Recombinant Photobacterium profundum Pimelyl-[acyl-carrier protein] methyl ester esterase (bioH)

What is the physiological role of Pimelyl-[acyl-carrier protein] methyl ester esterase in bacterial biotin biosynthesis?

Pimelyl-[acyl-carrier protein] methyl ester esterase (bioH) plays a critical role in biotin biosynthesis by catalyzing the last biosynthetic step of the pimelate moiety, a key intermediate in the pathway. The enzyme's primary function is to remove the methyl group introduced by BioC when the pimeloyl moiety synthesis is complete. This allows the synthesis of pimeloyl-ACP via the fatty acid synthetic pathway through hydrolysis of the ester bonds of pimeloyl-ACP esters .

In the well-characterized E. coli system, bacteria employ a methylation and demethylation strategy to enable elongation of a temporarily disguised malonate moiety to a pimelate moiety using the fatty acid synthesis machinery. The BioC methyltransferase initiates the process by methylating the free carboxyl group of malonyl-ACP, allowing it to enter the fatty acid synthesis pathway. After two elongation cycles, BioH removes this methyl group, effectively acting as a "gatekeeper" that prevents further elongation and produces pimeloyl-ACP for subsequent steps in biotin synthesis .

How can I determine if Photobacterium profundum bioH utilizes the same catalytic mechanism as E. coli bioH?

To determine if P. profundum bioH utilizes the same catalytic mechanism as its E. coli counterpart, researchers should:

• Sequence alignment and structural prediction: Analyze the protein sequence to identify the conserved catalytic triad (typically Ser-His-Asp) found in E. coli bioH. For example, in the Moraxella catalytic triad (Ser117-His254-Asp287), these residues are essential for the hydrolysis of pimeloyl-ACP methyl ester .

• Site-directed mutagenesis: Create point mutations at the predicted catalytic residues in the P. profundum bioH gene and express these recombinant variants. Loss of function in these mutants would confirm the importance of these residues in the catalytic mechanism.

• Enzymatic assays with varying substrates: Test purified recombinant P. profundum bioH with substrates of differing acyl chain lengths to determine substrate preference. E. coli bioH shows preference for short chain fatty acid esters (acyl chain length up to 6 carbons) and short chain p-nitrophenyl esters .

• In vitro reconstitution assays: Establish a reconstituted desthiobiotin synthesis system similar to that used for BtsA characterization in Moraxella catarrhalis to confirm the enzyme's function in the context of the complete biosynthetic pathway .

What are the optimal growth conditions for Photobacterium profundum as a precursor to bioH studies?

P. profundum is a piezophilic bacterium with specific growth requirements that must be carefully maintained to ensure proper protein expression and function:

• Pressure conditions: P. profundum grows optimally at 28 MPa (high pressure), though it can grow under a wide range of pressures including atmospheric pressure (0.1 MPa). For physiologically relevant studies of bioH, cultivation at both atmospheric and high pressure conditions is recommended to observe potential pressure-dependent effects .

• Temperature: Optimal growth occurs at 15-17°C. All cultures should be maintained at this temperature range regardless of pressure conditions .

• Media composition: Use 75% strength 2216 Marine Medium (28 g/l) supplemented with 20 mM glucose and 100 mM HEPES buffer (pH 7.5) for optimal growth. For anaerobic conditions, exclude air from culture vessels .

• Culture method: For high-pressure cultivation, inoculate media into sterile vessels (such as plastic Pasteur pipettes) excluding air, seal them properly, and incubate in a water-cooled pressure vessel at 28 MPa and 17°C. For atmospheric pressure growth, use the same media and temperature but maintain at 0.1 MPa .

What expression systems are most effective for producing functional recombinant P. profundum bioH?

Based on available information about recombinant protein expression for similar enzymes:

• E. coli expression systems: E. coli offers the best yields and shorter turnaround times for recombinant bioH expression. Consider using BL21(DE3) or other expression strains with tightly controlled induction systems. The T7 promoter-based expression vectors are generally effective for bioH enzymes .

• Yeast expression systems: Saccharomyces cerevisiae or Pichia pastoris can also provide high yields with proper post-translational modifications that may be necessary for maintaining correct folding and activity of P. profundum bioH .

• Cold-adapted expression protocols: Since P. profundum is adapted to growth at 15-17°C, consider using cold-induction protocols (16-18°C) during the expression phase in E. coli to facilitate proper folding of this psychrophilic enzyme.

• Codon optimization: Given the different codon usage between P. profundum and expression hosts, codon optimization of the bioH gene should be considered to enhance expression levels.

• Fusion tags: Consider testing multiple fusion tags (His6, MBP, GST) to identify optimal solubility and activity preservation. For pressure-adapted enzymes, solubility tags often help maintain proper folding at atmospheric pressure conditions.

What analytical methods should be employed to verify the catalytic activity of purified recombinant P. profundum bioH?

To confirm that purified recombinant P. profundum bioH maintains its catalytic function:

• Esterase activity assays: Measure the hydrolysis of model substrates such as p-nitrophenyl esters, which would produce a colorimetric signal upon hydrolysis. This provides a rapid assessment of general esterase activity .

• LC-MS analysis: Develop an assay to directly monitor the conversion of pimeloyl-ACP methyl ester to pimeloyl-ACP using liquid chromatography-mass spectrometry. This approach can detect the mass difference between substrate and product.

• In vitro reconstitution: Establish a reconstituted biotin synthesis pathway similar to that used for BtsA characterization, where purified P. profundum bioH is combined with other biotin synthesis enzymes and precursors to monitor the complete pathway function .

• Complementation assays: Test whether the P. profundum bioH can complement an E. coli bioH deletion strain (ΔbioH), restoring biotin prototrophy. This provides functional evidence of the enzyme's activity in vivo .

• Pressure-dependent activity measurements: Develop specialized high-pressure bioreactor systems to measure enzyme kinetics under varying pressure conditions, which is particularly relevant for enzymes from piezophilic organisms.

How can protein stability of P. profundum bioH be maintained during purification to preserve native activity?

Preserving the stability of piezophilic enzymes during purification requires special considerations:

• Temperature control: Maintain cold conditions (4-10°C) throughout all purification steps to prevent denaturation of this cold-adapted enzyme.

• Buffer optimization: Include osmolytes (such as glycerol, sorbitol) and stabilizing agents that mimic the high-pressure native environment. Consider testing different buffer compositions with varying ionic strengths.

• Rapid purification protocols: Minimize the time between cell disruption and final purification to reduce exposure to potentially denaturing conditions.

• Activity monitoring: Regularly assess enzyme activity during purification to ensure the native conformation is maintained throughout the process.

• Storage conditions: Determine optimal storage conditions (temperature, buffer composition, additives) that preserve activity for extended periods. Consider flash-freezing aliquots in liquid nitrogen and storing at -80°C with cryoprotectants.

How does high hydrostatic pressure affect the expression and activity of bioH in P. profundum?

To investigate pressure effects on bioH expression and activity:

• Comparative proteomic analysis: Similar to studies performed on P. profundum under different pressure conditions, conduct proteomic analyses comparing bioH expression levels at atmospheric pressure (0.1 MPa) versus high pressure (28 MPa) .

• Transcriptional analysis: Perform qRT-PCR or RNA-Seq to quantify bioH transcript levels under varying pressure conditions to determine if expression is pressure-regulated.

• Enzyme kinetics under pressure: Develop specialized high-pressure enzyme assay systems to measure bioH kinetic parameters (Km, Vmax, kcat) at different pressures. This would require specialized equipment capable of maintaining pressure while allowing spectrophotometric measurements.

• Structural stability studies: Utilize techniques such as circular dichroism or fluorescence spectroscopy under pressure to examine conformational changes in the protein structure at different pressures.

Proteomic studies of P. profundum have shown that many metabolic enzymes are differentially expressed in response to pressure. For example, proteins involved in glycolysis/gluconeogenesis are up-regulated at high pressure, while several proteins involved in oxidative phosphorylation are up-regulated at atmospheric pressure . Similar pressure-dependent regulation might occur for bioH.

To investigate structural adaptations to high pressure:

• X-ray crystallography: Determine the crystal structure of P. profundum bioH and compare with structures from non-piezophilic organisms to identify potential pressure-adaptive features.

• Molecular dynamics simulations: Perform simulations under varying pressure conditions to predict conformational changes and flexibility differences.

• Hydrogen-deuterium exchange mass spectrometry: This technique can identify regions of differential flexibility or solvent accessibility under varying pressure conditions.

• Site-directed mutagenesis validation: Based on structural predictions, create mutants reverting potential pressure-adaptive residues to non-piezophilic counterparts and test their activity and stability under pressure.

• Comparative sequence analysis: Analyze amino acid composition between piezophilic and non-piezophilic bioH enzymes, focusing on features known to contribute to pressure adaptation (e.g., increased glycine content, reduced hydrophobic core packing).

How can we identify and validate the complete biotin synthesis pathway in P. profundum genome?

To characterize the complete biotin synthesis pathway:

• Genome mining: Search the P. profundum genome for homologs of known biotin synthesis genes (bioA, bioB, bioC, bioD, bioF, bioH) using bioinformatic approaches.

• Complementation assays: Clone candidate P. profundum biotin synthesis genes and test their ability to complement corresponding E. coli deletion mutants.

• Transcriptional analysis: Perform RNA-Seq under biotin-limited and biotin-replete conditions to identify co-regulated genes in the biotin synthesis pathway.

• Gene deletion studies: Create targeted gene deletions in P. profundum biotin synthesis genes and assess for biotin auxotrophy.

• Metabolomic profiling: Use LC-MS/MS to measure intermediates in the biotin synthesis pathway under various conditions to validate pathway function.

The biotin synthesis pathway involves multiple enzymes, with the late steps (BioF, BioA, BioD, and BioB) being well-conserved across bacteria, while the early steps responsible for synthesis of the pimelate moiety show considerable diversity .

What is the relationship between bioH function and bacterial adaptation to extreme environments?

To investigate the connection between bioH function and environmental adaptation:

• Comparative genomics: Analyze bioH sequence conservation across bacteria from diverse environments (piezophilic, psychrophilic, mesophilic, thermophilic) to identify environment-specific adaptations.

• Phenotypic characterization: Compare biotin synthesis efficiency under various stress conditions (pressure, temperature, salinity) between wild-type and bioH mutant strains.

• Metabolic profiling: Examine how biotin-dependent metabolic pathways function under diverse environmental conditions, particularly focusing on carboxylation reactions essential for carbon fixation in extreme environments.

• Evolutionary rate analysis: Calculate evolutionary rates of bioH compared to other biotin synthesis genes to identify signals of positive selection or conservation.

Recent research has begun to establish connections between biotin biosynthesis and bacterial virulence. For example, in Moraxella catarrhalis, the biotin synthesis enzyme BtsA is involved in bacterial invasion of lung epithelial cells and survival within macrophages . Similar connections might exist for P. profundum in its adaptation to deep-sea environments.

How do the various isoforms of pimeloyl-ACP methyl ester esterase differ across bacterial species?

Multiple isoforms of pimeloyl-ACP methyl ester esterase have been identified in different bacteria, each catalyzing the same reaction but with distinct evolutionary origins:

• BioH: The paradigm esterase found in E. coli and many other bacteria .

• BioG, BioK, BioJ, and BioV: Alternative esterases discovered in bacteria that encode BioC but not BioH. Each has been demonstrated to catalyze the cleavage of pimeloyl-ACP methyl ester in vitro and to rescue biotin synthesis in E. coli ΔbioH strains .

• BtsA: A novel pimeloyl-ACP methyl ester esterase identified in Moraxella catarrhalis that uses a Ser117-His254-Asp287 catalytic triad but lacks sequence homology with other known isozymes .

To investigate these differences:

• Phylogenetic analysis: Construct phylogenetic trees of all known bioH-like enzymes to understand their evolutionary relationships.

• Structural comparison: Compare crystal structures or homology models to identify conservation and divergence in tertiary structure despite sequence dissimilarity.

• Catalytic mechanism comparison: Determine if all isoforms use the same catalytic mechanism (typically a Ser-His-Asp catalytic triad) despite sequence divergence.

• Regulatory context: Examine the genomic context and regulatory elements associated with each isoform to understand their differential expression.

What are the major challenges in studying pressure effects on enzyme activity and how can they be overcome?

Studying enzyme activity under pressure presents unique technical challenges:

• Specialized equipment requirements: Develop or acquire high-pressure reaction vessels with optical windows or fiber optic probes that allow spectroscopic measurements during pressurization.

• Reaction initiation under pressure: Design pressure vessels that allow for compartmentalization of enzyme and substrate, with mixing capabilities after pressure stabilization.

• Real-time monitoring limitations: Establish methods for sampling or continuous monitoring of reaction progress under pressure without depressurization.

• Data interpretation complexities: Account for pressure effects on pH, substrate solubility, and equipment response when analyzing enzyme kinetic data.

• Control experiments: Design appropriate controls to distinguish direct pressure effects on the enzyme from other pressure-induced physical changes in the reaction system.

Potential solutions include:

• Collaborative work with specialized deep-sea research institutions that have high-pressure equipment

• Development of pressure-cycling approaches where reactions are briefly depressurized for measurement

• Use of pressure-stable fluorescent substrates or probes for continuous monitoring

• Mathematical modeling to account for known pressure effects on reaction parameters

How can contradictory results in bioH functional studies be reconciled and validated?

When faced with contradictory results in bioH research:

• Strain and condition verification: Confirm the exact P. profundum strain being used, as genomic variations can exist. Verify growth conditions, particularly pressure and temperature parameters.

• Protein purification assessment: Evaluate protein purity, folding status, and potential co-purifying factors that might influence activity.

• Substrate quality control: Verify the integrity and purity of substrates, particularly synthetic pimeloyl-ACP methyl ester.

• Methodological standardization: Develop standardized assays with appropriate controls that can be reproduced across laboratories.

• Cross-validation using multiple techniques: Apply orthogonal methods to test the same hypothesis. For example, combine in vitro biochemical assays with in vivo complementation studies and structural analyses.

• Collaborative verification: Establish collaborations between laboratories to independently verify key findings using identical materials and protocols.

The complexity of working with pressure-adapted enzymes often requires integration of multiple lines of evidence before conclusive interpretations can be made.