Recombinant Bradyrhizobium japonicum Histidinol-phosphate aminotransferase 2 (hisC2)

What is the basic function of histidinol-phosphate aminotransferase in B. japonicum?

Histidinol-phosphate aminotransferase (HspAT) in Bradyrhizobium japonicum functions primarily in the histidine biosynthesis pathway by catalyzing the conversion of histidinol phosphate to histidinol. As part of the subfamily Iβ aminotransferases, HspAT specifically recognizes amino-organo phosphates, particularly histidinol phosphate (Hsp) . Similar to other PLP (pyridoxal 5'-phosphate)-dependent enzymes, it exhibits a characteristic ping-pong bi-bi mechanism where the first half of the reaction involves conversion of PLP into pyridoxamine 5'-phosphate (PMP) . This enzymatic activity is critical for bacterial metabolism and ultimately supports the nitrogen-fixing capabilities of B. japonicum.

How does the structure of HspAT in B. japonicum differ from other aminotransferases?

Structurally, B. japonicum HspAT belongs to the aminotransferase subfamily Iβ, which shares structural similarities with aromatic amino acid aminotransferases (ArATs) but possesses distinct substrate specificities . The key structural differences lie in the residues lining the substrate binding pocket and the N-terminal lid, which are the primary determinants of substrate specificity . In HspAT, hydrophilic residues in the substrate binding pocket and N-terminal lid facilitate the entry and binding of its preferential substrate, histidinol phosphate (Hsp) . This differs from the hydrophobic nature of both the substrate binding pocket and N-terminal lid found in ArATs, which prevents binding of polar substrates like Hsp while facilitating binding of aromatic residues such as phenylalanine, tyrosine, and tryptophan .

What are the recommended methods for expressing recombinant B. japonicum hisC2?

Based on established protocols for similar B. japonicum proteins, recombinant hisC2 can be effectively expressed in Escherichia coli expression systems. Drawing from the methodology used for B. japonicum FixK2 protein, the expression can be optimized using N-terminally histidine-tagged constructs . The gene should be cloned into an appropriate expression vector under the control of an inducible promoter. For optimal expression, cultures should be grown to mid-logarithmic phase before induction, with special attention to temperature control as B. japonicum proteins may form inclusion bodies at higher temperatures. Post-induction growth conditions typically require optimization of temperature (often reduced to 25-30°C), duration (4-24 hours), and inducer concentration to balance yield with proper protein folding .

What purification strategies yield the highest purity and activity for recombinant B. japonicum hisC2?

Purification of recombinant B. japonicum hisC2 can be accomplished efficiently using metal chelate affinity chromatography (MCAC), particularly Ni²⁺-charged columns if a histidine tag is incorporated . Based on purification strategies for other B. japonicum proteins, a single-step MCAC purification can yield highly pure protein . For enhanced purity, additional chromatographic steps such as ion-exchange chromatography or size-exclusion chromatography may be employed. During purification, it's essential to maintain reducing conditions (typically using DTT or β-mercaptoethanol) to prevent disulfide bond formation and protein aggregation. Additionally, including PLP in purification buffers may help maintain the enzyme's active conformation. Activity assessments should be performed immediately after purification as prolonged storage might affect enzymatic function .

How can researchers determine the specific binding site residues that confer substrate specificity to B. japonicum hisC2?

To determine the specific binding site residues conferring substrate specificity to B. japonicum hisC2, researchers should employ a combined structural and mutagenesis approach. The substrate binding pocket and N-terminal lid regions should be the primary focus, as these regions determine substrate specificity in aminotransferases . Site-directed mutagenesis of residues lining the substrate binding pocket can systematically transform hydrophilic residues into hydrophobic ones (and vice versa) to assess changes in substrate specificity. The mutant proteins should be analyzed through enzyme kinetics studies comparing binding affinity (Km) and catalytic efficiency (kcat/Km) toward histidinol phosphate and other potential substrates.

For structural analysis, X-ray crystallography of the enzyme in complex with its substrate or substrate analogues can provide direct visualization of binding interactions. Computational approaches such as molecular docking and molecular dynamics simulations can complement experimental data by predicting binding modes and energetics. Additionally, hydrogen-deuterium exchange mass spectrometry (HDX-MS) can identify regions that undergo conformational changes upon substrate binding, further elucidating the mechanism of substrate recognition .

What techniques can reveal the ligand-induced conformational changes in B. japonicum hisC2?

To investigate ligand-induced conformational changes in B. japonicum hisC2, researchers should employ multiple biophysical techniques. X-ray crystallography provides the most detailed structural information, especially when structures are determined in both apo and ligand-bound states . Cryo-electron microscopy (cryo-EM) offers an alternative approach for structural determination, particularly valuable if crystallization proves challenging.

Solution-based techniques provide complementary information about protein dynamics. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) can identify regions that become more or less solvent-accessible upon ligand binding. Fluorescence spectroscopy, particularly if intrinsic tryptophan residues are near the binding site, can detect conformational changes through alterations in the local environment. Circular dichroism (CD) spectroscopy monitors changes in secondary structure elements upon ligand binding.

For more detailed dynamics assessment, nuclear magnetic resonance (NMR) spectroscopy can track specific residue movements in solution, though this may be challenging for larger proteins. Small-angle X-ray scattering (SAXS) provides information about global conformational changes and can be particularly useful for detecting domain movements. These methodologies collectively provide a comprehensive view of the plasticity of aminotransferases and their adaptations to different ligands .

How does genetic variation in the hisC2 gene affect B. japonicum's symbiotic efficiency with legumes?

To investigate the relationship between hisC2 genetic variations and symbiotic efficiency, researchers should employ a systematic approach combining molecular genetics and physiological assessments. First, naturally occurring hisC2 variants should be identified through sequencing of diverse B. japonicum isolates from different field sites . These variants can be classified based on sequence polymorphisms, particularly those affecting the substrate binding pocket and catalytic residues.

Knockout and complementation studies are essential to establish the direct role of hisC2 in symbiosis. This involves creating hisC2 deletion mutants and complementing them with different hisC2 variants to assess functional restoration. For field applications, controlled experiments can be designed similar to the methodology used in EPA-approved field trials for B. japonicum strains (0.25-0.5 acre plots) . In these trials, soybean seeds should be inoculated with different B. japonicum strains carrying hisC2 variants, and multiple parameters should be measured:

Statistical analyses should account for environmental variables and field site differences to isolate the specific effects of hisC2 variations on symbiotic performance .

What is the relationship between hisC2 expression and the highly reiterated sequence (HRS) phenomenon in B. japonicum?

The relationship between hisC2 expression and the highly reiterated sequence (HRS) phenomenon in B. japonicum represents an intriguing research question at the intersection of genomic architecture and metabolic function. HRS isolates of B. japonicum contain numerous copies of repeated sequences (RSα and RSβ), with some isolates possessing extremely high numbers (86-175 copies) of RSα . These repeated sequences are often clustered around regions containing nitrogen-fixation and nodulation genes .

To investigate potential relationships, researchers should first determine whether the hisC2 gene is located near these RS clusters by genome mapping. If proximity exists, the next step would be to quantify hisC2 expression levels in normal versus HRS isolates using RT-qPCR or RNA-Seq. Since HRS isolates exhibit slower growth than normal isolates , metabolic profiling using liquid chromatography-mass spectrometry (LC-MS) could reveal whether histidine biosynthesis is affected.

The genomic rearrangements in HRS isolates may also impact gene regulation. Chromatin immunoprecipitation sequencing (ChIP-seq) could identify any altered transcription factor binding patterns at the hisC2 promoter in HRS versus normal isolates. Additionally, the tandem repeat RSα dimer structure found in some HRS isolates, similar to the (IS30)2 structure that causes transpositional rearrangements in E. coli , might affect hisC2 expression if transposition events occur near the gene. Long-read sequencing technologies would be valuable for characterizing these complex genomic arrangements and their potential impact on hisC2 expression.

How does B. japonicum hisC2 interact with other enzymes in the histidine biosynthesis pathway?

Investigating protein-protein interactions of hisC2 with other enzymes in the histidine biosynthesis pathway requires multiple complementary approaches. Pull-down assays using tagged recombinant hisC2 can identify interaction partners from B. japonicum cell lysates, followed by mass spectrometry identification. For validation, techniques such as bimolecular fluorescence complementation (BiFC) or Förster resonance energy transfer (FRET) can confirm these interactions in vivo.

To characterize metabolic channeling (the direct transfer of intermediates between enzymes), researchers should analyze the kinetics of coupled enzymatic reactions with purified proteins. This would involve comparing the efficiency of sequential reactions when enzymes are free in solution versus when they form complexes. Cross-linking mass spectrometry (XL-MS) can provide structural insights into interaction interfaces, while hydrogen-deuterium exchange mass spectrometry (HDX-MS) can reveal conformational changes upon complex formation.

The historical understanding of aminotransferases suggests they typically function as dimers , but higher-order complexes with pathway enzymes may exist. Size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) can determine the stoichiometry of these complexes. Finally, cryo-electron microscopy may be employed to visualize the architecture of multi-enzyme assemblies if they exist in the histidine biosynthesis pathway.

What role does the FixLJ-FixK2 regulatory cascade play in controlling hisC2 expression in B. japonicum?

The FixLJ-FixK2 regulatory cascade is a key control system in B. japonicum that regulates genes required for microaerobic, anaerobic, or symbiotic growth . To investigate its potential role in controlling hisC2 expression, researchers should first analyze the hisC2 promoter region for the presence of CRP/FNR box motifs, which are recognized by FixK2. These palindromic DNA motifs are typically associated with FixK2-regulated promoters .

If putative FixK2 binding sites are identified, electrophoretic mobility shift assays (EMSA) using purified FixK2 protein and the hisC2 promoter fragment can confirm direct binding. DNase I footprinting can precisely map the binding site. For functional validation, in vitro transcription assays should be performed using purified B. japonicum σ80-RNA polymerase holoenzyme and FixK2 with the hisC2 promoter .

In vivo studies would involve creating reporter gene fusions (e.g., lacZ) to the hisC2 promoter and measuring expression in wild-type and fixK2 mutant backgrounds under different oxygen concentrations. RNA-seq analysis comparing gene expression profiles in wild-type and fixK2 mutant strains under various growth conditions would provide a global view of regulation. Additionally, chromatin immunoprecipitation sequencing (ChIP-seq) using antibodies against FixK2 could identify genome-wide binding sites, including potential association with the hisC2 promoter.

The position of any FixK2 binding site relative to the transcription start site is critical - if located at approximately -41.5, it would classify the hisC2 promoter as a class II CRP/FNR-type promoter, similar to other FixK2-regulated genes in B. japonicum .

How can researchers maintain the stability and activity of purified recombinant B. japonicum hisC2?

Maintaining stability and activity of purified recombinant B. japonicum hisC2 requires careful attention to buffer composition and storage conditions. PLP-dependent enzymes like histidinol-phosphate aminotransferase typically require the presence of their cofactor to maintain proper folding and activity. Therefore, supplementation with pyridoxal 5'-phosphate (PLP) at concentrations of 50-100 μM in all purification and storage buffers is recommended. Additionally, reducing agents such as DTT (1-5 mM) or TCEP (0.5-1 mM) should be included to prevent oxidation of cysteine residues.

Buffer optimization is critical and should include screening different pH ranges (typically pH 7.0-8.0), salt concentrations (typically 100-300 mM NaCl), and stabilizing additives. Glycerol (10-20%) can significantly enhance stability during storage by preventing freeze-thaw damage. For long-term storage, flash-freezing aliquots in liquid nitrogen and storing at -80°C is recommended over storage at -20°C or 4°C.

Activity assays should be performed immediately after purification to establish a baseline, and then periodically during storage to monitor stability. If activity loss is observed over time, consider:

For activity measurement protocols, spectrophotometric assays tracking the formation or consumption of keto acid products or substrates (typically at 340 nm with NADH-coupled systems) provide convenient real-time monitoring of enzymatic activity .

What are effective strategies to overcome solubility issues when expressing recombinant B. japonicum hisC2?

Solubility challenges with recombinant B. japonicum hisC2 can be addressed through multiple strategies targeting the expression system, growth conditions, and protein design. Based on experiences with other B. japonicum proteins that exhibit solubility issues, consider the following approaches:

First, optimize the expression conditions: lower the induction temperature to 16-25°C and reduce the IPTG concentration to 0.1-0.5 mM to slow protein production and allow proper folding. Extended expression times (18-24 hours) at lower temperatures often yield more soluble protein. Alternatively, auto-induction media can provide gradual induction and improve solubility.

If these approaches are insufficient, fusion tags beyond the standard histidine tag may enhance solubility. MBP (maltose-binding protein), SUMO, or GST (glutathione S-transferase) fusions have proven effective for enhancing the solubility of recalcitrant proteins. These larger solubility tags can be removed post-purification using specific proteases if they interfere with downstream applications.

For proteins resistant to these approaches, co-expression with molecular chaperones (GroEL/GroES, DnaK/DnaJ/GrpE, or trigger factor) can facilitate proper folding. Additionally, supplementing growth media with chemical chaperones such as sorbitol (0.5-1 M) or glycine betaine (2.5-10 mM) may improve solubility.

If the protein remains insoluble despite these efforts, solubilization and refolding from inclusion bodies may be necessary. Gradual dialysis techniques with decreasing concentrations of mild detergents or chaotropic agents, coupled with the presence of the PLP cofactor during refolding, can recover active enzyme. Finally, if specific domains or regions contribute disproportionately to insolubility, targeted mutagenesis of hydrophobic surface residues to polar residues might improve solubility without compromising activity .

What assay methods are most suitable for measuring B. japonicum hisC2 enzymatic activity?

For measuring B. japonicum hisC2 enzymatic activity, several complementary assay methods can be employed, each with distinct advantages for different research questions. The primary reaction catalyzed by histidinol-phosphate aminotransferase involves the transfer of an amino group from histidinol phosphate to an α-ketoglutarate acceptor, producing an imidazole acetol phosphate and glutamate.

A continuous spectrophotometric assay offers real-time monitoring capabilities. This approach typically couples the aminotransferase reaction to a secondary reaction that involves the consumption or production of NADH, which can be measured at 340 nm. For hisC2, the glutamate produced can be measured using glutamate dehydrogenase and NAD+, with the reduction of NAD+ to NADH providing a proportional signal.

For more sensitive detection, HPLC-based assays can directly quantify substrate consumption and product formation. Reverse-phase HPLC with pre-column derivatization (using o-phthalaldehyde or dansyl chloride) allows for sensitive detection of the amino acid products. This method is especially valuable when determining kinetic parameters requiring precise quantification of reaction components.

Isothermal titration calorimetry (ITC) provides thermodynamic parameters of substrate binding and can detect heat released during catalysis, offering insights into reaction energetics. While less commonly used for routine activity measurements, ITC provides valuable complementary data on enzyme-substrate interactions.

For high-throughput screening applications, such as when testing multiple enzyme variants, colorimetric endpoint assays can be developed. These typically involve coupling the aminotransferase reaction to additional enzymes that ultimately produce a chromogenic product. Optimization should focus on reaction buffer composition (pH 7.5-8.0 typically optimal), PLP concentration (50-100 μM), substrate concentrations, and reaction temperature (30°C is often suitable for B. japonicum enzymes) .

How can researchers distinguish between the activities of different aminotransferases in B. japonicum extract?

Distinguishing between different aminotransferases in B. japonicum extract requires a systematic approach combining substrate specificity, inhibitor sensitivity, and chromatographic separation. Histidinol-phosphate aminotransferase (hisC2) has distinctive substrate preferences compared to other aminotransferases such as aromatic amino acid aminotransferases (ArATs), which can be exploited for selective activity measurement .

The primary approach involves substrate selectivity. HspAT specifically recognizes histidinol phosphate, while ArATs preferentially bind aromatic amino acids like phenylalanine . By measuring activity with histidinol phosphate versus aromatic amino acids, researchers can create activity profiles that differentiate between these enzymes. Additionally, the differential response to pH can help distinguish aminotransferases - activity assays across a pH range (6.0-9.0) may reveal optimal pH windows specific to each enzyme class.

For definitive separation, chromatographic techniques such as ion-exchange chromatography can physically separate different aminotransferases based on their distinct surface charge properties. Each fraction can then be tested for activity with different substrates to create a comprehensive activity profile. Immunoprecipitation using antibodies specific to hisC2 can selectively remove this enzyme from the extract, allowing researchers to measure the remaining aminotransferase activities and calculate hisC2's contribution by difference.

Finally, genetic approaches involving the creation of deletion mutants lacking specific aminotransferase genes can provide definitive confirmation of enzyme activities when extracts from wild-type and mutant strains are compared .

How can recombinant B. japonicum hisC2 be engineered for enhanced catalytic efficiency or altered substrate specificity?

Engineering recombinant B. japonicum hisC2 for enhanced catalytic efficiency or altered substrate specificity requires a rational design approach informed by structural understanding. Based on the structural determinants of substrate specificity in aminotransferases, researchers should focus on modifying residues in the substrate binding pocket and N-terminal lid region . For enhanced catalytic efficiency with the native substrate, targeted mutations can be introduced to optimize substrate positioning relative to the PLP cofactor or to facilitate product release, which is often rate-limiting in aminotransferases.

To alter substrate specificity, researchers should consider the hydrophilic/hydrophobic balance of the binding pocket. Converting key hydrophilic residues to hydrophobic ones may shift specificity toward aromatic amino acids, while the opposite modification might enhance binding of more polar substrates . Semi-rational approaches combining computational design with directed evolution can be particularly effective. This involves using computational tools to identify promising mutation sites, creating focused libraries with mutations at these positions, and then screening for desired activities.

The protein engineering workflow should include:

• Structure determination or modeling of B. japonicum hisC2

• Computational identification of hotspot residues for mutagenesis

• Creation of variant libraries using site-directed mutagenesis or saturation mutagenesis

• High-throughput screening for desired catalytic properties

• Iterative rounds of optimization focusing on promising variants

For enhanced thermostability, which often correlates with longer shelf-life and industrial utility, researchers can introduce additional disulfide bonds or optimize surface charge distribution. Consensus design approaches, which identify conserved residues across homologous proteins from thermophilic organisms, can also guide stability-enhancing mutations.

Finally, methods that mimic natural protein evolution, such as DNA shuffling between hisC2 and related aminotransferases, can generate chimeric enzymes with novel properties not achievable through point mutations alone .

What potential applications exist for B. japonicum hisC2 in biocatalysis and synthetic biology?

B. japonicum hisC2, as a histidinol-phosphate aminotransferase, holds significant potential for various biocatalytic applications and synthetic biology projects. In biocatalysis, aminotransferases are valuable for their ability to perform regio- and stereoselective amination reactions, which are challenging for traditional chemical synthesis. The specific properties of hisC2, particularly its ability to recognize phosphorylated substrates, open unique opportunities for synthesizing phosphorylated amino compounds and chiral amines that serve as pharmaceutical precursors.

For synthetic biology applications, hisC2 can be incorporated into artificial metabolic pathways for the production of non-canonical amino acids. By engineering the substrate binding pocket to accommodate novel substrates while maintaining the core catalytic mechanism, researchers could develop pathways for producing unnatural amino acids with unique side chains containing imidazole or other heterocyclic groups. These compounds have potential applications as building blocks for peptidomimetics or bioactive molecules.

One particularly promising application involves exploiting the metal-binding properties of histidine-containing compounds in bioremediation. Drawing parallels with the metal-binding capabilities of histidine-rich proteins in B. japonicum like HypB (which binds multiple metal ions including Ni²⁺, Zn²⁺, Cu²⁺, Co²⁺, Cd²⁺, and Mn²⁺) , engineered metabolic pathways incorporating hisC2 could produce histidine-rich peptides or modified amino acids capable of chelating heavy metals from contaminated environments.

In agricultural biotechnology, engineered B. japonicum strains with optimized hisC2 expression could potentially enhance nitrogen fixation efficiency in soybean symbiosis . If histidine biosynthesis influences nodulation or nitrogen fixation capabilities, modulating hisC2 activity could contribute to developing improved biofertilizers with reduced reliance on chemical fertilizers.

These applications would require systematic protein engineering and metabolic pathway optimization, but the unique properties of hisC2 make it a valuable enzyme for expanding the biocatalytic toolkit in various sectors .

What computational approaches are most effective for predicting substrate binding and catalytic mechanisms of B. japonicum hisC2?

Computational approaches for predicting substrate binding and catalytic mechanisms of B. japonicum hisC2 should incorporate multiple methods at different scales of resolution. Molecular docking provides an efficient starting point for exploring substrate binding. Software packages such as AutoDock Vina, GOLD, or Glide can generate binding poses of histidinol phosphate and other potential substrates in the enzyme's active site. These docking studies should account for the presence of the PLP cofactor, which is covalently linked to a lysine residue in the active site during part of the catalytic cycle.

Molecular dynamics (MD) simulations offer more detailed insights by capturing the dynamic behavior of the enzyme-substrate complex. Long-timescale (100 ns - 1 μs) explicit-solvent MD simulations can reveal transient interactions, water-mediated hydrogen bonds, and conformational changes that occur upon substrate binding. Enhanced sampling techniques like metadynamics or umbrella sampling help overcome energy barriers to study the complete catalytic cycle.

For investigating the catalytic mechanism at the electronic level, quantum mechanics/molecular mechanics (QM/MM) calculations are essential. The QM region should include the substrate, PLP cofactor, and key catalytic residues, while treating the rest of the protein with MM force fields. This approach can elucidate proton transfer events, transition states, and energy barriers along the reaction coordinate.

Machine learning approaches are increasingly valuable, particularly when experimental data from similar enzymes exists. Specifically, graph neural networks can model the active site as a graph where atoms are nodes and bonds are edges, enabling prediction of catalytic properties based on structural features. These models can be trained on datasets of known aminotransferases to predict specificity and activity of hisC2 variants.

For integrating structural predictions with genomic information, coevolutionary analysis using methods like Direct Coupling Analysis (DCA) can identify co-evolving residue pairs that might be involved in substrate recognition or catalysis, providing additional targets for experimental validation .

How can molecular dynamics simulations inform the design of improved inhibitors for B. japonicum hisC2?

Molecular dynamics (MD) simulations provide critical insights for rational inhibitor design targeting B. japonicum hisC2 by capturing the dynamic behavior of the enzyme-inhibitor complex. Beginning with the finding that morpholine-ring scaffold compounds like 2-(N-morpholino)ethanesulfonic acid (MES) can specifically inhibit histidinol phosphate aminotransferases , MD simulations can elucidate the structural basis for this inhibition and guide optimization efforts.

The workflow for inhibitor design using MD simulations should include:

These computational predictions should guide the synthesis of a focused library of inhibitors for experimental validation, creating an iterative design process that efficiently leads to potent and selective hisC2 inhibitors .