Recombinant Acinetobacter sp. ATP phosphoribosyltransferase regulatory subunit (hisZ)

What is ATP phosphoribosyltransferase and what role does HisZ play in its function?

ATP phosphoribosyltransferase (ATPPRT) catalyzes the first reaction in histidine biosynthesis, condensing ATP and 5-phospho-α-D-ribosyl-1-pyrophosphate (PRPP) to generate N1-(5-phospho-β-D-ribosyl)-ATP (PRATP) and pyrophosphate . ATPPRT exists as a complex of two distinct proteins: the catalytic subunit (HisGS) and the regulatory subunit (HisZ) .

HisZ plays a dual regulatory role in this enzyme system:

• It allosterically activates HisGS, substantially enhancing its catalytic activity by increasing kcat

• It mediates allosteric inhibition by histidine, the end product of the biosynthetic pathway

This regulation occurs despite the HisZ:HisGS interface lying approximately 20 Å away from the active site, demonstrating the importance of long-distance allosteric effects in enzyme regulation .

How is the ATPPRT holoenzyme structured in Acinetobacter species?

In Acinetobacter species, including A. baumannii, the ATPPRT holoenzyme assembles as a hetero-octamer with a specific architectural arrangement :

• A tetrameric HisZ core forms the central component of the structure

• This core is sandwiched by two dimers of HisGS

• The complete hetero-octameric complex can be reconstituted in vitro by mixing purified recombinant HisGS and HisZ subunits

The crystal structure of A. baumannii ATPPRT (AbATPPRT) has been determined using X-ray crystallography . When comparing AbATPPRT with related structures from other organisms such as Lactococcus lactis and Thermotoga maritima, significant differences are observed with RMSDs of 31.64 Å over 1973 Cα atoms and 46.56 Å over 1833 Cα atoms, respectively . These substantial structural differences may reflect the distinct kinetic properties of AbATPPRT compared to other bacterial ATPPRTs.

What is the biological significance of HisZ in Acinetobacter baumannii?

In Acinetobacter baumannii, HisZ plays a critical role in bacterial survival and pathogenicity :

• The HisZ-encoding gene is essential for A. baumannii growth in rich medium

• The HisGS-encoding gene is necessary for the bacterium's persistence in the lung during pneumonia

This differential essentiality suggests that HisZ may have functions beyond its role in histidine biosynthesis regulation. The importance of HisZ for bacterial growth makes the ATPPRT system a promising target for antibiotic development aimed at combating A. baumannii infections . This pathogen is of particular concern in clinical settings due to its ability to acquire antibiotic resistance and cause severe hospital-acquired infections.

What experimental approaches can be used to express and purify recombinant A. baumannii HisZ?

Recombinant A. baumannii HisZ (AbHisZ) can be prepared using the following methodology :

• Cloning: Insert the AbHisZ-encoding gene into an appropriate expression vector

• Expression: Transform the construct into a suitable host (typically E. coli) and induce protein expression

• Purification: Perform multi-step chromatography to isolate the protein

• Buffer exchange: Transfer the purified protein into a suitable buffer (e.g., 20 mM Tris pH 7.0, 50 mM KCl, and 10 mM MgCl2)

• Concentration: Concentrate using devices such as Vivaspin (Millipore)

• Quantification: Determine concentration spectrophotometrically at 280 nm using the molar extinction coefficient

For reconstitution of the active holoenzyme, AbHisGS and AbHisZ are typically mixed in a 1:1 molar ratio . The hetero-octameric structure can then be verified using techniques such as size-exclusion chromatography or analytical ultracentrifugation.

How does histidine inhibit ATPPRT activity and what is the role of HisZ in this process?

Histidine inhibits ATPPRT through a feedback inhibition mechanism mediated by HisZ :

• Inhibition mechanism: Histidine binds to ATPPRT noncompetitively, meaning it can bind to both the free enzyme and enzyme-substrate complexes

• Binding kinetics: Histidine binds to free ATPPRT, ATPPRT:PRPP, and ATPPRT:ATP binary complexes with similar affinity through a two-step binding mechanism

• HisZ role: The histidine binding site is located on HisZ, which transmits the inhibitory signal to the catalytic subunit HisGS

This allosteric regulation ensures that histidine biosynthesis is shut down when cellular histidine levels are adequate, preventing wasteful energy expenditure on unnecessary synthesis of an already abundant amino acid.

What molecular mechanisms underlie the allosteric activation of HisGS by HisZ?

The allosteric activation of HisGS by HisZ involves complex molecular events that enhance catalytic efficiency through structural and dynamic changes :

• Active site preorganization: HisZ binding constrains the dynamics of HisGS to favor a preorganized active site where key catalytic residues (particularly Arg56 and Arg32) are optimally positioned to stabilize the transition state

• Electrostatic optimization: The preorganized active site minimizes the reorganization energy required for charge redistribution during catalysis

• Catalytic resilience: In HisGS mutants with impaired catalytic function (e.g., Arg56Ala-HisGS), HisZ modulates the dynamics of compensatory residues (like Arg32) to partially restore function

Molecular dynamics (MD) simulations have been instrumental in elucidating these mechanisms by revealing how HisZ binding affects protein dynamics and active site geometry . The allosteric activation accelerates the chemical step to such an extent that product release becomes rate-limiting in the holoenzyme, whereas the chemical step remains rate-limiting in HisGS alone .

What kinetic mechanism does A. baumannii ATPPRT follow and how does it differ from other bacterial ATPPRTs?

A. baumannii ATPPRT (AbATPPRT) follows a unique kinetic mechanism compared to other bacterial ATPPRTs :

This random kinetic mechanism means that either ATP or PRPP can bind first to AbATPPRT with equal probability, unlike other ATPPRTs that require a specific binding order . This unique property may reflect specific adaptations of A. baumannii to its ecological niche or pathogenic lifestyle.

How can pre-steady-state kinetics reveal the rate-limiting steps in HisZ-activated ATPPRT catalysis?

Pre-steady-state kinetics provides crucial insights into the individual steps of the catalytic cycle and reveals how HisZ activation changes the rate-limiting step in ATPPRT :

These findings demonstrate that HisZ activation accelerates the chemical step so dramatically that product release becomes the new bottleneck in catalysis .

What crystallographic approaches are most effective for studying the HisZ-HisGS interaction?

Crystallographic studies of the HisZ-HisGS interaction require specific methodological approaches to obtain high-quality structural data :

• Protein preparation:

  • Mix HisGS and HisZ in a 1:1 molar ratio

  • Buffer exchange into crystallization buffer (e.g., 20 mM Tris pH 7.0, 50 mM KCl, 10 mM MgCl2)

  • Concentrate to approximately 8 mg/mL (138 μM)

• Crystallization:

  • Hanging drop vapor diffusion

  • Optimal precipitant conditions: 0.2 M sodium nitrate, 0.1 M bis-tris propane pH 8.5, 20% polyethylene glycol 3350

  • Mix protein and precipitant in a 1:1 ratio

• Data collection and processing:

  • Cryoprotection with mother liquor containing 20% glycerol

  • X-ray diffraction using suitable radiation source (e.g., rotating anode X-ray generator)

  • Data processing with software such as iMosflm and Aimless

• Structure solution and refinement:

  • Molecular replacement using related structures as search models

  • Model building with COOT and refinement with Refmac

  • Careful interpretation of electron density at the HisZ-HisGS interface

These approaches have successfully yielded structural insights into AbATPPRT, although challenges remain in visualizing certain loop regions and interface details due to electron density limitations .

How do environmental factors affect HisZ-mediated regulation of ATPPRT activity?

Various environmental factors significantly influence the HisZ-mediated regulation of ATPPRT :

• pH effects:

  • Maximum catalysis occurs above pH 8.0

  • pH-rate profiles can reveal ionizable groups essential for catalysis or regulation

• Temperature influences:

  • Temperature affects both reaction rate and possibly regulatory interactions

  • At 5°C, pre-steady-state burst kinetics become observable for AbATPPRT

  • Temperature optima may vary between the HisGS alone and the HisZ-activated holoenzyme

• Metal ion dependencies:

  • Mg2+ is typically required for catalysis

  • Replacement with Mn2+ enhances kcat for HisGS alone but may have different effects on the holoenzyme

  • Metal ions may affect both the chemical step and the protein-protein interactions

• Substrate specificity:

  • At 25°C, kcat is higher when ADP replaces ATP as substrate for AbATPPRT but not for HisGS alone

  • This suggests that HisZ binding alters substrate recognition properties

These environmental dependencies must be considered when designing experiments to study HisZ function and when developing potential inhibitors targeting the ATPPRT system .

What approaches can be used to investigate allosteric rescue of catalytically impaired HisGS by HisZ?

Investigating allosteric rescue of catalytically impaired HisGS involves multiple complementary approaches :

• Site-directed mutagenesis:

  • Create HisGS variants with mutations at catalytically important residues (e.g., Arg56Ala)

  • Determine the effect on catalytic parameters both alone and in complex with HisZ

• Kinetic characterization:

  • Compare steady-state parameters (kcat, KM) for mutant HisGS alone versus in complex with HisZ

  • Perform pre-steady-state kinetic analysis to identify which step(s) are rescued by HisZ binding

• Structural studies:

  • Determine crystal structures of mutant HisGS alone and in complex with HisZ

  • Identify conformational changes induced by HisZ binding that might compensate for the mutation

• Molecular dynamics simulations:

  • Model the dynamics of mutant HisGS with and without HisZ

  • Analyze how HisZ binding affects the positioning and dynamics of key catalytic residues

This integrated approach has revealed that in the case of Arg56Ala-HisGS, HisZ binding constrains the dynamics of another arginine residue (Arg32) to partially compensate for the absence of Arg56, demonstrating the remarkable resilience of allosteric regulation .

How can molecular dynamics simulations provide insights into the allosteric mechanism of HisZ?

Molecular dynamics (MD) simulations offer powerful insights into the allosteric mechanisms of HisZ by capturing protein dynamics at atomic resolution :

• Methodological approaches:

  • Simulation of multiple protein states (HisGS alone, HisZ alone, HisZ:HisGS complex)

  • Analysis of conformational ensembles and transitions

  • Calculation of dynamic correlations between distant regions of the protein

• Key findings from MD studies:

  • HisZ binding constrains HisGS dynamics to favor active site preorganization

  • Long-range allosteric communication occurs through networks of dynamically coupled residues

  • In mutant HisGS variants, HisZ binding can restore catalytic function by modulating the dynamics of compensatory residues

• Computational challenges:

  • Simulating large protein complexes (hetero-octamer) requires substantial computational resources

  • Timescales of allosteric transitions may exceed accessible simulation times

  • Validating simulation predictions requires integration with experimental data

MD simulations complement experimental approaches by revealing dynamic aspects of allosteric regulation that are difficult to capture with static structural methods, providing a more complete understanding of how HisZ activates HisGS across a distance of ~20 Å .

What potential exists for developing antibiotics targeting the HisZ-HisGS interface in pathogenic Acinetobacter species?

The HisZ-HisGS system presents a promising target for novel antibiotic development against pathogenic Acinetobacter species :

• Target validation:

  • HisZ is essential for A. baumannii growth in rich medium

  • HisGS is necessary for persistence during pneumonia infection

  • The system is absent in humans, offering potential selectivity

• Strategic approaches:

  • Targeting the HisZ-HisGS interface to prevent activation of HisGS

  • Designing compounds that lock HisZ in an inhibitory conformation

  • Exploiting the unique kinetic mechanism of AbATPPRT for selective inhibition

• Potential inhibitor classes:

  • Peptide-based inhibitors (e.g., derivatives of histidine-proline dipeptide which shows competitive inhibition)

  • Small molecules that bind at the interface between subunits

  • Allosteric inhibitors that mimic the inhibitory effects of histidine

• Methodological considerations:

  • Structure-based design using crystal structures of AbATPPRT

  • High-throughput screening for compounds that disrupt HisZ-HisGS interaction

  • Kinetic analysis to characterize inhibition mechanisms

The fact that a related HisZ protein sharing 43% sequence identity with AbHisZ acts as a tight-binding inhibitor of AbHisGS suggests that protein-protein interactions could be exploited for inhibitor design . This represents a novel approach to antibiotic development against this clinically important pathogen.