Recombinant Phthiodiolone/phenolphthiodiolone dimycocerosates ketoreductase (Rv2951c, MT3025)

What is Rv2951c and what is its role in Mycobacterium tuberculosis?

Rv2951c is a gene in Mycobacterium tuberculosis that encodes phthiodiolone/phenolphthiodiolone dimycocerosates ketoreductase (PKR), a key enzyme in the biosynthesis pathway of phthiocerol dimycocerosates (PDIMs). This gene is located between positions 3303103 and 3304248 on the negative strand of the M. tuberculosis genome and encodes a protein of 381 amino acids . The enzyme catalyzes the reduction of phthiodiolones to phthiotriols, which are subsequently methylated to yield phthiocerols . This two-step process is crucial for the synthesis of PDIMs, which are important components of the mycobacterial cell envelope and contribute significantly to virulence and pathogenicity .

How does the Rv2951c-encoded enzyme affect M. tuberculosis pathogenicity?

The enzyme encoded by Rv2951c plays a critical role in M. tuberculosis pathogenicity through its contribution to the synthesis of phthiocerol dimycocerosates (PDIMs). These complex lipids protect the pathogen from the early innate immune response of infected hosts . Genetic studies with Rv2951c mutants have demonstrated that disruption of this gene prevents the formation of phthiocerol derivatives but leads to the accumulation of phthiodiolone dimycocerosates . Comparative analyses of wild-type strains and mutants have established that both phthiocerol and phthiodiolone dimycocerosates contribute to the pathogenicity of M. tuberculosis in mice, highlighting their functional redundancy in bacterial virulence .

What cofactor does phthiodiolone ketoreductase require for activity?

Phthiodiolone ketoreductase (PKR) is an F420H2-dependent enzyme, also designated as fPKR . F420 is a flavin-related cofactor universally present in mycobacteria but absent in humans, making it an attractive target for therapeutic intervention . The enzyme utilizes the reduced form of F420 (F420H2) to catalyze the reduction of phthiodiolones to phthiotriols. F420H2 is generated from F420 and glucose-6-phosphate by F420-dependent glucose-6-phosphate dehydrogenase (Fgd) . This cofactor dependency places PKR in a family of enzymes that includes several others that help M. tuberculosis evade killing by the host immune system .

What is the biochemical function of phthiodiolone ketoreductase in cell wall lipid synthesis?

The biochemical function of phthiodiolone ketoreductase is to catalyze the reduction of keto groups in phthiodiolone and phenolphthiodiolone molecules to hydroxyl groups, forming phthiotriol and phenolphthiotriol intermediates . This reaction follows the scheme:

Phthiodiolones + F420H2 → Phthiotriols + F420

These hydroxylated intermediates are subsequently methylated by the methyltransferase encoded by Rv2952 to produce phthiocerols and phenolphthiocerols . These modified lipids are incorporated into the complex cell envelope structure as dimycocerosate esters, contributing to the waxy barrier that protects mycobacteria from environmental stresses and host immune responses .

What are phthiocerol dimycocerosates (PDIMs) and how do they relate to Rv2951c?

Phthiocerol dimycocerosates (PDIMs or DIM A) are complex waxy lipids found in the cell envelope of M. tuberculosis and related pathogenic mycobacteria such as M. bovis and M. leprae . They exist alongside related compounds:

• Phthiodiolone dimycocerosates (DIM B)

• Glycosylated phenolphthiocerol dimycocerosates (PGL-tb)

• Glycosylated phenolphthiodiolone dimycocerosates

The structural distinction between these compounds involves the presence of either methoxy groups (in phthiocerol derivatives) or keto groups (in phthiodiolone derivatives) . Rv2951c encodes the ketoreductase that converts the keto-containing compounds to hydroxylated intermediates in the pathway toward methoxy-containing lipids . Disruption of Rv2951c blocks this conversion, causing accumulation of keto-containing lipids (DIM B) and elimination of methoxy-containing lipids (DIM A) .

What experimental approaches can be used to assess phthiodiolone ketoreductase activity in vitro?

Assessment of phthiodiolone ketoreductase activity requires specific consideration of its F420H2 cofactor dependency. Several methodological approaches are viable:

Spectrophotometric assay:
This approach exploits the absorption properties of F420H2, which absorbs at 420 nm while the oxidized form does not. The enzyme activity can be monitored by following the decrease in absorbance at 420 nm as the reaction proceeds.

Reconstituted pathway assay:
This more comprehensive system includes:

• F420

• Glucose-6-phosphate

• F420-dependent glucose-6-phosphate dehydrogenase (Fgd) for in situ F420H2 generation

• Phthiodiolone substrate

• Purified Rv2951c protein

The search results describe such a system where "the reaction mixture was competent in reducing phthiodiolones to phthiotriols, which were then methylated to phthiocerols" .

Product analysis:
Direct analysis of reaction products can be performed using:

• High-performance liquid chromatography (HPLC)

• Thin-layer chromatography (TLC)

• Mass spectrometry (MS)

These methods allow quantification of substrate consumption and product formation rates under various conditions.

How can recombinant Rv2951c be expressed and purified for structural and functional studies?

Expression and purification of recombinant Rv2951c requires careful consideration of protein stability and activity. Based on available information, the following methodological approach is recommended:

Expression system selection:

• Bacterial expression in E. coli (typically BL21(DE3) strain)

• Mycobacterial expression systems for native-like post-translational modifications

• Insect cell systems for potentially improved folding

Vector design:

• Include affinity tags (His6, GST, or MBP) for purification

• Consider codon optimization for the expression host

• Include TEV protease cleavage site for tag removal

Purification protocol:

• Cell lysis under optimized buffer conditions

• Initial purification by affinity chromatography

• Secondary purification by size-exclusion or ion-exchange chromatography

• Quality assessment by SDS-PAGE (target >85% purity)

Storage conditions:
Based on commercial product information, reconstitute purified protein to 0.1-1.0 mg/mL in deionized sterile water, followed by addition of glycerol to a final concentration of 5-50% . Store working aliquots at 4°C for up to one week and long-term storage at -20°C/-80°C .

What are the implications of Rv2951c mutations on M. tuberculosis virulence and cell envelope integrity?

Genetic studies with Rv2951c mutants have revealed significant implications for bacterial physiology and pathogenesis:

Lipid profile alterations:

• Elimination of phthiocerol dimycocerosates (DIM A) and phenolic glycolipids (PGL-tb)

• Accumulation of phthiodiolone dimycocerosates (DIM B) and glycosylated phenolphthiodiolone dimycocerosates

This data highlights the complexity of mycobacterial cell envelope composition and underscores the adaptability of the pathogen in maintaining virulence factors.

What is the evolutionary conservation of Rv2951c across mycobacterial species?

The evolutionary conservation of Rv2951c reflects its important role in mycobacterial lipid metabolism:

Distribution across species:

• In Mycobacterium tuberculosis: Annotated as Rv2951c

• In Mycobacterium bovis: Annotated as BCG_2972c

• In Mycobacterium leprae: Not specifically named in search results, but implied by the presence of "similar waxy lipids"

Conservation pattern:
The enzyme belongs to a family of F420-dependent enzymes found throughout mycobacteria. Phylogenetic analysis mentioned in the search results revealed "potential F420-dependent lipid-modifying enzymes in a broad range of mycobacteria" , suggesting this enzyme family extends beyond just tuberculosis-causing species.

Functional significance:
The conservation of this enzyme family correlates with the presence of complex cell envelope lipids across pathogenic mycobacteria. This conservation pattern underscores the importance of these enzymes in the distinctive lipid metabolism that characterizes mycobacteria as a genus.

How can F420H2-dependent activity of phthiodiolone ketoreductase be measured in comparative studies?

Measuring and comparing F420H2-dependent activity of phthiodiolone ketoreductase across different experimental conditions requires specialized methodological approaches:

Spectrophotometric assay optimization:

• Wavelength: Monitor at 420 nm (absorption maximum of F420H2)

• Temperature range: 25-37°C (physiological relevance)

• pH optimization: Typically 6.5-7.5 for mycobacterial enzymes

• Buffer selection: Phosphate or HEPES buffers with appropriate ionic strength

Reaction rate determination:

• Initial velocity measurements at varying substrate concentrations

• Calculation of kinetic parameters (Km, Vmax, kcat)

• Determination of pH and temperature optima

• Effects of potential inhibitors on reaction rates

Coupled enzyme system standardization:
When using the F420/G6P/Fgd system to generate F420H2, standardize:

• F420 concentration (typically 10-50 μM)

• G6P concentration (1-5 mM)

• Fgd enzyme activity (in excess to ensure non-limiting)

• Phthiodiolone substrate concentration range

Control reactions:
Include parallel reactions:

• Without enzyme (negative control)

• Without F420 (to confirm F420-dependence)

• Without G6P (when using the coupled system)

• With heat-inactivated enzyme

This methodological framework allows reliable comparison of enzyme activities across different experimental conditions, mutant forms, or homologs from different mycobacterial species.

What are potential inhibitors of phthiodiolone ketoreductase and their implications for TB drug development?

The unique features of phthiodiolone ketoreductase make it an attractive target for anti-tuberculosis drug development:

Target validation rationale:

• PDIMs are established virulence factors that "protect this pathogen from the early innate immune response"

• The enzyme relies on F420H2, a cofactor that is "universally present in mycobacteria and absent in humans," providing excellent selectivity potential

• The PDIM synthesis pathway represents a novel target space distinct from current TB drugs

Potential inhibitor classes:

• F420 analogs: Compounds that compete with the natural cofactor

• Substrate mimics: Molecules structurally similar to phthiodiolones but unable to undergo reduction

• Transition state analogs: Compounds that mimic the reaction intermediate

• Allosteric inhibitors: Molecules binding outside the active site to alter enzyme conformation

Screening strategies:

• High-throughput biochemical assays monitoring F420H2 oxidation

• Structure-based virtual screening if protein structure becomes available

• Fragment-based drug discovery approaches

• Repurposing screens of existing drug libraries

Implications for therapy:
The search results note that the enzyme is part of a group of F420-dependent enzymes in M. tuberculosis, "each of which helps the pathogen to evade killing by the host immune system" . This suggests that inhibitors targeting this enzyme family might have broader effects beyond just blocking PDIM synthesis, potentially increasing their therapeutic efficacy.

How do phthiocerol dimycocerosates and phthiodiolone dimycocerosates differ in their contribution to the mycobacterial cell envelope?

The structural and functional differences between these related lipid classes reveal important aspects of mycobacterial cell envelope biology:

Structural comparison:

Functional overlap:
Experimental comparisons between wild-type strains, Rv2951c mutants (producing only phthiodiolone derivatives), and mutants lacking both lipid types revealed:

• "Functional redundancy between phthiocerol and phthiodiolone dimycocerosates in both the protection of the mycobacterial cell and the pathogenicity of M. tuberculosis in mice"

• Similar profiles in resistance to detergents like SDS, indicating comparable contributions to the permeability barrier properties of the cell envelope

• Maintained virulence in mouse infection models despite the altered lipid profile