Recombinant Mannheimia succiniciproducens Imidazole glycerol phosphate synthase subunit HisH (hisH)

What is Mannheimia succiniciproducens and why is it significant in research?

Mannheimia succiniciproducens is a capnophilic (CO₂-loving) Gram-negative facultative anaerobic rumen bacterium that efficiently produces succinic acid from a wide range of carbon sources, including pentose sugars, hexose sugars, and disaccharides . The strain MBEL55E, originally isolated from the rumens of Korean cows, has been completely genome sequenced and extensively studied for its exceptional succinic acid production capabilities .

To study this organism, researchers employ specialized anaerobic culture techniques with CO₂-enriched environments. The significance of M. succiniciproducens lies in its natural capacity for high-yield succinic acid production, a four-carbon dicarboxylic acid with numerous industrial applications. Through metabolic engineering approaches, researchers have achieved succinic acid yields as high as 1.16 mol per mol glucose in fed-batch cultures, demonstrating its biotechnological potential .

What is the role of Imidazole glycerol phosphate synthase (HisFH) and its HisH subunit in metabolism?

Imidazole glycerol phosphate synthase (HisFH) is a heterodimeric bienzyme complex operating at a critical branch point of metabolism . The complex consists of two functional subunits: HisF (the cyclase) and HisH (the glutaminase) . This enzyme complex catalyzes the synthesis of the histidine precursor imidazole glycerol phosphate (ImGP) and the purine precursor 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) from N′-[(5′-phosphoribulosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (PrFAR) and glutamine .

The HisH subunit specifically functions as a glutaminase, hydrolyzing glutamine to glutamate and ammonia . This ammonia then migrates approximately 25 Å through a central tunnel in the HisF β-barrel structure to reach the cyclase active site, where it reacts with PrFAR . Notably, in the absence of HisF ligands, the glutaminase activity of HisH remains at basal levels, preventing wasteful glutamine turnover - this represents a sophisticated allosteric regulation mechanism .

How is the HisH glutaminase activity regulated in the HisFH complex?

The glutaminase activity of HisH is subject to elegant allosteric regulation within the HisFH complex. In the absence of HisF ligands, HisH exhibits only basal glutaminase activity, which prevents wasteful glutamine hydrolysis . The catalytically active HisFH conformation is only formed when substrates of both HisH and HisF are bound .

Studies have demonstrated that high concentrations of the cyclase reaction products (ImGP and AICAR) can stimulate the glutaminase activity, suggesting product-based regulation . Additionally, researchers have identified a correlation between HisF dynamics and the degree of HisH stimulation, with regions encompassing α-helices 1-3, β-strands 1-3, and their connecting loops in HisF being particularly important for transmitting the allosteric signal from HisF to HisH .

To study this regulation experimentally, researchers employ techniques including steady-state kinetics, hydrogen-deuterium exchange mass spectrometry, and site-directed mutagenesis targeting the interface between the subunits.

What expression systems are most effective for producing recombinant M. succiniciproducens HisH?

When expressing recombinant M. succiniciproducens HisH, researchers must select appropriate expression systems based on their experimental goals. Based on experiences with other recombinant proteins from M. succiniciproducens, the following approaches have proven effective:

Table 1: Comparison of Expression Systems for Recombinant M. succiniciproducens HisH

For initial characterization studies, E. coli-based expression is most commonly employed, often using vectors with T7 promoters and N-terminal affinity tags (His6 or GST) to facilitate purification . When expressing HisH, consider that it functions as part of a complex with HisF, so co-expression strategies may improve solubility and stability.

What are the key challenges in purifying active recombinant M. succiniciproducens HisH?

Purifying recombinant M. succiniciproducens HisH presents several challenges that must be addressed to obtain functionally active enzyme:

• Maintaining quaternary structure: Since HisH naturally functions as part of the HisFH complex, the isolated subunit may exhibit instability or altered activity. Co-purification with HisF or stabilizing buffers containing osmolytes may be necessary .

• Preventing aggregation: HisH may have hydrophobic regions that become exposed when separated from HisF, leading to aggregation. Buffer optimization with mild detergents or stabilizing agents can help maintain solubility.

• Preserving enzymatic activity: The glutaminase activity of HisH is allosterically regulated and may require specific conditions to remain active during purification . Activity assays should be performed at each purification step.

A typical purification protocol might include:

• Immobilized metal affinity chromatography (IMAC) for His-tagged proteins

• Ion exchange chromatography to separate different oligomeric states

• Size exclusion chromatography as a final polishing step

• Activity assays throughout to monitor functional integrity

The kinetic properties of purified MDHs from different organisms have been extensively characterized, providing a methodological template for enzyme purification and characterization .

How can researchers verify the enzymatic activity of recombinant M. succiniciproducens HisH?

Verifying the enzymatic activity of recombinant M. succiniciproducens HisH requires appropriate assays that account for its glutaminase function and allosteric regulation:

Table 2: Methods for Assessing Recombinant M. succiniciproducens HisH Activity

When assessing HisH activity, researchers should consider:

• Testing activity both in isolation and in the presence of HisF to observe allosteric effects

• Including appropriate controls (heat-inactivated enzyme, no-substrate controls)

• Optimizing assay conditions (pH, temperature, substrate concentration)

• Determining enzyme kinetic parameters (Km, Vmax, kcat) under various conditions

• Comparing activity to other characterized HisH proteins from model organisms

Similar methodological approaches have been successfully applied to characterizing other enzymes from M. succiniciproducens, such as malate dehydrogenase (MDH), where detailed kinetic analyses revealed substrate inhibition patterns .

How does the substrate specificity and inhibition profile of M. succiniciproducens HisH compare to HisH from other organisms?

The substrate specificity and inhibition profile of M. succiniciproducens HisH likely exhibits distinct characteristics compared to HisH proteins from other organisms, similar to what has been observed with other M. succiniciproducens enzymes.

Drawing parallels from studies of malate dehydrogenase (MDH), researchers discovered significant differences in substrate specificity and inhibition patterns between M. succiniciproducens MDH (MsMDH) and MDHs from other organisms . For instance, MsMDH showed lower specific activity at physiological pH and stronger uncompetitive inhibition toward oxaloacetate compared to Corynebacterium glutamicum MDH (CgMDH), with ki values of 67.4 μM and 588.9 μM, respectively .

To characterize M. succiniciproducens HisH specificity and inhibition:

• Comparative kinetic analysis: Determine kinetic parameters (Km, kcat, Ki) for glutamine and potential inhibitors across a range of pH values and compare with HisH from model organisms like T. maritima .

• Structural analysis: Identify key residues influencing substrate binding and catalysis through homology modeling, sequence alignment, and site-directed mutagenesis.

• Inhibition studies: Test product inhibition (by glutamate) and feedback inhibition (by histidine pathway intermediates) to understand regulatory mechanisms.

Understanding these differences could provide insights into the evolutionary adaptations of M. succiniciproducens to its natural rumen environment and guide enzyme engineering efforts.

What structural and functional modifications might enhance HisH activity for biotechnological applications?

Based on insights from detailed enzyme characterization studies, several strategies could be employed to enhance M. succiniciproducens HisH activity for biotechnological applications:

Table 3: Potential HisH Engineering Strategies Based on Enzyme Characterization Data

Drawing from successful engineering of M. succiniciproducens MDH, where researchers identified a key residue influencing specific activity and substrate inhibition through structural comparison , similar approaches could be applied to HisH. Researchers should focus on:

• Conducting structural comparisons between M. succiniciproducens HisH and well-characterized HisH proteins to identify crucial residues for mutagenesis

• Employing directed evolution to screen for variants with enhanced properties

• Testing co-expression of engineered HisH with its partner HisF to ensure proper complex formation and function

Successful engineering efforts should ultimately aim to enhance glutamine utilization efficiency, which could have broader implications for metabolic pathways beyond histidine biosynthesis.

How might the allosteric regulation of HisH impact broader metabolic engineering strategies for M. succiniciproducens?

The allosteric regulation of HisH within the HisFH complex presents both challenges and opportunities for metabolic engineering of M. succiniciproducens. Understanding and manipulating this regulation could have significant implications:

• Nitrogen metabolism optimization: As HisH controls ammonia release from glutamine, engineering its allosteric regulation could influence nitrogen distribution across competing metabolic pathways. This might be particularly relevant in succinic acid production strains where nitrogen metabolism must be balanced with carbon flux .

• Integration with existing metabolic engineering approaches: M. succiniciproducens has been successfully engineered for enhanced succinic acid production through deletion of genes involved in competing pathways (ldhA, pflB, pta, ackA) . Complementing these deletions with optimized HisFH activity could further improve metabolic flux toward desired products.

• Cross-pathway effects: The products of the HisFH reaction (ImGP and AICAR) impact both histidine and purine biosynthesis pathways . Engineering HisH regulation could therefore have ripple effects across multiple biosynthetic pathways, potentially affecting cell growth and productivity.

• CO2 utilization: M. succiniciproducens is capnophilic, with CO2 fixation via PEP carboxykinase being crucial for succinic acid production . Engineering HisH to optimize nitrogen metabolism could indirectly affect carbon flux through central metabolic pathways, potentially enhancing CO2 utilization efficiency.

Metabolic modeling approaches, including flux balance analysis incorporating allosteric regulation, would be valuable for predicting the system-wide effects of HisH engineering and guiding strain development strategies.

What methodological approaches are most effective for studying the kinetics of the M. succiniciproducens HisFH complex?

Studying the kinetics of the M. succiniciproducens HisFH complex requires sophisticated methodological approaches to capture the allosteric communication between subunits and the multi-step reaction mechanism.

Table 4: Advanced Methodological Approaches for HisFH Kinetic Analysis

Drawing from approaches used to characterize other M. succiniciproducens enzymes, researchers should consider:

• Integrated experimental design: Combining multiple techniques to build a comprehensive understanding of the complex kinetic behavior

• Physiologically relevant conditions: Conducting experiments under conditions that mimic the cellular environment of M. succiniciproducens

• Mathematical modeling: Developing kinetic models that incorporate allosteric regulation and substrate/product inhibition

These approaches have been successfully applied to characterize the kinetic properties of M. succiniciproducens MDH, revealing important details about substrate inhibition patterns across different pH values , and could be adapted for the more complex HisFH system.

How might recombinant M. succiniciproducens HisH be leveraged for biocatalytic applications beyond its native function?

While primarily studied for its role in histidine biosynthesis, recombinant M. succiniciproducens HisH has potential applications in biocatalysis beyond its native function, particularly in glutamine transformation processes:

• Glutaminase activity applications: As a glutaminase, engineered HisH variants could be developed for:

  • Production of glutamate for food applications

  • Deamination reactions in pharmaceutical synthesis

  • Glutamine level reduction in certain foods or biological samples

• Ammonia production control: The regulated release of ammonia through HisH activity could be harnessed for:

  • Controlled amination reactions in chemical synthesis

  • Nitrogen transfer in cascade biocatalytic processes

  • In situ ammonia generation for pH control in enzymatic reactions

• Biosensor development: The allosteric regulation of HisH by HisF and its substrates could be leveraged to develop biosensors for:

  • Detection of metabolites that affect the HisFH complex

  • Monitoring of glutamine/glutamate levels in biological samples

  • Screening systems for drug discovery targeting allosteric enzyme regulation

Similar to how various MDHs have been evaluated for their specific activities and substrate inhibition profiles to enhance succinic acid production , engineered HisH variants could be screened for novel activities or improved properties for specific biocatalytic applications.