Recombinant Prochlorococcus marinus subsp. pastoris Urease subunit beta (ureB)

What is the molecular structure of the UreB subunit in Prochlorococcus marinus?

The UreB (β) subunit of P. marinus urease has an apparent molecular mass of 13 kDa as determined by gel electrophoresis, with a predicted molecular mass of 12 kDa based on its amino acid sequence . This subunit forms part of the complete urease enzyme, which has a total molecular mass of 168 kDa as determined by gel filtration . The native enzyme consists of three different subunits—UreA (γ, 11 kDa), UreB (β, 13 kDa), and UreC (α, 63 kDa)—arranged in a quaternary structure of (αβγ)₂ . This makes it the smallest characterized urease known to date, which is consistent with the minimalistic nature of Prochlorococcus species.

How does UreB contribute to the quaternary structure and function of the complete urease enzyme?

UreB plays an essential structural role in forming the active urease enzyme in P. marinus. The quaternary structure of (αβγ)₂ indicates that two copies of each subunit, including UreB, come together to form the functional enzyme . While the specific interactions between UreB and the other subunits haven't been fully elucidated for P. marinus urease, structural studies of ureases from other organisms suggest that the β subunit contributes to the stability of the enzyme complex and helps maintain the proper conformation of the active site, which is primarily located in the UreC (α) subunit . The compact arrangement of these subunits contributes to the remarkably small size of the complete urease enzyme while maintaining its catalytic efficiency.

How does the UreB subunit from P. marinus compare to urease beta subunits from other organisms?

The UreB subunit from P. marinus is notably smaller than urease β subunits found in many other organisms, consistent with the minimal genome and proteome of Prochlorococcus species . Immunoblot analysis has shown distinct differences between the urease subunits of P. marinus and those of Anabaena/Nostoc PCC 7120 . Unlike some other cyanobacteria like Synechocystis sp. strain PCC 6803 where urease genes are scattered throughout the genome, the ureB gene in P. marinus is part of a gene cluster (ureDABC) that is divergently oriented from another urease gene cluster (ureEFG) . This genomic organization is similar to that found in Synechococcus sp. strain WH 7805, suggesting potential evolutionary relationships or functional advantages of this arrangement in marine cyanobacteria.

What is the genomic organization of the ureB gene in P. marinus?

The ureB gene in Prochlorococcus marinus strain PCC 9511 is located within the ureDABC gene cluster . Sequence analysis has revealed that this cluster is divergently oriented from another gene cluster, ureEFG, which encodes urease accessory proteins . This organization differs from some other cyanobacteria, such as Synechocystis sp. strain PCC 6803, where urease genes are scattered throughout the genome . The genomic arrangement in P. marinus may facilitate coordinated expression of the structural subunits, potentially providing an advantage in nitrogen-limited marine environments.

How is the expression of ureB regulated in response to nitrogen availability?

The expression of ureB in P. marinus appears to be regulated in response to nitrogen availability. A putative NtcA-binding site has been identified upstream from the ureEFG cluster, suggesting that the urease genes are under nitrogen control . NtcA is a transcriptional regulator that plays a central role in nitrogen assimilation in cyanobacteria. While the NtcA-binding site was found specifically upstream of the ureEFG cluster, it likely influences the expression of both gene clusters due to their divergent organization. This regulatory mechanism would allow P. marinus to adjust urease production based on nitrogen availability in its typically oligotrophic marine environment, utilizing urea as an alternative nitrogen source when necessary.

What cloning strategies have been used to express recombinant UreB from P. marinus?

For the initial characterization of P. marinus urease, researchers employed a cloning strategy that began with designing degenerate primers to amplify a conserved region of the ureC gene . The amplification product was then used as a probe to clone a 5.7 kbp fragment of the P. marinus genome, which contained both the ureDABC and ureEFG gene clusters . For expressing recombinant UreB specifically, researchers would typically:

• Amplify the ureB gene using PCR with specific primers designed based on the known sequence

• Clone the amplified gene into an appropriate expression vector (typically with a histidine tag for purification)

• Transform the construct into a suitable expression host (E. coli or other bacteria)

• Induce protein expression under optimized conditions

• Purify the recombinant protein using affinity chromatography

It's important to note that for proper folding and function of recombinant UreB, co-expression with the other urease subunits and accessory proteins may be necessary, as proper assembly of the urease complex requires coordinated action of multiple components .

What are the optimal conditions for purifying recombinant UreB from P. marinus?

The purification of recombinant UreB from P. marinus would typically follow protocols similar to those used for the native enzyme, with modifications to account for expression system differences. For the native urease complex from P. marinus PCC 9511, researchers achieved a 900-fold purification to a specific activity of 94.6 μmol urea min⁻¹ (mg protein)⁻¹ through a combination of heat treatment and liquid chromatography methods .

A typical purification protocol for recombinant UreB would include:

• Cell lysis under conditions that maintain protein solubility

• Heat treatment (if the protein is sufficiently heat-stable)

• Ammonium sulfate precipitation

• Ion exchange chromatography (DEAE-Sepharose or similar)

• Size exclusion chromatography

• Mono-Q column chromatography with a linear NaCl gradient

For recombinant protein with affinity tags, simplified protocols using affinity chromatography (e.g., nickel columns for His-tagged proteins) would be employed. The purified protein should be stored with glycerol (approximately 30% v/v) at -20°C to maintain stability .

How can researchers assess the activity and structural integrity of recombinant UreB?

Assessing the activity and structural integrity of recombinant UreB involves several complementary approaches:

• SDS-PAGE and Western blotting: To confirm the molecular weight (approximately 13 kDa) and immunoreactivity of the purified protein. Polyclonal antibodies raised against urease from related organisms can often cross-react with P. marinus UreB .

• Native PAGE: To assess whether UreB can form appropriate complexes with other urease subunits when co-expressed.

• Functional assays: Since UreB alone is not catalytically active, functional assessment requires reconstitution with the other urease subunits (UreA and UreC). The complete urease complex can then be assayed for activity by measuring ammonia production from urea hydrolysis.

• Circular dichroism spectroscopy: To assess the secondary structure of the recombinant protein and compare it to predicted models.

• Mass spectrometry: For precise molecular weight determination and confirmation of post-translational modifications if present.

When assessing the reconstituted urease complex containing UreB, researchers should expect a Km value of approximately 0.23 mM urea, and inhibition by HgCl₂, acetohydroxamic acid, and EDTA, but not by boric acid or L-methionine-DL-sulfoximine .

What biophysical techniques are most informative for characterizing UreB structure and interactions?

Several biophysical techniques provide valuable insights into UreB structure and interactions:

These techniques, used in combination, can provide complementary information about UreB structure and function that would not be apparent from biochemical assays alone.

What expression systems are most effective for producing functional recombinant UreB?

For producing functional recombinant UreB from P. marinus, several expression systems can be considered, each with specific advantages:

• E. coli expression systems: The most common approach, using vectors like pET or pBAD series. For optimal results:

  • Use E. coli strains optimized for membrane or difficult proteins (e.g., C41(DE3), C43(DE3), or Rosetta)

  • Express at lower temperatures (16-20°C) to improve folding

  • Consider co-expression with urease accessory proteins (UreD, UreE, UreF, UreG) for proper assembly

• Cyanobacterial expression systems: Expression in related cyanobacteria like Synechocystis sp. PCC 6803 might provide a more native-like environment for proper folding.

• Cell-free protein synthesis: For rapid screening of conditions and mutations without the complications of cell culture.

For functional studies of the complete urease, co-expression of all three structural subunits (UreA, UreB, UreC) along with the accessory proteins is recommended, as the individual UreB subunit lacks catalytic activity on its own. Expression conditions should mimic the natural growth conditions of P. marinus (18-20°C in PCR-Tu liquid medium) .

How can researchers generate site-directed mutations in UreB to study structure-function relationships?

To study structure-function relationships in P. marinus UreB through site-directed mutagenesis, researchers should follow these steps:

• Identify targets for mutation:

  • Conserved residues identified through alignment with UreB sequences from other organisms

  • Residues predicted to be at interfaces with other subunits

  • Residues potentially involved in nickel incorporation or substrate binding

• Design mutagenesis primers following standard principles:

  • 25-45 nucleotides in length

  • Mutation site in the middle of the primer

  • GC content of 40-60%

  • Terminating with one or more G or C bases

• Perform site-directed mutagenesis using established methods such as:

  • QuikChange protocol (Agilent)

  • Q5 Site-Directed Mutagenesis (New England Biolabs)

  • Overlap extension PCR

• Screen and verify mutants by:

  • Restriction digestion (if the mutation creates or eliminates a restriction site)

  • Sanger sequencing to confirm the desired mutation and absence of unwanted changes

• Express and purify mutant proteins using the same protocols as for wild-type UreB

• Characterize mutant phenotypes by:

  • Assessing ability to form complexes with other urease subunits

  • Measuring enzyme activity of reconstituted complexes

  • Determining structural stability through thermal denaturation assays

This approach will provide insights into residues critical for UreB function within the urease complex, potentially revealing unique adaptations in the minimalistic P. marinus enzyme.

What protocols are most reliable for assessing interactions between UreB and other urease subunits?

Several complementary approaches can reliably assess interactions between UreB and other urease subunits:

• Co-immunoprecipitation (Co-IP):

  • Express tagged versions of UreB and other subunits

  • Perform pull-down experiments using antibodies against the tag

  • Analyze precipitated complexes by Western blotting with antibodies against the other subunits

• Yeast two-hybrid assays:

  • Construct fusion proteins of UreB and other urease subunits with DNA-binding and activation domains

  • Test interactions through reporter gene activation

  • Useful for initial screening of potential interaction partners

• Bacterial two-hybrid systems:

  • Similar to yeast two-hybrid but performed in bacterial cells

  • May be more appropriate for prokaryotic proteins like UreB

• Biolayer interferometry (BLI) or surface plasmon resonance (SPR):

  • Immobilize purified UreB on sensor surfaces

  • Measure binding kinetics with other purified subunits in real-time

  • Determine association and dissociation constants

• Native PAGE and gel filtration chromatography:

  • Mix purified UreB with other subunits and analyze complex formation

  • Compare migration patterns or elution profiles with those of individual proteins

• Microscale thermophoresis (MST):

  • Label UreB with a fluorescent dye

  • Measure changes in thermophoretic mobility upon interaction with other subunits

  • Determine binding affinities under near-native conditions

• Cross-linking coupled with mass spectrometry:

  • Treat reconstituted complexes with chemical cross-linkers

  • Digest and analyze by mass spectrometry

  • Identify cross-linked peptides to map interaction interfaces

These methods, particularly when used in combination, provide robust evidence for specific interactions between UreB and other components of the urease complex.

How does the sequence and structure of UreB contribute to the unique properties of P. marinus urease?

The UreB subunit from P. marinus contributes significantly to the unique properties of this urease, particularly its remarkably small size (the smallest known urease at 168 kDa) . Sequence analysis of the P. marinus urease genes has revealed that while the enzyme maintains the essential catalytic capabilities, it appears to have undergone evolutionary streamlining consistent with the minimalistic genome of Prochlorococcus species .

The key contributions of UreB to the unique properties of P. marinus urease likely include:

Comparative sequence analysis of P. marinus UreB with urease beta subunits from other organisms would likely reveal conserved regions essential for function alongside unique adaptations that contribute to the enzyme's minimalistic character.

What insights can structural modeling of UreB provide for understanding nitrogen metabolism in marine cyanobacteria?

Structural modeling of P. marinus UreB can provide several important insights into nitrogen metabolism in marine cyanobacteria:

• Evolutionary adaptations: Comparing modeled structures of UreB from P. marinus with those from other cyanobacteria can reveal structural adaptations specific to oligotrophic marine environments, where efficient nitrogen utilization is critical .

• Functional conservation: Despite its small size, structural modeling would likely reveal conservation of key structural elements needed for proper assembly with UreA and UreC, suggesting fundamental constraints on urease architecture even in highly streamlined organisms.

• Environmental specialization: Models of the complete urease complex could help explain the kinetic properties observed (Km of 0.23 mM urea) , which may reflect adaptation to typical urea concentrations in oceanic environments.

• Regulatory mechanisms: Combining structural models with information about the NtcA-binding site upstream of the ureEFG cluster could provide insights into how nitrogen limitation triggers structural changes in regulatory proteins that control urease expression.

• Metabolic integration: Structural modeling of interactions between the urease complex and other nitrogen metabolism proteins could illuminate how P. marinus integrates multiple nitrogen sources in its natural environment.

These insights are particularly important given that Prochlorococcus species dominate in oligotrophic ocean regions where efficient nitrogen utilization provides a competitive advantage.

How do the biochemical properties of UreB from P. marinus compare across different ecotypes?

While specific comparative data on UreB across different Prochlorococcus ecotypes is limited in the provided search results, we can infer several potential areas of comparison based on known ecological adaptations of Prochlorococcus:

Table 1: Hypothesized UreB Variations Across Prochlorococcus Ecotypes

Research comparing urease activity across Prochlorococcus ecotypes would likely reveal:

• Ecotype-specific adaptations: Different UreB sequences optimized for the specific environmental niches of each ecotype.

• Expression pattern differences: Variation in how UreB expression responds to nitrogen limitation across ecotypes adapted to different ocean depths and regions.

• Structural variations: Subtle differences in UreB structure that contribute to different urease kinetic properties across ecotypes.

• Gene cluster conservation: Analysis of whether the divergently oriented ureDABC and ureEFG gene clusters are conserved across all ecotypes or show lineage-specific arrangements.

These comparisons would provide valuable insights into how different Prochlorococcus lineages have adapted their nitrogen metabolism to specific oceanic niches.

What are the most promising approaches for studying UreB function in environmental samples?

Studying UreB function in environmental samples presents unique challenges due to the low biomass and complex microbial communities in oceanic samples. Several promising approaches include:

• Metatranscriptomics and metaproteomics:

  • Analyzing expression patterns of ureB genes across different ocean regions and depths

  • Identifying post-translational modifications that might regulate UreB function in situ

  • Correlating expression with environmental parameters like nitrogen availability

• Single-cell genomics and transcriptomics:

  • Isolating individual Prochlorococcus cells from environmental samples

  • Sequencing to identify strain-specific ureB variants

  • Correlating genotypes with specific ocean regions or conditions

• Environmental enzyme assays:

  • Developing sensitive methods to measure urease activity in filtered seawater samples

  • Using activity-based protein profiling to capture active urease complexes

  • Correlating activity with urea cycling in marine ecosystems

• Stable isotope probing:

  • Using ¹⁵N-labeled urea to track nitrogen flow through Prochlorococcus populations

  • Combining with cell sorting to identify specific ecotypes actively utilizing urea

• Biosensor development:

  • Creating reporter systems based on the P. marinus ureB promoter

  • Deploying in environmental monitoring systems to track nitrogen utilization patterns

These approaches, especially when combined, can provide insights into how UreB functions within the complex nitrogen cycling of marine ecosystems dominated by Prochlorococcus species.

How might structural knowledge of UreB inform bioengineering applications?

The unique structural properties of UreB from P. marinus, particularly its contribution to the smallest known urease enzyme , offer several promising bioengineering applications:

• Enzyme miniaturization templates:

  • Using P. marinus UreB as a model for minimizing enzyme size while maintaining function

  • Applying similar design principles to reduce the size of other multi-subunit enzymes

  • Creating more efficient biocatalysts for industrial applications

• Biosensor development:

  • Engineering UreB-based sensors for detecting urea in environmental or clinical samples

  • Developing systems with improved sensitivity based on the efficient catalytic properties (Km of 0.23 mM)

  • Creating field-deployable sensors for monitoring nitrogen cycling in marine environments

• Bioremediation tools:

  • Engineering modified ureases based on the P. marinus model for environmental applications

  • Developing systems for removing excess urea from aquaculture or coastal environments

  • Creating immobilized enzyme systems with enhanced stability

• Nitrogen-use efficiency in transgenic organisms:

  • Introducing optimized urease systems based on P. marinus UreB into plants or microorganisms

  • Enhancing nitrogen scavenging abilities in organisms used for bioremediation

  • Improving growth under nitrogen-limited conditions

• Protein engineering platforms:

  • Using the minimalistic structure of UreB as a scaffold for designing novel protein functions

  • Developing more efficient multi-enzyme complexes based on the streamlined design principles

The compact, efficient design of P. marinus UreB represents an excellent example of natural optimization that could inspire numerous bioengineering applications focused on efficiency and minimal resource utilization.

What experimental approaches would best elucidate the role of UreB in nitrogen acquisition under oceanic conditions?

To elucidate the specific role of UreB in nitrogen acquisition under realistic oceanic conditions, researchers should consider the following experimental approaches:

• Mesocosm experiments:

  • Create controlled ocean water environments mimicking natural conditions

  • Introduce wild-type and UreB-modified Prochlorococcus strains

  • Monitor growth, urea utilization, and competitive fitness under various nitrogen regimes

• Comparative genomics and transcriptomics:

  • Analyze ureB sequence variations across Prochlorococcus ecotypes from different ocean regions

  • Correlate sequence differences with urease activity and nitrogen utilization efficiency

  • Identify regulatory differences in ureB expression across ecotypes

• In situ gene expression studies:

  • Develop methods to measure ureB expression in natural Prochlorococcus populations

  • Correlate expression with environmental parameters like depth, temperature, and nitrogen availability

  • Track diel patterns of expression to understand temporal regulation

• Isotope tracer experiments:

  • Use ¹⁵N-labeled urea in laboratory cultures and mesocosm experiments

  • Track nitrogen flow through various metabolic pathways

  • Compare assimilation rates between wild-type and engineered strains with modified UreB

• Structural biology under relevant conditions:

  • Determine UreB structure and urease complex assembly under oceanic salt concentrations

  • Measure enzyme kinetics at environmentally relevant temperatures and pH

  • Assess the impact of trace metal availability on complex formation and activity

These approaches would provide a comprehensive understanding of how this remarkably small urease enzyme, with its UreB component, contributes to the ecological success of Prochlorococcus as the most abundant photosynthetic organism in oligotrophic oceans.