Recombinant Photobacterium profundum Biotin synthase (bioB)

What is the basic function of biotin synthase from P. profundum?

Biotin synthase (BioB) from P. profundum, like other BioB enzymes, catalyzes the final step in biotin biosynthesis by converting dethiobiotin (DTB) to biotin. This reaction involves introducing a sulfur atom between the C6 and C9 positions of dethiobiotin to complete the thiophane ring of biotin . The reaction proceeds through a stepwise formation of two carbon-sulfur bonds and requires radical chemistry initiated by S-adenosylmethionine (SAM) . As a deep-sea enzyme from a piezophilic organism, P. profundum BioB likely exhibits pressure-adapted structural and catalytic properties compared to mesophilic homologs.

What iron-sulfur clusters are present in BioB and what roles do they play?

BioB contains two distinct iron-sulfur clusters essential for catalysis:

The [2Fe-2S]²⁺ cluster has been shown through isotope labeling studies to be the direct source of the sulfur atom incorporated into biotin, making BioB a "suicide enzyme" that requires cluster reassembly after each catalytic cycle .

How does P. profundum's piezophilic nature potentially influence BioB structure and function?

P. profundum grows optimally at 28 MPa (approximately 280 times atmospheric pressure) and can grow across a wide pressure range (0.1-90 MPa) . This adaptation likely influences its BioB enzyme in several ways:

• Protein flexibility: The enzyme likely has structural adaptations that maintain conformational flexibility under high pressure

• Active site architecture: Possibly altered to accommodate volume changes during catalysis under pressure

• Protein-protein interactions: The interactions with chaperones like HscA may be pressure-optimized

• Cluster stability: Iron-sulfur clusters may have pressure-adapted coordination environments

Proteomic studies of P. profundum have shown differential expression of various metabolic enzymes under high versus atmospheric pressure, indicating sophisticated pressure-responsive regulation systems .

What are the optimal conditions for recombinant expression of P. profundum BioB?

Based on established protocols for other BioB enzymes and considering P. profundum's unique characteristics, the following expression system is recommended:

When expressing P. profundum BioB, researchers should consider that the organism's ability to grow at atmospheric pressure makes standard expression systems viable, but pressure treatment post-purification may be necessary to observe native-like activity .

What methodologies are critical for effective iron-sulfur cluster reconstitution in recombinant P. profundum BioB?

Iron-sulfur cluster reconstitution is essential for BioB activity. The following protocol is recommended:

• [4Fe-4S]²⁺ cluster reconstitution: This cluster can be reconstituted using Fe³⁺, S²⁻, and dithiothreitol under strict anaerobic conditions .

• [2Fe-2S]²⁺ cluster reconstitution: This cluster is more challenging to reconstitute chemically and may require:

  • Biological scaffolds such as the ISC (Iron-Sulfur Cluster) assembly system components

  • The molecular chaperone HscA, which significantly improves the efficiency of [2Fe-2S]²⁺ cluster assembly in vivo

  • IscU, which serves as a scaffold for cluster assembly before transfer to BioB

• Pressure considerations: Reconstitution may need to be performed at elevated pressures to achieve maximum occupancy for the P. profundum enzyme.

For in vivo cluster formation, co-expression with ISC system proteins (especially HscA) has been shown to improve cluster assembly, with HscA binding with increased affinity to BioB missing one or both FeS clusters .

How can researchers assess iron-sulfur cluster integrity in purified P. profundum BioB?

Multiple complementary techniques should be employed:

Electron paramagnetic resonance (EPR) has been particularly valuable in characterizing BioB reaction intermediates, as demonstrated in studies that captured the structure of a paramagnetic intermediate generated during catalysis .

What is the detailed mechanism of the BioB-catalyzed reaction?

The BioB reaction proceeds through the following steps:

• Reductive cleavage of AdoMet by the [4Fe-4S]²⁺ cluster, generating a 5'-deoxyadenosyl radical and methionine

• Hydrogen atom abstraction from dethiobiotin by the 5'-deoxyadenosyl radical, creating a substrate-centered carbon radical

• Formation of the first C-S bond at C9, creating 9-mercaptodethiobiotin as a discrete intermediate

• A second round of AdoMet cleavage and radical generation

• Formation of the second C-S bond at C6, completing the thiophane ring

• Dissociation of biotin and the remnants of the [2Fe-2S]²⁺ cluster

This mechanism has been elucidated through multiple experimental approaches, including EPR studies that captured key paramagnetic intermediates . The sulfur inserted between C6 and C9 is derived from the [2Fe-2S]²⁺ cluster, as demonstrated by isotope labeling studies with ³⁴S .

How does the cysteine desulfurase activity of BioB relate to its biotin synthase activity?

BioB displays significant cysteine desulfurase activity, which may be connected to its biotin synthase function:

• BioB can mobilize sulfur from free cysteine through a PLP-dependent mechanism

• This reaction proceeds through a protein-bound persulfide intermediate

• Two conserved cysteines, Cys97 and Cys128, are critical for this desulfuration activity

• Biotin synthase activity is significantly enhanced by PLP and cysteine

This suggests that cysteine desulfuration might provide an alternative pathway for sulfur insertion or play a role in regenerating the [2Fe-2S]²⁺ cluster after catalysis . The phenomenon raises intriguing questions about whether P. profundum BioB has evolved pressure-specific adaptations in this auxiliary activity.

What factors limit the turnover number of biotin synthase and how might this differ in P. profundum?

BioB has a limited turnover number due to the destruction of the [2Fe-2S]²⁺ cluster during catalysis:

In E. coli, each BioB polypeptide chain is capable of up to 20 turnovers prior to degradation . The pressure adaptation of P. profundum might influence this turnover limit, as proteins differentially expressed under pressure include those involved in key metabolic pathways .

What experimental setup is required to study pressure effects on recombinant P. profundum BioB activity?

A comprehensive pressure-enzyme activity study requires specialized equipment:

• High-pressure reaction vessels:

  • Stainless steel vessels with pressure ratings up to 100 MPa

  • Temperature control systems (optimal temperature for P. profundum is 15°C)

  • Mixing mechanisms compatible with high pressure

• Activity measurement approaches:

  • Endpoint analysis: Pressurize reaction mixture, incubate, release pressure, measure biotin formation

  • Real-time monitoring: Specialized vessels with optical windows for spectroscopic measurements under pressure

  • Quench-flow systems adapted for high pressure

• Control experiments:

  • Parallel assays with E. coli BioB as a non-piezophilic control

  • Pressure stability tests for all reaction components

  • Activity measurements across the full viable pressure range (0.1-90 MPa)

This experimental design should be complemented with structural studies to correlate activity changes with potential conformational changes under pressure.

How can researchers distinguish pressure effects on protein structure versus catalytic mechanism?

This requires a multi-technique approach:

By distinguishing between effects on substrate binding (Km) versus turnover rate (kcat), researchers can determine whether pressure primarily affects the enzyme conformation or specific catalytic steps. Comparing wild-type with site-directed mutants can further pinpoint pressure-sensitive regions of the protein.

What are the hypothesized molecular adaptations in P. profundum BioB that could confer pressure tolerance?

Based on known pressure adaptations in other proteins from piezophiles and the specific requirements of BioB catalysis:

• Amino acid composition:

  • Increased glycine content in flexible regions

  • Reduced number of surface-exposed hydrophobic residues

  • Strategic placement of charged residues to maintain hydration under pressure

• Iron-sulfur cluster coordination:

  • Potentially altered coordination geometry of the [2Fe-2S]²⁺ cluster

  • Modified protein environment around clusters to accommodate pressure-induced electronic changes

• Active site architecture:

  • Larger active site volume to accommodate pressure-induced compression

  • Modified substrate binding interactions that are less sensitive to pressure

• Protein-protein interactions:

  • Pressure-optimized interaction surfaces for HscA binding

  • Enhanced interaction with IscU for more efficient cluster transfer under pressure

Proteomic studies have shown that P. profundum differentially expresses proteins involved in key metabolic pathways depending on pressure conditions, suggesting sophisticated pressure adaptation mechanisms .

How can comparative genomics and structural biology elucidate pressure adaptation in P. profundum BioB?

Advanced comparative approaches can reveal evolutionary adaptations to pressure:

• Multi-species sequence analysis:

  • Compare BioB sequences from piezophilic, piezotolerant, and piezosensitive organisms

  • Identify conserved substitutions specific to piezophiles

  • Construct phylogenetic trees to trace adaptation evolution

• Structural comparison:

  • Solve high-resolution structures of BioB from multiple species at various pressures

  • Map pressure-specific substitutions onto structural models

  • Identify regions with altered flexibility or packing

• Ancestral sequence reconstruction:

  • Infer ancestral BioB sequences before pressure adaptation

  • Express and characterize ancestral proteins

  • Identify key mutations that conferred pressure tolerance

This multi-faceted approach can identify both conserved pressure adaptation mechanisms and those unique to the biotin synthesis pathway.

What experimental strategies can address the hypothesis that P. profundum BioB has evolved pressure-optimized FeS cluster assembly mechanisms?

Testing this hypothesis requires several complementary approaches:

• In vivo cluster assembly studies:

  • Express BioB under various pressure conditions

  • Quantify cluster incorporation efficiency

  • Compare with E. coli BioB as control

• Protein-protein interaction analysis under pressure:

  • Examine BioB interactions with HscA and IscU at various pressures

  • Determine if the BioB:HscA:IscU complex is pressure-stable

  • Measure binding affinities as a function of pressure

• Domain swap experiments:

  • Create chimeric proteins with domains from piezophilic and non-piezophilic BioB

  • Identify domains critical for pressure-optimized cluster assembly

• Site-directed mutagenesis:

  • Target residues near cluster binding sites

  • Introduce mutations that mimic piezophilic adaptations into E. coli BioB

  • Examine pressure effects on mutant activity

These experiments would provide insights into whether P. profundum has evolved specialized mechanisms for maintaining iron-sulfur cluster integrity under deep-sea conditions.

How can researchers investigate the interplay between pressure and temperature adaptation in P. profundum BioB?

P. profundum is both piezophilic (pressure-loving) and psychrophilic (cold-loving), growing optimally at 28 MPa and 15°C . This dual adaptation creates a complex evolutionary landscape:

• Pressure-temperature activity matrices:

  • Measure BioB activity across a grid of pressure (0.1-90 MPa) and temperature (4-37°C) conditions

  • Generate 3D activity landscapes

  • Identify potential synergistic or antagonistic effects

• Computational analysis:

  • Molecular dynamics simulations incorporating both pressure and temperature variables

  • Identify residues responding to pressure, temperature, or both

  • Model energy landscapes under various conditions

• Comparative studies with BioB from:

  • Piezophilic-psychrophilic organisms (like P. profundum)

  • Piezophilic-mesophilic organisms

  • Piezotolerant-psychrophilic organisms

  • Mesophilic-mesothermal organisms (like E. coli)

This research could reveal whether P. profundum has evolved independent mechanisms for pressure and temperature adaptation or whether these adaptations are mechanistically linked.