Recombinant Phenolphthiocerol synthesis polyketide synthase type I Pks15/1 (pks15/1), partial

What is Pks15/1 and what is its role in mycobacterial metabolism?

Pks15/1 is a 6-domain protein with homology to reducing iterative type I polyketide synthases (PKSs) that plays a critical role in the biosynthesis of phenolic glycolipids (PGLs) in several pathogenic mycobacteria, including Mycobacterium tuberculosis strains and Mycobacterium leprae. Specifically, Pks15/1 works in cooperation with FadD22 (a coenzyme A-independent stand-alone didomain initiation module) to produce p-hydroxyphenylalkanoate (PHPA) intermediates during phenolphthiocerol (PPOL) biosynthesis. These intermediates serve as precursors for the phenolphthiocerol moiety of PGLs, which are important virulence factors that play key roles in pathogenicity and host-pathogen interactions .

The Pks15/1 system functions by accepting a p-hydroxybenzoyl starter unit from FadD22 and extending it through multiple rounds of catalysis using malonyl-CoA as an extender unit. The enzyme is characterized by having a relaxed control of catalytic cycle iterations, which explains the characteristic alkyl chain length variability seen in mycobacterial PGLs .

How does the domain structure of Pks15/1 relate to its function?

Pks15/1 consists of two gene components with distinct functional domains that work together as an iterative type I polyketide synthase:

• pks15: Encodes the ketoacyl synthase (KS) domain

• pks1: Encodes multiple functional domains including:

  • Keto reductase (KR) domain

  • Dehydratase (DH) domain

  • Enoyl reductase (ER) domain

  • Acyltransferase (AT) domain

  • Acyl carrier protein (ACP) domain

This modular arrangement allows for the sequential catalytic activities required for polyketide chain elongation and modification. The KS domain catalyzes carbon-carbon bond formation through decarboxylative Claisen condensation reactions, while the reductive domains (KR, DH, ER) modify the β-keto group of the growing polyketide chain. The AT domain selects and transfers the appropriate extender unit (malonyl-CoA), and the ACP domain serves as the attachment point for the growing polyketide chain via its phosphopantetheinyl arm .

Unlike non-reducing iterative type I PKSs that contain product template (PT) and thioesterase (TE) domains for controlling chain length and cyclization, Pks15/1 lacks these domains, suggesting a distinct mechanism for controlling the structure of its final products .

What is the significance of the 7 bp deletion in the pks15/1 locus?

The 7 bp deletion in the pks15/1 locus is a significant polymorphism that separates different lineages of the Mycobacterium tuberculosis complex. This deletion functions as a valuable molecular marker with important epidemiological implications:

• Lineage identification: The presence or absence of this deletion enables researchers to distinguish between Euro-American, Indo-Oceanic, and Asian lineages of M. tuberculosis. Specifically:

  • Isolates with the 7 bp deletion typically belong to Euro-American or Indo-Oceanic lineages

  • Isolates with the intact (wild-type) pks15/1 locus are characteristic of Asian lineages, including the Beijing genotype

• Functional consequences: The 7 bp deletion results in a frameshift mutation that splits pks15 and pks1 into separate genes, potentially affecting the functionality of the enzyme complex and subsequently the production of phenolic glycolipids .

• Epidemiological surveillance: The polymorphism serves as a useful tool for tracking the spread of different M. tuberculosis lineages, especially in regions where the prevalence of Asian lineages (including W-Beijing) remains poorly characterized .

Studies in Mexico have confirmed the utility of this locus as a molecular marker, finding that all Asian lineage isolates tested possessed the wild-type pks15/1 locus, while other lineages carried the 7 bp deletion .

How is pks15/1 conserved across mycobacterial species that produce phenolic glycolipids?

Sequence analysis indicates that Pks15/1 is well-conserved among PGL-producing mycobacteria. The enzyme consists of 2104-2118 amino acid residues with 79-100% sequence identity across PGL-producing mycobacterial species . This high degree of conservation reflects the essential role of Pks15/1 in the biosynthesis of phenolic glycolipids, which are important virulence factors in these pathogens.

The conservation pattern aligns with functional studies demonstrating that the pks15/1 gene is required for PGL biosynthesis. Genetic studies have confirmed that disruption of pks15/1 leads to a loss of PGL production, further emphasizing its critical role in this biosynthetic pathway .

How can researchers amplify and sequence the pks15/1 locus for genotypic characterization?

A standardized protocol for amplifying and sequencing the pks15/1 locus involves the following steps:

• DNA extraction:

  • Collect one loop of cultured mycobacteria

  • Extract DNA according to established protocols (e.g., Van Soolingen et al. method)

  • Resuspend DNA in nuclease-free water

  • Measure concentration by spectrophotometry

  • Store DNA at -20°C until use

• PCR amplification:

  • Target a 405 bp fragment that includes the 7 bp polymorphic region

  • Use primers PKR (5'-CTGCCCAGGAAACACGAC-3') and PKF (5'-GTGTCCTCCTTTGGGATCAG-3')

  • Prepare PCR reaction mixture containing:

    • 10 mM Tris pH 8

    • 1.5 mM MgCl₂

    • 0.2 mM of each deoxynucleotide triphosphate

    • 10 nmoles of PKF and PKR primers

    • 1.25 U Taq polymerase

    • 5% glycerol

    • 200 ng DNA template

    • Nuclease-free water to a final volume of 25 μL

• Amplification parameters:

  • Initial denaturation: 95°C for 5 min

  • 30 cycles of:

    • 95°C for 45 s (denaturation)

    • 60°C for 45 s (annealing)

    • 72°C for 45 s (extension)

  • Final extension: 72°C for 8 min

• PCR product purification:

  • Electrophoretically separate products in a 1.5% agarose gel

  • Purify using centrifugal filters

  • Determine final DNA concentration by comparison with DNA ladder

• Sequencing:

  • Perform sequencing reactions in forward directions using Big Dye Terminator Cycle Sequencing Kit

  • Analyze sequences to identify the presence or absence of the 7 bp region

This methodology allows researchers to reliably characterize the pks15/1 locus for genomic studies and epidemiological surveillance.

What biochemical assays can be used to study Pks15/1 enzymatic activity in vitro?

Several biochemical assays have been developed to study the enzymatic activity of Pks15/1 in vitro:

• Protein phosphopantetheinylation analysis:

  • Use Bacillus subtilis phosphopantetheinyl transferase Sfp to phosphopantetheinylate Pks15/1 in vitro

  • Standard reaction mixture contains:

    • 4 μM Pks15/1 (wild-type or mutant)

    • 100 mM sodium phosphate buffer (pH 7.2)

    • 10% glycerol

    • 2 mM TCEP

    • 0.5 mM MgCl₂

    • 50 nM Sfp

    • 50 μM biotinyl-CoA

  • Incubate at 30°C for 15 minutes

  • Analyze by SDS-PAGE and Western blot using antibiotin-alkaline phosphatase conjugated antibody

  • Detect biotinylated proteins using alkaline phosphatase BCIP/NBT

• Radioactive labeling assays:

  • Study incorporation of covalently bound [¹⁴C]malonyl-CoA-derived label onto Pks15/1

  • The standard reaction mixture includes:

    • 50 μM [¹⁴C]malonyl-CoA

    • 100 mM sodium phosphate buffer (pH 7.2)

    • 10% glycerol

    • 1 mM TCEP

    • 0.5 mM MgCl₂

    • 1 mM ATP

    • 8 μM Pks15/1 (wild-type or mutant)

  • Incubate at 30°C for 3 minutes

  • Analyze by SDS-PAGE

  • Detect ¹⁴C-labeled proteins using storage phosphor screens and a scanner

• FadD22-Pks15/1 reconstituted systems:

  • Establish in vitro systems that combine purified FadD22 and Pks15/1

  • Monitor the formation of p-hydroxyphenylalkanoate intermediates

  • Study the functional cooperation between these enzymes for the production of PGL precursors

These assays provide valuable insights into the enzymatic mechanisms of Pks15/1 and its interactions with other components of the PGL biosynthetic pathway.

How does Pks15/1 contribute to mycobacterial virulence?

Pks15/1 contributes to mycobacterial virulence primarily through its role in the biosynthesis of phenolic glycolipids (PGLs), which are important virulence factors in several pathogenic mycobacteria:

Understanding the precise mechanisms by which Pks15/1-derived PGLs enhance virulence remains an active area of research and may provide insights into novel therapeutic approaches targeting this pathway.

What is the proposed biosynthetic pathway involving Pks15/1 for phenolphthiocerol production?

The biosynthetic pathway for phenolphthiocerol (PPOL) production involving Pks15/1 follows a systematic sequence of enzymatic reactions:

• Initiation: FadD22, a coenzyme A-independent stand-alone didomain initiation module, activates p-hydroxybenzoic acid (pHBA) using ATP to form a p-hydroxybenzoyl-S-ArCP (aryl carrier protein) thioester intermediate .

• Transfer to Pks15/1: The p-hydroxybenzoyl group is transferred from the ArCP domain of FadD22 to the ketosynthase (KS) domain active-site cysteine residue of Pks15/1, forming a p-hydroxybenzoyl-KS transient intermediate .

• Chain elongation: The p-hydroxybenzoyl-KS intermediate serves as a substrate for Pks15/1-dependent acyl chain elongation:

  • The acyltransferase (AT) domain loads malonyl-CoA onto the acyl carrier protein (ACP) domain

  • The KS domain catalyzes the decarboxylative condensation of the malonyl unit with the p-hydroxybenzoyl starter

  • The keto reductase (KR), dehydratase (DH), and enoyl reductase (ER) domains perform reductive processing of the β-keto group

  • This cycle repeats multiple times (iterative catalysis)

• Intermediate formation: This iterative process results in the production of p-hydroxyphenylalkanoate (PHPA) intermediates with varying chain lengths .

• Further processing: The PHPA intermediates are likely further extended by the PpsA-E noniterative type I PKS system to complete PPOL biosynthesis .

• Completion of PGL: Finally, PPOL is esterified with characteristic long-chain multimethyl-branched fatty acids by the acyltransferase PapA5 to form complete PGLs .

The relaxed control of catalytic cycle iterations by Pks15/1 explains the characteristic alkyl chain length variability seen in mycobacterial PGLs. For example, in M. marinum, the PPOL with the largest carbon chain would require 11 elongation cycles by Pks15/1 to produce 23-(4-hydroxyphenyl)tricosanoic acid as the PHPA intermediate .

How can transcriptome analysis be used to study pks15/1 expression under different growth conditions?

Transcriptome analysis provides valuable insights into pks15/1 expression patterns under various growth conditions, offering a deeper understanding of its regulation and role in mycobacterial physiology:

• RNA-seq approaches: Modern RNA-seq techniques can be applied to study pks15/1 expression across different growth conditions. The search results mention the availability of "publicly available datasets of transcriptome data (RNA-seq) from more than 100 MTBC experiments in 40 growth conditions" that can be leveraged to analyze pks15/1 expression patterns .

• Differential expression analysis: By comparing pks15/1 expression levels across various growth conditions (such as nutrient limitation, hypoxia, acidic pH, or exposure to host factors), researchers can identify specific environmental triggers that modulate pks15/1 transcription.

• Co-expression networks: Analysis of genes that are co-expressed with pks15/1 can reveal regulatory networks and functional associations, potentially identifying previously unknown participants in the PGL biosynthetic pathway.

• Experimental design considerations:

  • Use appropriate reference genes for normalization

  • Include biological and technical replicates

  • Consider time-course experiments to capture dynamic expression changes

  • Compare wild-type strains with mutants lacking specific regulatory elements

• Integration with other omics data: Combining transcriptome data with proteomics, metabolomics, and chromatin immunoprecipitation (ChIP-seq) can provide a more comprehensive understanding of pks15/1 regulation and function.

By systematically analyzing pks15/1 expression across different conditions, researchers can identify the regulatory mechanisms controlling PGL biosynthesis and potentially discover novel intervention targets that could disrupt this virulence-associated pathway.

What are the challenges in expressing and purifying recombinant Pks15/1 for structural studies?

Expressing and purifying recombinant Pks15/1 for structural studies presents several significant challenges that researchers must address:

• Large protein size: Pks15/1 is a massive protein (2104-2118 amino acids), making expression of the full-length protein extremely challenging in conventional expression systems .

• Multiple catalytic domains: The presence of six distinct catalytic domains (KS, AT, DH, ER, KR, and ACP) increases the complexity of proper protein folding during recombinant expression .

• Post-translational modifications: The ACP domain requires post-translational phosphopantetheinylation to be functionally active, necessitating co-expression with a phosphopantetheinyl transferase (such as Sfp from Bacillus subtilis) or an additional modification step during purification .

• Protein solubility: Type I PKSs are often prone to aggregation and poor solubility when overexpressed, requiring optimization of:

  • Expression temperature (typically lowered to 16-18°C)

  • Induction conditions (lower IPTG concentrations)

  • Specialized solubility tags (such as MBP, SUMO, or TrxA)

  • Co-expression with chaperones

• Purification strategy:

  • Multi-step purification is typically required

  • Buffer optimization to maintain stability

  • Potential need for detergents or amphipathic additives

  • Consideration of domain boundaries for domain-by-domain approach

• Protein stability: Once purified, maintaining the stability of the large multi-domain protein during concentration and crystallization attempts represents another major challenge.

• Domain-based approach alternative: Due to these challenges, many researchers opt for a domain-by-domain approach, expressing and studying individual domains or combinations of functionally linked domains rather than the entire protein.

Based on the search results, researchers have successfully produced and purified recombinant Pks15/1 for functional studies , suggesting that some of these challenges can be overcome with careful optimization of expression and purification conditions.

What mechanisms control the iterative catalysis of Pks15/1 and determine the final product structure?

The mechanisms controlling iterative catalysis in Pks15/1 and determining the final product structure remain incompletely understood and represent an exciting frontier in PKS research:

• Distinct control mechanisms: Unlike non-reducing iterative type I PKSs that contain product template (PT) and thioesterase (TE) domains involved in product chain length control and release, reducing iterative type I PKSs like Pks15/1 lack these domains. This suggests a distinct mechanism for controlling the structure of the final products .

• Relaxed iteration control: Evidence indicates that Pks15/1 has a relaxed control of catalytic cycle iterations, which explains the characteristic alkyl chain length variability seen in mycobacterial PGLs. This property distinguishes it from more strictly programmed PKS systems .

• Programming hypothesis: Du and colleagues proposed that "the final size of highly reduced polyketide products of iterative type I PKSs is not determined by the PKSs alone." This suggests that in the case of Pks15/1, the complete information for final product size may be "programmed" via interactions with other enzymes in the PGL pathway .

• Partner enzyme interactions: The functional partnerships between Pks15/1 and downstream enzymes (such as the PpsA-E system) likely play a critical role in defining the final products of the pathway. These interactions may provide contextual cues that influence Pks15/1's catalytic programming .

• Research opportunities: The FadD22-Pks15/1 reconstituted systems described in the literature provide "a first foundation for future efforts to unveil the mechanism of iterative catalysis control" and to examine the functional partnerships that define the final products .

Understanding these control mechanisms would not only advance our knowledge of PKS enzymology but could also enable the engineering of these systems for the production of novel bioactive compounds with potential therapeutic applications.

How could targeting Pks15/1 contribute to novel therapeutic approaches against mycobacterial infections?

Targeting Pks15/1 offers promising avenues for developing novel therapeutic approaches against mycobacterial infections, particularly for drug-resistant strains:

• Virulence factor inhibition: Since PGLs are important virulence factors but not essential for basic mycobacterial survival, inhibiting Pks15/1 could reduce pathogenicity without directly killing the bacteria. This approach might face less selective pressure for resistance development compared to traditional antibiotics .

• Anti-virulence strategy: Compounds that inhibit Pks15/1 could serve as anti-virulence agents that work by:

  • Disrupting host-pathogen interactions

  • Enhancing immune clearance of infection

  • Potentially working synergistically with conventional antibiotics

• Rational drug design targets: The multiple catalytic domains of Pks15/1 provide several potential druggable sites:

  • The KS domain's active site cysteine is critical for catalysis

  • The AT domain's substrate binding pocket determines extender unit specificity

  • The interfaces between FadD22 and Pks15/1 represent protein-protein interaction targets

• Alternative defense strategies: As noted in the search results, "elucidation of the PGL biosynthetic pathway will not only expand our understanding of natural product biosynthesis, but may also illuminate routes to novel therapeutics to afford alternative lines of defense against mycobacterial infections" .

• Lineage-specific approaches: Given that the intact pks15/1 gene is particularly associated with Asian lineages (including Beijing strains) of M. tuberculosis, inhibitors targeting this enzyme might be especially effective against these lineages, which are often associated with drug resistance in certain geographical regions .

Developing inhibitors of Pks15/1 would require a deep understanding of its structure-function relationships and catalytic mechanisms. The reconstituted enzymatic systems described in the literature provide valuable platforms for screening potential inhibitors and understanding their mechanisms of action .