Recombinant Phthiocerol synthesis polyketide synthase type I PpsE (ppsE), partial

What is PpsE and what role does it play in Mycobacterium tuberculosis?

PpsE is a polyketide synthase (PKS) that forms part of the phenolphthiocerol synthase cluster in Mycobacterium tuberculosis. It functions as the final enzyme in the series of Pps proteins (PpsA-PpsE) responsible for the synthesis of phthiocerol dimycocerosate (PDIM), a key virulence determinant in M. tuberculosis pathogenesis. PpsE contains both enoyl-reductase and dehydrase domains and is responsible for the addition of saturated alkyl units derived from malonyl or methyl-malonyl-CoA during PDIM synthesis . The enzyme plays a crucial role in completing the phthiocerol chain that will subsequently be coupled with mycocerosic acids to form the final PDIM structure that contributes to bacterial virulence.

How is PpsE structurally organized compared to other polyketide synthases?

PpsE belongs to the type I modular polyketide synthase family, characterized by multiple functional domains organized within a single large polypeptide. Its structure includes:

• An acyl carrier protein (ACP) domain for tethering growing polyketide chains

• A ketosynthase (KS) domain for carbon-carbon bond formation

• An acyltransferase (AT) domain for substrate selection and loading

• A dehydratase (DH) domain for introducing unsaturation

• An enoyl-reductase (ER) domain for reducing double bonds

Unlike some other Pps proteins in the same pathway, PpsE contains functional dehydratase and enoyl-reductase domains, allowing it to produce saturated alkyl extensions to the growing phthiocerol chain . This modular organization enables the coordinated synthesis of complex lipids that are essential for M. tuberculosis virulence.

What experimental evidence confirms PpsE's enzymatic function?

Biochemical and genetic studies have confirmed PpsE's enzymatic function through several approaches:

• Protein-protein interaction studies using two-hybrid systems demonstrated that PpsE interacts with type II thioesterase TesA, confirming its involvement in the PDIM synthetic pathway .

• GST pull-down assays with purified proteins validated the PpsE-TesA interaction .

• Interaction studies with MmpL7 domain 2 indicated PpsE's role in the final step of phthiocerol synthesis .

• Genetic knockout experiments in M. tuberculosis demonstrated altered PDIM production when the PpsE function was disrupted.

These multiple lines of evidence collectively establish PpsE's crucial enzymatic role in phthiocerol synthesis and subsequent PDIM formation.

How does PpsE interact with TesA and what is the functional significance of this interaction?

PpsE interacts directly with the type II thioesterase TesA of M. tuberculosis, as demonstrated through both two-hybrid system experiments and GST pull-down assays with purified proteins . This interaction serves several critical functions:

• Quality control: TesA likely removes aberrant intermediates from the PpsE active site to prevent stalling of the PDIM synthetic machinery.

• Catalytic efficiency: The interaction may enhance PpsE's catalytic rate by ensuring proper substrate positioning and product release.

• Pathway coordination: TesA-PpsE interaction may help synchronize the various enzymatic steps in the complex PDIM biosynthetic pathway.

This interaction represents an important regulatory mechanism in polyketide biosynthesis, allowing for efficient and accurate production of these complex lipids essential for M. tuberculosis virulence .

What is known about the interaction between PpsE and MmpL7, and how does this relate to PDIM transport?

Research has identified a direct interaction between PpsE and domain 2 of the MmpL7 transporter in M. tuberculosis . This interaction suggests a coupling mechanism between PDIM synthesis and transport:

• PpsE, which catalyzes the final step of phthiocerol synthesis, interacts with the cytoplasmic domain 2 of MmpL7.

• This interaction suggests that MmpL7 domain 2 is accessible to the cytoplasm where PDIM synthesis occurs .

• The interaction likely facilitates the immediate transport of newly synthesized PDIM molecules across the cell membrane.

Experimental evidence from dominant negative studies showed that overexpression of MmpL7 domain 2 affected both PDIM synthesis and transport, indicating that the PpsE-MmpL7 interaction creates a functional complex that coordinates these processes . This represents an elegant example of coupled synthesis and transport, maximizing efficiency in cell wall component production.

What are the optimal conditions for handling recombinant PpsE protein?

Based on manufacturer recommendations for recombinant PpsE, the following handling conditions are optimal :

Prior to opening, vials should be briefly centrifuged to bring contents to the bottom. For long-term storage, aliquoting with glycerol addition (50% final concentration is standard) is recommended to maintain protein stability and prevent activity loss from repeated freeze-thaw cycles .

How can researchers effectively express and purify functional PpsE for in vitro studies?

For successful expression and purification of functional PpsE:

• Expression system selection: Baculovirus expression systems have proven effective for producing recombinant PpsE with proper folding and post-translational modifications .

• Purification strategy:

  • Affinity chromatography using appropriate tags (determined during the manufacturing process)

  • Size exclusion chromatography to separate aggregates and ensure homogeneity

  • Activity-based verification assays to confirm functional integrity

• Quality control assessments:

  • SDS-PAGE analysis (target purity >85%)

  • Western blot confirmation of identity

  • Mass spectrometry to verify intact protein mass

• Functional validation:

  • Enzymatic activity assays using appropriate substrates

  • Protein-protein interaction studies (e.g., with TesA or MmpL7)

These approaches ensure the production of high-quality PpsE protein suitable for detailed biochemical and structural studies.

What methods are most effective for studying PpsE interactions with other proteins?

Several complementary methods have proven effective for studying PpsE's protein-protein interactions:

• Two-hybrid system: Successfully used to identify the interaction between PpsE and TesA . This approach is valuable for initial discovery of protein interactions.

• GST pull-down assays: Employed to confirm the PpsE-TesA interaction after purification of both proteins . This biochemical approach provides direct evidence of physical interaction.

• Co-immunoprecipitation: Useful for validating interactions in more native contexts, particularly when studying endogenous protein complexes.

• Surface plasmon resonance (SPR): Provides quantitative binding parameters including association/dissociation rates and binding affinities.

• Crosslinking coupled with mass spectrometry: Identifies specific interaction interfaces and contact residues between PpsE and its binding partners.

When studying PpsE interactions, researchers should implement multiple complementary methods to establish robust evidence for protein-protein interactions, as exemplified by the combined use of two-hybrid and GST pull-down approaches in confirming the PpsE-TesA interaction .

How does PpsE coordinate with other Pps proteins in the PDIM biosynthetic pathway?

PpsE functions as part of a coordinated multi-enzyme system for PDIM biosynthesis. The pathway progression involves:

• PpsA initiates the pathway, accepting a starter unit and introducing a hydroxyl group.

• PpsB receives the product from PpsA, extends it, and introduces a second hydroxyl group, creating a 1,3-diol structure .

• PpsC, PpsD, and PpsE sequentially extend the growing chain by adding saturated alkyl units derived from malonyl or methyl-malonyl-CoA .

• PpsE performs the final extension and modification in the phthiocerol backbone synthesis.

The coordinated action requires precise protein-protein recognition and substrate channeling between the Pps proteins. Evidence suggests that these enzymes may form a multiprotein complex to facilitate efficient transfer of the growing polyketide chain. Additionally, PpsE's interaction with TesA suggests that quality control mechanisms are integrated into this biosynthetic assembly line to ensure proper product formation.

What is the relationship between PpsE and the FAS-II pathway in mycobacteria?

Research indicates a novel interaction linking the Polyketide Synthase (PKS) pathway involving PpsE and the Fatty Acid Synthase II (FAS-II) pathway in mycobacteria:

• The FAS-II enzyme KasA has been shown to interact with PpsB and PpsD in the acyl carrier domain regions .

• This interaction suggests potential lipid transfer between the PDIM biosynthetic pathway (involving PpsE) and the FAS-II pathway.

• Studies with purified proteins and radiolabeled lipids demonstrated that fatty acids loaded onto PpsB could be transferred to KasA and incorporated into long-chain fatty acids synthesized in a Mycobacterium smegmatis lysate .

How do trans-acting enzyme partners influence PpsE function?

Trans-acting enzyme partners play crucial roles in modulating PpsE function in the PDIM biosynthetic pathway:

These trans-acting partners collectively create a sophisticated regulatory network that ensures proper functionality of the PDIM biosynthetic machinery. The interactions highlight that PpsE does not function in isolation but rather as part of an integrated multiprotein system coordinating lipid synthesis in mycobacteria.

Why is PpsE considered a viable target for tuberculosis drug development?

PpsE represents a promising target for tuberculosis drug development for several key reasons:

• Essentiality for virulence: PpsE is critical for the synthesis of PDIM, a known virulence factor in M. tuberculosis. Disruption of PDIM synthesis attenuates bacterial virulence in infection models.

• Absence in humans: As a bacterial polyketide synthase with no human homolog, PpsE inhibitors would likely show selective toxicity against M. tuberculosis without affecting human enzymes.

• Structural distinctiveness: The modular nature and specific domain architecture of PpsE offer multiple sites for selective inhibitor binding.

• Validated pathway importance: The phthiocerol biosynthetic pathway has been genetically validated as important for M. tuberculosis pathogenesis, with disruptions in this pathway leading to attenuated virulence .

• Synergistic potential: Inhibitors targeting PpsE could potentially be used in combination with existing TB drugs, enhancing efficacy through multi-target approaches.

The development of PpsE inhibitors would represent a novel approach to tuberculosis therapy by targeting a bacterial virulence mechanism rather than essential processes, potentially reducing selection pressure for resistance development.

What methodological approaches can identify potential inhibitors of PpsE activity?

Several methodological approaches can be employed to identify potential PpsE inhibitors:

• Structure-based virtual screening:

  • Molecular docking of compound libraries against PpsE active sites

  • Fragment-based screening to identify initial binding scaffolds

  • Structure-activity relationship analysis to optimize lead compounds

• Biochemical assays:

  • Development of high-throughput enzymatic assays measuring PpsE catalytic activity

  • Thermal shift assays to identify compounds that bind and stabilize PpsE

  • ATPase activity assays to monitor inhibition of energy consumption

• Cell-based approaches:

  • Whole-cell screening with PDIM production as an endpoint

  • Reporter systems linked to PDIM biosynthesis pathway activity

  • Phenotypic screens for compounds that mimic PpsE genetic knockout

• Protein-protein interaction disruption:

  • Screening for molecules that disrupt the PpsE-TesA interaction

  • Compounds that interfere with the PpsE-MmpL7 interaction

• Rational design based on substrates:

  • Development of substrate analogs that competitively inhibit PpsE

  • Transition-state mimics targeting the catalytic mechanism

These diverse approaches can be implemented in parallel to identify multiple chemical starting points for PpsE inhibitor development.

How might disruption of PpsE function affect M. tuberculosis survival in host environments?

Disruption of PpsE function would have several significant consequences for M. tuberculosis survival in host environments:

• Altered cell envelope integrity: Loss of PDIM production would alter the mycobacterial cell envelope structure, potentially increasing susceptibility to host defense mechanisms including antimicrobial peptides.

• Impaired host cell entry: PDIM has been implicated in facilitating efficient mycobacterial entry into host cells; disruption of PpsE would likely impair this process, reducing bacterial colonization efficiency.

• Enhanced susceptibility to immune clearance: M. tuberculosis lacking PDIM shows increased susceptibility to reactive nitrogen intermediates and other host defense mechanisms, resulting in enhanced bacterial clearance.

• Reduced intracellular persistence: The absence of PDIM affects bacterial ability to persist within macrophages, a key niche for M. tuberculosis during infection.

• Pathway interference effects: Disrupting PpsE could lead to accumulation of biosynthetic intermediates, potentially causing metabolic perturbations that further compromise bacterial fitness.