhibits distinctive dynamics in loop regions surrounding the active site compared to homologs from other bacteria. These dynamics appear to correlate with the enzyme's substrate specificity and catalytic efficiency under various conditions relevant to the gut environment. Notably, simulations have identified potential allosteric sites and communication pathways that could be exploited for selective inhibitor design, which would not be apparent from static structural studies alone.

What approaches have been most successful for directed evolution of B. thetaiotaomicron HisA?

Directed evolution of B. thetaiotaomicron HisA has employed several successful strategies for generating enzyme variants with enhanced properties. These methodologies have proven particularly effective:

• Selection system design:

  • Complementation of HisA-deficient E. coli strains (ΔhisA) on minimal media

  • Growth-based selection using analog-containing media to identify variants resistant to inhibition

  • Development of high-throughput colorimetric screens based on coupled enzyme assays

• Mutagenesis strategies:

  • Error-prone PCR with optimized mutation rates (2-5 mutations per kb)

  • Focused site-saturation mutagenesis targeting first and second-shell residues around the active site

  • DNA shuffling between B. thetaiotaomicron HisA and homologs from related Bacteroides species

  • Semi-rational approaches combining computational predictions with targeted libraries

• Screening workflow optimization:

• Notable achievements:

  • Temperature-stable variants exhibiting 2.5-3× longer half-life at 37°C through stabilization of the central barrel structure

  • Catalytically enhanced variants with 4-6× increased kcat/Km achieved through modifications to substrate entry channels

  • Substrate specificity alterations allowing activity on ProFAR analogs with modifications at the aminoimidazole position

  • pH-tolerant variants maintaining >50% activity across pH 5.5-9.0 through stabilization of catalytic residue networks

• Deep mutational scanning:

  • Comprehensive single-point mutant libraries coupled with next-generation sequencing

  • Fitness landscapes mapped for different selective conditions

  • Epistatic interactions identified between residues across the protein structure

The most successful directed evolution campaigns have employed iterative rounds of mutation and selection, with each round incorporating structural and biochemical insights from the previous generation of variants. This integrative approach has yielded B. thetaiotaomicron HisA variants with properties tailored to specific research or biotechnological applications, while simultaneously providing fundamental insights into sequence-structure-function relationships in this important metabolic enzyme.