Recombinant Thermus thermophilus (3R)-hydroxymyristoyl-[acyl-carrier-protein] dehydratase (fabZ)

What is the role of FabZ in bacterial fatty acid biosynthesis?

FabZ is a crucial enzyme in the type II fatty acid biosynthesis (FAS) pathway, which is essential for bacterial membrane formation. It catalyzes the third step in the elongation cycle, specifically the dehydration of (3R)-hydroxyacyl-ACP to trans-2-acyl-ACP. Unlike FabA (another isoform), FabZ is universally expressed in bacteria with type II FAS systems . In T. thermophilus, FabZ plays a vital role in maintaining membrane integrity at extremely high temperatures (65-80°C), making it particularly interesting for studying thermostable enzymes in essential metabolic pathways .

Methodologically, researchers investigating FabZ function should consider:

• The entire type II FAS pathway context when studying FabZ activity

• The interplay between FabZ and other fatty acid synthesis enzymes

• The specialized adaptations in thermophilic variants compared to mesophilic homologs

How does T. thermophilus FabZ differ structurally from mesophilic homologs?

T. thermophilus FabZ exhibits several structural adaptations that contribute to its exceptional thermostability:

• Higher salt bridge density: Analysis of thermostable proteins from T. thermophilus reveals an extensive network of salt bridges that stabilize the tertiary structure at elevated temperatures

• Compact hydrophobic core: The protein shows a more tightly packed hydrophobic core with fewer cavities than mesophilic homologs

• Rigid active site architecture: The catalytic His/Glu dyad is positioned within a thermally stable environment

• Hexameric assembly: Forms a hexamer (trimer of dimers) similar to other FabZ proteins, but with enhanced intersubunit interactions

The structure reveals a deep, narrow tunnel formed at the dimer interface where the catalytic residues reside, similar to the P. aeruginosa FabZ structure determined at 2.5 Å resolution .

What are the optimal conditions for recombinant expression of T. thermophilus FabZ?

Successful expression of recombinant T. thermophilus FabZ requires careful optimization of several parameters:

For homologous expression in T. thermophilus, researchers have observed significantly higher yields of correctly folded, active enzyme. The thermophilic host provides the proper cellular machinery for thermostable protein folding .

What purification strategy is most effective for obtaining high-purity T. thermophilus FabZ?

A multi-step purification protocol is recommended:

• Heat treatment (70-75°C, 20 minutes): Exploits the thermostability of FabZ to eliminate most host proteins. This step alone can achieve >80% purity when expressed in E. coli

• Ion exchange chromatography: Anionic exchange (Q-Sepharose) at pH 8.0 with gradient elution (0-500 mM NaCl)

• Affinity chromatography: If using a His-tagged construct, Ni-NTA affinity purification (elution with 250 mM imidazole)

• Size exclusion chromatography: Final polishing step to achieve >95% purity and remove aggregates

The heat treatment step is particularly effective and distinctive for T. thermophilus proteins, allowing for significant purification without complex chromatography . When expressed with a His-tag, the recombinant protein can be purified to homogeneity with just heat treatment followed by affinity chromatography .

How can the enzymatic activity of T. thermophilus FabZ be reliably measured?

Several assay methods can be employed to measure FabZ activity:

• Direct spectrophotometric assay: Monitoring the formation of trans-2-enoyl-ACP at 260 nm (ε = 6,700 M⁻¹cm⁻¹). This requires careful temperature control and baseline correction due to high assay temperatures.

• Coupled enzyme assay: Using FabI (enoyl-ACP reductase) and NADH to couple the FabZ reaction to NADH oxidation, which can be monitored at 340 nm. The reaction mixture typically contains:

  • 100 μM (3R)-hydroxy-acyl-ACP substrate

  • 200 μM NADH

  • 1-10 nM purified FabZ

  • 50 mM phosphate buffer, pH 7.5

  • FabI coupling enzyme (1-5 μM)

• Mass spectrometry-based assay: Quantitative MS to monitor substrate conversion, particularly useful for specificity studies. This approach has shown that T. thermophilus FabZ efficiently processes C6-(3R)-hydroxy substrates within 1 minute at enzyme concentrations as low as 10 nM .

For optimal results, assays should be conducted at 65-90°C, and reaction components must be thermostable. Controls without enzyme are essential due to potential spontaneous dehydration of substrates at high temperatures .

What is the substrate specificity profile of T. thermophilus FabZ?

T. thermophilus FabZ exhibits distinct substrate preference profiles:

The enzyme exhibits strong stereoselectivity, exclusively processing the (3R)-hydroxy configuration with negligible activity toward (3S)-hydroxy substrates. Unlike some bacterial FabZ enzymes, T. thermophilus FabZ shows higher activity with shorter chain substrates (C6-C8), which may reflect adaptation to the membrane composition requirements at high temperatures .

The substrate specificity can be determined through mass spectrometry analysis of enzyme-catalyzed reactions, monitoring the conversion of hydroxy-ACP substrates to the corresponding trans-2-enoyl-ACP products .

What molecular features contribute to the thermostability of T. thermophilus FabZ?

Several structural elements contribute to the exceptional thermostability of T. thermophilus FabZ:

• Electrostatic interactions network: Increased number of salt bridges and charged residue networks compared to mesophilic homologs

• Hexameric quaternary structure: The enzyme forms a trimer of dimers, with the active site located at the dimer interface. This oligomeric arrangement provides significant stabilization

• Optimized hydrophobic packing: Enhanced core packing with reduced cavity volume

• Reduced loop flexibility: Shorter loop regions and proline residues in strategic positions reduce conformational entropy

• Strategic hydrogen bonding: Increased number of hydrogen bonds, particularly around catalytic residues

Comparative molecular dynamics simulations at different temperatures (303K, 333K, and 363K) reveal that T. thermophilus proteins maintain a static salt bridge/charged residue network that plays a fundamental role in temperature resistance by enhancing both electrostatic interactions and entropic energy dispersion .

How does T. thermophilus FabZ maintain catalytic efficiency at high temperatures?

T. thermophilus FabZ maintains catalytic efficiency at elevated temperatures through several adaptations:

• Protected active site architecture: The catalytic His/Glu dyad resides in a deep, narrow tunnel that shields it from denaturation

• Optimized electrostatic environment: The pKa values of catalytic residues are tuned for function at high temperatures

• Conformational stability near active site: Reduced flexibility in regions surrounding the catalytic residues ensures proper substrate positioning

• Substrate binding adaptations: Modifications in the substrate binding tunnel accommodate the increased molecular motion at high temperatures

Kinetic studies show that T. thermophilus FabZ exhibits optimal activity at 90°C, significantly higher than the optimal growth temperature (65-75°C) of the organism itself. This is a common characteristic of enzymes from Thermus species and allows the bacteria to maintain metabolic activity during temperature fluctuations .

How can T. thermophilus FabZ be engineered for enhanced catalytic properties?

Advanced protein engineering strategies for T. thermophilus FabZ include:

• Rational design approaches:

  • Structure-guided mutations of substrate binding tunnel residues to alter specificity

  • Modifying the active site to introduce FabA-like isomerization activity

  • Engineering the entrance/exit channels to accommodate alternative substrates

• Directed evolution strategies:

  • Development of high-throughput assays compatible with thermostable enzymes

  • Selection systems in T. thermophilus using CRISPR-Cas9 based genome editing

  • Compartmentalized self-replication (CSR) adapted for thermophilic conditions

Recently developed tools for genetic manipulation of T. thermophilus, including a thermostable CRISPR-Cas9 system that functions at 65°C, provide new opportunities for in vivo engineering. This system has demonstrated about 90% efficiency in generating knockout mutants, facilitating the creation of modified strains expressing engineered FabZ variants .

What potential biotechnological applications exist for T. thermophilus FabZ?

T. thermophilus FabZ has several promising biotechnological applications:

• Biocatalysis at elevated temperatures: The extreme thermostability (active up to 90°C) makes it suitable for industrial processes requiring high temperatures to improve reaction rates, substrate solubility, or prevent microbial contamination

• PCR enhancement: Similar to how T. thermophilus RecA enhances PCR signals for DNA viruses, thermostable FabZ could potentially be employed in molecular biology applications requiring stable proteins at high temperatures

• Metabolic engineering of fatty acid biosynthesis: Incorporation into synthetic pathways for production of specialized fatty acids or biofuels that benefit from high-temperature bioprocessing

• Structural templates for designing thermostable enzymes: The molecular features conferring thermostability can inform rational design of other thermostable biocatalysts

• Antibiotic drug discovery: As type II fatty acid biosynthesis is essential for bacterial membrane formation, thermostable FabZ offers a unique model for developing inhibitors targeting this pathway in thermophilic pathogens

The integration of T. thermophilus FabZ into high-temperature bioprocesses could enable new approaches to challenging chemical transformations while minimizing contamination risks and potentially reducing cooling costs in industrial settings.

How does the catalytic mechanism of T. thermophilus FabZ compare to FabA?

The catalytic mechanisms of FabZ and FabA share similarities but have crucial differences:

Key differences:

• FabA possesses isomerase activity (trans-2 to cis-3) that FabZ lacks

• The substrate binding tunnel in FabZ is narrower and more linear compared to FabA's kinked and elongated binding pocket

• FabZ efficiently processes a wider range of substrate chain lengths

• T. thermophilus FabZ shows distinct stereoselectivity profiles optimized for thermophilic environments

The structural differences in the substrate binding channels between FabA and FabZ control the conformation and positioning of bound substrates, allowing FabA to catalyze isomerization while FabZ cannot. Site-directed mutagenesis studies have shown that the obvious differences in active site residues between the two enzyme families do not fully account for FabA's unique isomerization ability .

Understanding these mechanistic differences provides insights into the evolutionary divergence of these enzyme families and offers opportunities for rational design of novel biocatalysts with tailored activities.