Recombinant Pseudomonas syringae pv. tomato (3R)-hydroxymyristoyl-[acyl-carrier-protein] dehydratase (fabZ)

What is (3R)-hydroxymyristoyl-[acyl-carrier-protein] dehydratase (fabZ) and what reaction does it catalyze?

(3R)-hydroxymyristoyl-[acyl-carrier-protein] dehydratase, commonly known as fabZ, is an enzyme that belongs to the hydro-lyase activity class (GO:0016836) . This enzyme catalyzes a critical dehydration reaction in bacterial fatty acid biosynthesis: (3R)-3-hydroxytetradecanoyl-[acyl-carrier protein] = tetradecenoyl-[acyl-carrier protein] + H2O .

During this reaction, fabZ removes the hydroxyl group from the β-carbon (C-3) and a hydrogen from the α-carbon (C-2) to form a trans-2 carbon-carbon double bond in the acyl chain. This dehydration step is essential in the bacterial fatty acid elongation cycle, occurring after the reduction of β-ketoacyl-ACP by fabG (β-ketoacyl-ACP reductase) and before further modifications of the acyl chain.

How does fabZ differ structurally and functionally from fabA?

While both fabZ and fabA catalyze the dehydration of 3-hydroxyacyl-ACP intermediates, they have distinct functional roles and structural features:

Where is fabZ located in the Pseudomonas fab gene cluster?

In Pseudomonas and related bacteria, the fabZ gene typically exists within a clustered arrangement of fatty acid biosynthesis genes. While the search results don't specifically detail the exact position of fabZ in P. syringae pv. tomato, we can draw parallels from related Pseudomonas species.

In Pseudomonas aeruginosa, the fab gene cluster includes fabD (encoding malonyl-CoA:ACP transacylase), fabG (encoding β-ketoacyl-ACP reductase), acpP (encoding ACP), and fabF (encoding β-ketoacyl-ACP synthase II) . This cluster is delimited by the plsX and pabC genes .

Interestingly, the fabH gene (encoding β-ketoacyl-ACP synthase III), which is typically located between plsX and fabD in most gram-negative bacteria, is absent from this gene cluster in P. aeruginosa . The organization in P. syringae pv. tomato DC3000, a model plant-pathogenic gram-negative bacterium , likely follows a similar pattern, but specific genomic analysis would be required to confirm the exact arrangement.

How do specific residues in the substrate-binding tunnel of fabZ affect substrate specificity?

The substrate specificity of fabZ is significantly influenced by the architecture of its hydrophobic substrate-binding tunnel. Research on S. brodae amxFabZ has provided valuable insights into this structure-function relationship:

These findings suggest that targeted mutations in the substrate-binding tunnel could be a strategic approach for engineering fabZ variants with altered substrate preferences for biotechnological applications.

What is the catalytic mechanism of fabZ at the molecular level?

The catalytic mechanism of fabZ involves several precise molecular steps:

• Initial substrate positioning: The acyl chain of the fatty acid substrate binds in the hydrophobic substrate-binding tunnel, positioning the 3-hydroxy group and adjacent carbon atoms appropriately for catalysis .

• Proton abstraction: A conserved histidine residue (H48 in S. brodae amxFabZ) functions as a catalytic base, abstracting a proton from the C2 atom of the substrate . This is a critical step that initiates the dehydration reaction.

• Substrate stabilization: A conserved glutamate or aspartate residue helps maintain the substrate in the correct conformation during this process .

• Hydroxyl protonation: The 3-hydroxy group is then protonated, likely by the same catalytic histidine residue that initially abstracted the proton from C2 .

• Water elimination: This protonation facilitates the elimination of water, resulting in the formation of a (2E)-carbon-carbon double bond in the substrate .

• Product release: The dehydrated product (trans-2-enoyl-ACP) is then released from the enzyme.

The importance of the catalytic histidine is highlighted by studies on the H48N mutant of S. brodae amxFabZ, which showed dramatically reduced activity compared to the wild-type enzyme. This mutant could only achieve appreciable dehydration of C6 and C8 substrates at enzyme concentrations 100 times higher than required for the wild-type .

How do mutations in the catalytic residues of fabZ impact substrate conversion rates?

Mutations in catalytic residues can significantly alter the enzymatic efficiency of fabZ. Studies with S. brodae amxFabZ provide quantitative insights into these effects:

H48N mutation (catalytic histidine):

• With wild-type enzyme, 10 nM concentration was sufficient to achieve nearly complete dehydration of 100 μM (3R)-3-hydroxy-C6-ACP within 30 minutes .

• In contrast, the H48N mutant required 1 μM enzyme concentration (100-fold higher) to achieve appreciable dehydration of the same substrate within 120 minutes .

• The H48N mutant showed no measurable activity against longer-chain C10 and C12 substrates even after 20 hours of incubation .

This dramatic reduction in catalytic efficiency results from disrupting the proton abstraction capability of the histidine residue, which is essential for initiating the dehydration reaction. The residual activity observed with shorter-chain substrates suggests that either:

• The asparagine substitution retains minimal ability to function in proton transfer

• An alternative catalytic mechanism becomes accessible, albeit much less efficiently

• The spatial arrangement of shorter substrates in the binding pocket allows limited reaction progress even with compromised catalytic machinery

These findings demonstrate the critical importance of the conserved histidine residue in the fabZ catalytic mechanism while also revealing the substrate-dependent nature of catalytic impairment caused by mutations.

What are the most effective strategies for recombinant expression of Pseudomonas syringae fabZ?

Based on successful approaches with related bacterial enzymes, the following strategies are recommended for recombinant expression of P. syringae fabZ:

Expression system selection:

• E. coli BL21(DE3) or similar strains designed for high-level protein expression are typically most effective

• Expression vectors with strong, inducible promoters (T7, trc, or araBAD) provide controlled production

• Fusion tags such as hexahistidine (His6) facilitate purification and can enhance solubility

Optimization parameters:

• Temperature: Lower induction temperatures (16-25°C) often improve solubility of recombinant enzymes

• Induction time: Extended expression periods (overnight) at lower temperatures may yield more soluble protein

• Media composition: Rich media (like TB or 2×YT) can increase biomass and protein yield

• Inducer concentration: Optimizing IPTG concentration (typically 0.1-1.0 mM) to balance expression level and solubility

Co-expression considerations:

• Since fabZ functions in a pathway involving acyl-carrier protein (ACP), co-expression or separate purification of ACP may be necessary for functional studies

• For ACP purification, an ACP-intein-chitin binding domain fusion approach has proven effective for P. aeruginosa ACP and could be adapted for P. syringae

What purification methods yield the highest purity and activity of recombinant fabZ?

A multi-step purification strategy is recommended to obtain highly pure and active recombinant fabZ:

Initial capture:

• Immobilized metal affinity chromatography (IMAC) using Ni-NTA or similar resins is highly effective for His-tagged fabZ

• Buffer conditions should include 20-50 mM Tris-HCl (pH 7.5-8.0), 200-500 mM NaCl, and potentially low concentrations of imidazole (10-20 mM) to reduce non-specific binding

Secondary purification:

• Size exclusion chromatography separates aggregates and different oligomeric states

• Ion exchange chromatography can remove remaining impurities based on charge differences

Tag removal considerations:

• If the fusion tag affects activity, it can be removed using specific proteases (TEV, thrombin, etc.)

• A second IMAC step can separate the cleaved tag from the native protein

Quality assessment:

• SDS-PAGE to confirm purity

• Mass spectrometry to verify intact mass and detect post-translational modifications

• Circular dichroism to assess proper folding

• Activity assays to confirm enzymatic function

For ACP purification, which is often needed alongside fabZ for functional studies, the ACP-intein-chitin binding domain approach used for P. aeruginosa ACP yields native protein with properly modified 4′-phosphopantetheine functional group, no acylation, and removal of the amino-terminal methionine .

How can the enzymatic activity of purified recombinant fabZ be accurately measured?

Accurate measurement of fabZ activity requires sensitive detection methods and careful experimental design:

Mass spectrometry-based assays:

• Quantitative MS can track the conversion of loaded ACP substrates by detecting the 18 Da mass decrease corresponding to water elimination during dehydration

• This approach allows direct monitoring of substrate depletion and product formation without relying on indirect spectroscopic methods

Substrate preparation:

• ACP must be correctly loaded with various (3R)-3-hydroxyacyl substrates

• Both stereochemistry (3R vs. 3S) and chain length (C6-C12) variations are important for comprehensive characterization

• Control assays should confirm substrate stability under assay conditions (some spontaneous dehydration of shorter-chain substrates may occur over extended periods)

Standard assay conditions:

• 100 μM substrate concentration with enzyme concentrations ranging from 10 nM to 1 μM, depending on expected activity

• Temperature: 30°C is typically suitable

• Buffer: Mild buffer conditions (pH 7.0-7.5) with potential addition of reducing agents

• Time points: Multiple measurements over 0-120 minutes to determine initial rates

Data analysis:

• Quantify percent conversion at each time point

• Calculate initial rates for kinetic parameter determination

• Compare different substrates to establish specificity profiles

• Use appropriate enzyme concentrations for different substrates to ensure measurable activity within the linear range

This methodology revealed, for example, that S. brodae amxFabZ efficiently dehydrated (3R)-3-hydroxy-C6-ACP (nearly complete conversion within 30 minutes with 10 nM enzyme) but showed dramatically reduced activity with longer-chain substrates requiring 100-fold higher enzyme concentrations .

What crystallization conditions have been most successful for fabZ enzymes?

While the search results don't specifically detail crystallization conditions for P. syringae fabZ, successful approaches with related FabZ enzymes provide valuable guidance:

Protein preparation:

• High purity (>95% by SDS-PAGE) is essential

• Monodisperse protein samples (verified by dynamic light scattering)

• Concentration typically between 10-20 mg/mL in a stabilizing buffer

• Fresh preparation is preferable to freeze-thawed samples

Crystallization techniques:

• Sitting or hanging drop vapor diffusion methods are most commonly successful

• Initial screening using commercial sparse matrix screens (Hampton Research, Molecular Dimensions)

• Optimization of promising conditions by varying precipitant concentration, pH, additives, and temperature

Successful conditions for related FabZ enzymes:

• Precipitants: PEG 3350-8000 (12-25%) often yields well-diffracting crystals

• Buffer pH range: 6.5-8.0, often with Tris, HEPES, or phosphate buffers

• Salt additives: 0.1-0.3 M salts (particularly ammonium sulfate, sodium chloride)

• Temperature: 4°C or 18°C, with slower crystal growth generally producing better diffraction quality

Co-crystallization considerations:

• For mechanistic studies, co-crystallization with substrate analogs or inhibitors may be attempted

• These compounds are typically added at 2-5× molar excess prior to crystallization setup

The successful crystallization of both wild-type and mutant (H48N and I69G) forms of S. brodae amxFabZ demonstrates that fabZ enzymes are amenable to structural studies, even when containing mutations that significantly alter enzymatic activity.

What structural features distinguish fabZ from other dehydratase enzymes?

FabZ possesses several distinctive structural features that differentiate it from other dehydratases:

• FabZ typically forms a hexameric assembly (trimer of dimers) in solution

• Each monomer adopts an α+β "hot dog" fold characteristic of this enzyme family

• The active site is formed at the dimer interface, with residues from both monomers contributing to substrate binding and catalysis

Catalytic machinery:

• A conserved histidine residue (H48 in S. brodae amxFabZ) serves as the catalytic base

• A conserved acidic residue (Glu or Asp) positions the substrate correctly for dehydration

• Unlike some other dehydratases, fabZ lacks isomerase activity found in related enzymes like fabA

Substrate binding tunnel:

• The hydrophobic tunnel accommodates the acyl chain of the substrate

• The tunnel's dimensions contribute to substrate specificity, with residues like I69 in S. brodae amxFabZ influencing chain length preference

• The tunnel is more restricted than in fabA, which helps explain the differing substrate preferences

Interaction with ACP:

• FabZ must interact with ACP to access its substrate

• The ACP interaction surface includes positively charged residues that complement the negatively charged surface of ACP

How can molecular dynamics simulations enhance understanding of fabZ substrate specificity?

Molecular dynamics (MD) simulations offer powerful insights into fabZ function that complement experimental approaches:

Dynamic substrate binding analysis:

• MD simulations can reveal transient interactions and conformational changes during substrate binding that are not captured in static crystal structures

• The dynamic behavior of the substrate-binding tunnel can be visualized, including fluctuations that may accommodate different substrate chain lengths

• Water molecule movements within the active site can be tracked to understand the dehydration mechanism

Substrate specificity predictions:

• Simulations with different acyl chain lengths can predict binding energies and stability

• Free energy calculations can quantify the energetic preference for different substrates

• Results can guide experimental design, suggesting specific mutations to alter substrate specificity

Mutation effects prediction:

• Virtual mutations (like the I69G mutation in S. brodae amxFabZ ) can be tested in silico before experimental validation

• Structural perturbations caused by mutations can be assessed dynamically rather than just statically

• Long-range effects of mutations on protein dynamics can be identified

Catalytic mechanism elucidation:

• Combined quantum mechanics/molecular mechanics (QM/MM) approaches can model transition states and reaction barriers

• Proton transfer pathways between the conserved histidine and substrate can be mapped

• Alternative mechanistic hypotheses can be evaluated computationally

ACP-fabZ interaction modeling:

• Molecular docking followed by MD simulation can model the transient complex between fabZ and ACP

• These simulations can reveal how substrate transfer occurs between the proteins

Molecular dynamics has already proven valuable in studying substrate binding in both FabZ and FabA enzymes , providing insights that complement and extend crystallographic data.

How does fabZ activity influence bacterial membrane composition and fluidity?

FabZ activity directly impacts bacterial membrane properties through its role in fatty acid biosynthesis:

Chain length determination:

• FabZ substrate specificity influences the distribution of fatty acid chain lengths in the membrane

• In S. brodae amxFabZ, the preference for shorter-chain substrates (C6, C8) over longer ones (C10, C12) would impact the resulting fatty acid profile

Saturation level influence:

• FabZ processes predominantly saturated acyl chains, while fabA introduces desaturation

• The relative activities of these enzymes determine the ratio of saturated to unsaturated fatty acids

• This ratio is a key determinant of membrane fluidity, with more unsaturated fatty acids increasing fluidity

Temperature adaptation:

• Bacteria modulate fabZ activity in response to temperature changes

• At lower temperatures, increased production of unsaturated fatty acids (requiring both fabZ and fabA) maintains appropriate membrane fluidity

Physiological consequences:

• Alterations in fabZ activity can affect:

  • Membrane permeability to antibiotics and other compounds

  • Function of membrane-embedded proteins

  • Resistance to environmental stresses

  • Bacterial morphology and cell division

In Pseudomonas syringae pv. tomato DC3000, a model plant-pathogenic bacterium , membrane composition likely plays important roles in environmental adaptation, host interaction, and pathogenesis, making fabZ an important enzyme for bacterial fitness.

How do fabZ inhibitors exert antimicrobial effects?

FabZ inhibitors can disrupt bacterial growth through several mechanisms:

Fatty acid biosynthesis disruption:

• Direct inhibition of fabZ prevents the dehydration step in fatty acid elongation

• This creates a bottleneck in the type II fatty acid synthesis (FAS) pathway

• Accumulation of 3-hydroxyacyl-ACP intermediates may occur, potentially triggering feedback inhibition of earlier steps

Membrane dysfunction:

• Inhibited fabZ activity leads to altered membrane fatty acid composition

• Resulting membranes may have compromised integrity and abnormal fluidity

• Membrane protein function may be impaired, affecting various cellular processes

Selective toxicity basis:

• Bacteria utilize the type II FAS system with discrete enzymes like fabZ

• Mammals employ a type I FAS system with a single multifunctional enzyme complex

• This structural and organizational difference provides the basis for selective targeting of bacterial fabZ

Resistance considerations:

Potential synergistic effects:

• FabZ inhibitors may show synergy with other antibiotics that target cell membranes

• Combined inhibition of multiple enzymes in the FAS pathway could prevent resistance development

What gene knockout or mutation strategies can reveal the physiological importance of fabZ?

Several genetic approaches can elucidate fabZ function in Pseudomonas syringae:

Conditional knockout systems:

• Temperature-sensitive mutants: Site-directed mutagenesis can create temperature-sensitive fabZ variants, similar to the W258Q mutation created in P. aeruginosa fabD

• Inducible promoter control: Placing fabZ under control of an inducible promoter allows tunable expression

• CRISPR interference (CRISPRi): Reversible repression of fabZ expression through targeted dCas9-mediated gene silencing

Point mutations:

• Catalytic residue mutations: Modifying the conserved histidine (analogous to H48 in S. brodae amxFabZ ) can create strains with reduced fabZ activity

• Substrate tunnel mutations: Alterations similar to the I69G mutation in S. brodae amxFabZ can modify substrate specificity

• These mutations should be introduced chromosomally to maintain natural expression levels

Phenotypic analyses:

• Growth rate determination under various conditions (temperature, pH, osmotic stress)

• Membrane composition analysis via lipid extraction and gas chromatography-mass spectrometry

• Antibiotic susceptibility testing

• Microscopic examination of cell morphology

• Virulence assays for plant infection by P. syringae pv. tomato

Complementation studies:

• Wild-type fabZ expression should restore normal phenotype in mutants

• Heterologous fabZ genes from other bacteria can reveal functional conservation and specialization

• Mutant complementation with fabZ variants can provide in vivo structure-function insights

Experience with related genes suggests successful approaches - for example, a chromosomal temperature-sensitive fabD mutant was obtained through site-directed mutagenesis in P. aeruginosa, and a fabF insertion mutant was generated that showed reduced cis-vaccenic acid levels . Notably, multiple attempts to disrupt the chromosomal fabG gene in P. aeruginosa were unsuccessful , suggesting this gene may be essential - fabZ might show similar characteristics.