Recombinant Nitrosomonas europaea 8-amino-7-oxononanoate synthase (bioF)

How does the genomic context of bioF in Nitrosomonas europaea compare to other bacteria?

Based on genomic analyses of Nitrosomonas europaea, the bioF gene exists within its single circular chromosome of 2,812,094 bp . Unlike some other genes in N. europaea that show duplication (such as AMO, HAO, and cytochrome c554), bioF likely exists as a single copy, similar to ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) genes . The genomic organization of biotin synthesis genes in N. europaea may differ from model organisms like E. coli and B. subtilis, potentially reflecting adaptations to its specialized chemolithoautotrophic lifestyle where biosynthetic pathways are particularly important given its limited ability to catabolize organic compounds .

What are the challenges in expressing recombinant Nitrosomonas europaea bioF?

Expression of recombinant N. europaea bioF presents several challenges stemming from the organism's unique physiology. N. europaea is an obligate chemolithoautotroph with specialized metabolism , which may result in codon usage patterns that are suboptimal for expression in common host systems like E. coli. Additionally, as a pyridoxal 5′-phosphate (PLP)-dependent enzyme, proper folding and cofactor incorporation is critical for activity. Researchers must optimize expression conditions including temperature, induction parameters, and host strain selection to overcome potential issues with protein solubility and activity. Purification strategies must also account for the enzyme's potential membrane association or interaction with other cellular components, as N. europaea has evolved specialized mechanisms for handling its unique metabolic requirements.

What is the substrate specificity of Nitrosomonas europaea bioF compared to homologous enzymes from other bacteria?

While definitive experimental data on N. europaea bioF substrate specificity is limited in the provided search results, we can make educated inferences based on characterized homologs. E. coli bioF can utilize either pimeloyl-CoA or pimeloyl-ACP (acyl carrier protein) as acyl chain donors, whereas B. subtilis bioF specifically requires pimeloyl-CoA . Given N. europaea's unique metabolism as an obligate chemolithoautotroph , its bioF may exhibit distinctive substrate preferences adapted to its specialized biochemistry.

The substrate specificity would likely be influenced by the predominant form of activated pimelate available in N. europaea cells. Considering that N. europaea has plentiful genes encoding transporters for inorganic ions but limited transporters for organic molecules , the mechanism of pimelate acquisition or synthesis in this organism may differ from heterotrophic bacteria, potentially influencing bioF substrate preferences.

How does the active site architecture of Nitrosomonas europaea bioF accommodate its catalytic mechanism?

The active site of N. europaea bioF would be expected to contain the canonical pyridoxal 5′-phosphate (PLP) binding motif essential for its decarboxylative condensation mechanism. As a PLP-dependent enzyme catalyzing the condensation of L-alanine with a pimelate thioester, its active site likely includes:

• A lysine residue that forms a Schiff base with PLP

• Basic residues that interact with the carboxylate group of L-alanine

• A hydrophobic pocket to accommodate the pimelate moiety

• Residues that stabilize the transition state during decarboxylation

The reaction proceeds through formation of a PLP-alanine adduct, decarboxylation, and nucleophilic attack on the thioester carbonyl of the pimelate donor. The specific amino acid residues involved in N. europaea bioF may show adaptations reflecting its substrate preference and the biochemical environment of this specialized chemolithoautotroph .

What expression systems are most effective for producing active recombinant Nitrosomonas europaea bioF?

For optimal expression of recombinant N. europaea bioF, a dual-approach strategy is recommended:

E. coli-based expression systems:

• BL21(DE3) or derivatives with pET vector systems under control of T7 promoter

• Co-expression with chaperones (GroEL/GroES) to enhance proper folding

• Supplementation with pyridoxal 5′-phosphate (0.1-0.2 mM) in the growth medium

• Expression at reduced temperature (16-20°C) following induction with low IPTG concentrations (0.1-0.5 mM)

Alternative expression systems:

• B. subtilis expression may be advantageous given the closer phylogenetic relationship to gram-positive bacteria

• Insect cell/baculovirus expression systems for cases where E. coli expression results in insoluble or inactive protein

The effectiveness of each system should be evaluated through activity assays measuring the conversion of pimeloyl-CoA and L-alanine to 8-amino-7-oxononanoate, with optimization of expression conditions based on enzyme activity rather than simply protein yield .

What purification strategy yields the highest specific activity for recombinant Nitrosomonas europaea bioF?

A multi-step purification strategy optimized for preserving the catalytic activity of N. europaea bioF should include:

• Initial capture: Immobilized metal affinity chromatography (IMAC) using a histidine tag (preferably C-terminal to minimize interference with folding)

• Intermediate purification: Ion exchange chromatography

  • Anion exchange (Q-Sepharose) at pH 8.0 (based on theoretical pI)

  • Salt gradient elution (0-500 mM NaCl)

• Polishing step: Size exclusion chromatography

  • Superdex 200 column equilibrated with 50 mM potassium phosphate buffer (pH 7.5) containing 100 mM NaCl, 5% glycerol, and 0.1 mM PLP

Throughout purification, buffer composition should include:

• 50 mM potassium phosphate buffer (pH 7.5)

• 100-150 mM NaCl (to maintain solubility)

• 0.1 mM pyridoxal 5′-phosphate (to maintain cofactor saturation)

• 1 mM DTT or 2 mM β-mercaptoethanol (to protect cysteine residues)

• 5-10% glycerol (to enhance stability)

This strategy typically yields enzyme with ≥95% purity and specific activity comparable to that observed for bioF enzymes from model organisms such as E. coli and B. subtilis .

What are the recommended methods for measuring Nitrosomonas europaea bioF enzymatic activity?

Several complementary approaches can be used to assess N. europaea bioF activity:

Spectrophotometric assay:

• Monitor release of CoA using DTNB (5,5'-dithiobis-(2-nitrobenzoic acid)) at 412 nm

• Reaction mixture: 50 mM HEPES (pH 7.5), 0.2 mM pimeloyl-CoA, 5 mM L-alanine, 0.1 mM PLP, 0.5 mM DTNB

• Initial rates measured at 30°C

HPLC-based assay:

• Reverse-phase HPLC separation of 8-amino-7-oxononanoate product

• Pre-column derivatization with o-phthalaldehyde for fluorescence detection

• C18 column with gradient of acetonitrile in 50 mM sodium phosphate (pH 6.5)

Coupled enzyme assay:

• Link 8-amino-7-oxononanoate production to 7,8-diaminopelargonic acid synthase (bioA) and NADPH oxidation

• Monitor absorbance decrease at 340 nm

• Advantages: continuous monitoring and increased sensitivity

For all assays, proper controls for substrate specificity should include testing both pimeloyl-CoA and pimeloyl-ACP to elucidate the preferred acyl donor for N. europaea bioF, similar to the comparative studies performed with E. coli and B. subtilis enzymes .

How can researchers determine the kinetic parameters of recombinant Nitrosomonas europaea bioF?

To determine comprehensive kinetic parameters for N. europaea bioF, researchers should:

• Establish steady-state kinetics:

  • Vary concentration of pimeloyl-CoA (1-200 μM) while maintaining L-alanine at saturation (10 mM)

  • Vary concentration of L-alanine (0.1-20 mM) while maintaining pimeloyl-CoA at saturation (200 μM)

  • Plot initial velocity data using Michaelis-Menten, Lineweaver-Burk, and Eadie-Hofstee transformations

• Analyze bisubstrate kinetics to determine reaction mechanism:

  • Perform product inhibition studies with CoA and 8-amino-7-oxononanoate

  • Construct double-reciprocal plots at different fixed concentrations of the second substrate

  • Pattern of lines will distinguish between ping-pong, ordered sequential, or random sequential mechanisms

• Determine pH and temperature dependence:

  • Measure activity across pH range 6.0-9.0 using appropriate buffers

  • Assess temperature dependence (15-45°C) and calculate activation energy

Expected kinetic values based on homologous enzymes:

These analyses would reveal whether N. europaea bioF follows similar kinetic patterns to E. coli bioF or more closely resembles B. subtilis bioF in terms of substrate specificity and catalytic efficiency .

How does Nitrosomonas europaea bioF substrate specificity compare with that of E. coli and B. subtilis?

The substrate specificity of bioF enzymes varies significantly between bacterial species, as demonstrated by the well-characterized differences between E. coli and B. subtilis:

For N. europaea, its specialized metabolism as an obligate chemolithoautotroph suggests that the bioF enzyme may have evolved specific adaptations. Given that N. europaea has abundant transporters for inorganic ions but limited transporters for organic molecules , the mechanism for obtaining or synthesizing the pimelate precursor might influence its substrate specificity. The genome analysis of N. europaea revealed genes for biosynthetic pathways , but the specific route for pimelate production remains to be characterized, which would provide insight into the likely acyl-donor for its bioF enzyme.

What structural adaptations might Nitrosomonas europaea bioF have evolved given its unique chemolithoautotrophic lifestyle?

N. europaea bioF likely exhibits several structural adaptations reflecting its host's specialized metabolism:

• Cofactor binding site modifications: As an obligate chemolithoautotroph deriving energy solely from ammonia oxidation , N. europaea faces unique challenges in maintaining proper cofactor (PLP) levels. The bioF enzyme may have evolved enhanced PLP binding affinity or structural elements that protect the cofactor from oxidative damage resulting from the ammonia oxidation process.

• Substrate channel architecture: The enzyme might possess a substrate channel optimized for the predominant form of activated pimelate available in N. europaea. This could include adaptations in the hydrophobic pocket accommodating the pimelate moiety.

• Oligomeric state stabilization: N. europaea experiences significant pH fluctuations during ammonia oxidation to nitrite . The bioF enzyme may have evolved enhanced subunit interfaces to maintain proper oligomeric structure across varying pH conditions.

• Surface charge distribution: The ionic environment within N. europaea differs from heterotrophic bacteria due to its specialized metabolism . bioF may present a distinct surface charge distribution to optimize protein-protein interactions within this environment.

• Regulatory sites: Given the limited carbon sources available to N. europaea , biotin synthesis regulation is likely critical. The bioF enzyme might contain unique allosteric regulatory sites absent in homologs from heterotrophic bacteria.

These adaptive features would reflect the evolutionary pressure to optimize biotin synthesis within the context of N. europaea's specialized metabolic niche .

How can structural studies of Nitrosomonas europaea bioF contribute to understanding substrate specificity in PLP-dependent enzymes?

Structural studies of N. europaea bioF offer several opportunities to advance our understanding of PLP-dependent enzyme substrate specificity:

• Comparative structural biology: N. europaea bioF represents a unique model for studying how PLP-dependent enzymes adapt to specialized metabolic contexts. Structural comparisons with E. coli and B. subtilis bioF enzymes, which have different substrate specificities , would reveal key determinants of acyl-donor recognition.

• Substrate binding pocket analysis: Crystallographic studies of N. europaea bioF in complex with different pimelate thioesters (pimeloyl-CoA and pimeloyl-ACP) would provide direct evidence of substrate preference and binding mechanisms. This would complement the genetic and biochemical data available for E. coli and B. subtilis bioF enzymes .

• Dynamic structural elements: Molecular dynamics simulations based on crystal structures could reveal flexible regions involved in substrate binding and catalysis. These studies could identify structural elements that adapt to different substrates, explaining the molecular basis for the distinct substrate specificities observed among bioF enzymes from different bacterial species .

• Structure-guided mutagenesis: Identification of key residues through structural studies would enable targeted mutagenesis to alter substrate specificity, potentially converting N. europaea bioF between pimeloyl-CoA-specific and dual-specificity forms. Such experiments would provide fundamental insights into substrate recognition in this enzyme family.

These structural insights would not only advance our understanding of bioF enzymes but would contribute to the broader field of PLP-dependent enzyme evolution and substrate specificity determination.

What is the potential role of Nitrosomonas europaea bioF in engineering biotin production pathways?

N. europaea bioF presents several opportunities for engineering enhanced biotin production systems:

• Alternative expression hosts: As noted in search result , biotin is currently produced primarily by chemical synthesis rather than biotechnology. N. europaea bioF could potentially offer advantages in certain expression systems due to its adaptation to a unique metabolic background . Its incorporation into engineered biotin production pathways might provide benefits in specific contexts, particularly in hosts with limited capacity for organic molecule transport.

• Hybrid pathway engineering: Given the different substrate specificities observed between E. coli and B. subtilis bioF enzymes , N. europaea bioF could represent a third distinct variant with unique properties. Hybrid pathways incorporating the most efficient enzymes from different organisms could potentially enhance biotin production. For example:

  • A pathway combining B. subtilis BioW (pimeloyl-CoA synthetase) with the most efficient bioF variant

  • Optimization of the complete pathway from pimelate production through final biotin synthesis

• Adaptation to extreme conditions: N. europaea naturally functions in environments with fluctuating nitrogen compounds and produces reactive nitrogen species like NO and N2O . Its bioF enzyme may have evolved stability features that could be valuable in industrial biotin production processes that encounter similar stresses.

• Co-production systems: The integration of biotin production with ammonia oxidation processes (where N. europaea naturally excels ) could create synergistic production systems that efficiently convert ammonia to multiple valuable products including biotin.

Engineering efforts would need to address the current knowledge gaps regarding N. europaea bioF properties, but the enzyme represents a promising component for next-generation biotin production systems that could potentially compete with chemical synthesis .

How might the nitrogen metabolism of Nitrosomonas europaea influence bioF expression and activity?

N. europaea has a complex nitrogen metabolism centered around ammonia oxidation to nitrite, with production of reactive nitrogen species including NO and N2O . This specialized metabolism likely influences bioF in several ways:

• Regulation in response to nitrogen availability: bioF expression might be coordinated with nitrogen metabolism through regulatory networks. The genome analysis of N. europaea revealed genes necessary for CO2 and NH3 assimilation , suggesting coordinated regulation of carbon and nitrogen pathways that would include biotin synthesis.

• Protection from reactive nitrogen species: The normal metabolism of N. europaea produces reactive nitrogen species that could potentially damage enzymes . bioF may have evolved structural features that protect catalytic residues and the PLP cofactor from modification by these compounds.

• Adaptation to pH fluctuations: Ammonia oxidation to nitrite produces protons, potentially causing local pH changes . The bioF enzyme might exhibit broader pH optima compared to homologs from neutralophilic bacteria to maintain activity across fluctuating pH conditions.

• Energetic constraints: N. europaea derives all its energy from ammonia oxidation , which may impose constraints on biotin synthesis. bioF might show kinetic adaptations reflecting the need to operate efficiently within these energetic limitations.

• Integration with electron transport: N. europaea has a complex electron transport chain centered around ammonia oxidation . The regulation and activity of bioF might be indirectly influenced by the redox state of the cell, which is primarily determined by the nitrogen oxidation processes.

Research examining bioF expression and activity under different nitrogen availability conditions would provide valuable insights into how this critical biosynthetic enzyme is integrated with the specialized metabolism of N. europaea .

What techniques could help resolve contradictory data about Nitrosomonas europaea bioF substrate specificity?

To resolve potential contradictions regarding N. europaea bioF substrate specificity, a multi-faceted experimental approach is recommended:

• Direct biochemical comparison:

  • Express recombinant N. europaea bioF alongside E. coli and B. subtilis bioF enzymes

  • Conduct parallel assays with identical substrates (pimeloyl-CoA and pimeloyl-ACP)

  • Use multiple detection methods to confirm results (spectrophotometric, HPLC, and mass spectrometry)

• Structural biology approaches:

  • Determine crystal structures of N. europaea bioF in complex with both potential substrates

  • Use computational docking and molecular dynamics to assess binding energetics

  • Identify key residues that might determine substrate preference

• In vivo complementation studies:

  • Construct E. coli bioF knockout strains complemented with N. europaea bioF

  • Test growth on media supplemented with different pimelate forms

  • Compare with parallel complementation using E. coli and B. subtilis bioF

• Genetic modifications and chimeric proteins:

  • Create chimeric enzymes swapping key domains between N. europaea, E. coli, and B. subtilis bioF

  • Analyze substrate preference changes in these chimeric enzymes

  • Use site-directed mutagenesis of predicted specificity-determining residues

• Systems biology approach:

  • Examine the genomic context of bioF in N. europaea

  • Identify genes involved in pimelate synthesis/acquisition

  • Map metabolic pathways to predict the likely physiological substrate

This comprehensive approach would provide multiple lines of evidence to resolve contradictions and definitively characterize the substrate specificity of N. europaea bioF, contributing to our understanding of how this enzyme functions within the unique metabolic context of this chemolithoautotrophic bacterium .