Recombinant Rhodopirellula baltica Bifunctional enzyme CysN/CysC (cysNC), partial

What is the bifunctional enzyme CysN/CysC and what role does it play in R. baltica metabolism?

The CysN/CysC enzyme in R. baltica (annotated as RB7941 in the genome) is a bifunctional protein that catalyzes two sequential reactions in the sulfate assimilation pathway:

• The CysN domain functions as sulfate adenylyltransferase (EC 2.7.7.4), catalyzing the reaction:
  ATP + SO₄²⁻ → APS (adenosine 5'-phosphosulfate) + PPi

• The CysC domain functions as adenylyl-sulfate kinase (EC 2.7.1.25), catalyzing:
  ATP + APS → PAPS (3'-phosphoadenosine 5'-phosphosulfate) + ADP

This enzyme constitutes a critical part of R. baltica's sulfur metabolism, which has been identified as containing "a fascinating set of carbohydrate-active enzymes" and "a conspicuous C1-metabolism pathway" . The fusion of these two enzymatic functions into a single protein represents an adaptation that likely enhances the efficiency of sulfate assimilation in this marine bacterium.

What expression systems are most effective for producing functional recombinant R. baltica CysN/CysC?

Multiple expression systems have been successfully employed for R. baltica CysN/CysC production:

What purification strategies yield the highest purity and activity for recombinant R. baltica CysN/CysC?

Based on general practices for similar enzymes and information from the literature on R. baltica proteins, an effective purification strategy would include:

• Affinity chromatography using His-tag or GST-tag (as indicated in available recombinant protein products)

• Size-exclusion chromatography to remove aggregates and ensure homogeneity

• Ion-exchange chromatography as a polishing step if necessary

When purifying the enzyme, consider these critical factors:

• Maintain divalent cations (particularly Mg²⁺) in all buffers, as they are essential for enzymatic activity

• Include stabilizing agents like glycerol (10-20%) to prevent activity loss during storage

• Use reducing agents (DTT or β-mercaptoethanol) to maintain cysteine residues in reduced state

• Avoid EDTA in purification buffers as it would chelate essential metal ions

From studies with similar enzymes, purification yields of 0.013 ± 0.1 μmol/min.mg specific activity have been reported for related phosphatases from R. baltica .

What are the optimal conditions for assaying both enzymatic activities of R. baltica CysN/CysC?

Based on characterization of similar enzymes from R. baltica and the conditions reported for other sulfur metabolism enzymes:

For CysN activity (ATP sulfurylase):

• Buffer: 50 mM Tris-HCl, pH 7.5-8.0, 20 mM KCl

• Essential cofactor: 10 mM Mg²⁺

• Substrates: ATP (1-2 mM) and Na₂SO₄ (1-5 mM)

• Temperature: 25-30°C (likely optimal around 30°C based on R. baltica's marine origin)

• Detection methods: Coupled assay measuring PPi release or direct HPLC analysis of APS formation

For CysC activity (APS kinase):

• Similar buffer conditions as CysN

• Substrates: APS (produced by CysN or added externally) and ATP

• Detection: HPLC analysis of PAPS formation or coupled enzymatic assay

Analysis methods similar to those used for other R. baltica enzymes would apply: "Activity assays carried out with recombinant enzymes showed synthesis by measuring formation of products, which were confirmed in assays carried out with R. baltica cell extracts" .

How do temperature, pH, and ionic conditions affect the stability and activity of R. baltica CysN/CysC?

While specific data for CysN/CysC from R. baltica is limited in the search results, we can draw insights from other R. baltica enzymes:

Temperature effects:
R. baltica enzymes typically show maximum activity at temperatures between 30-40°C, with significant activity (64%) retained at 25°C, which corresponds to the organism's optimal growth temperature. Little to no activity may be observed below 15°C .

pH dependence:
The optimal pH for R. baltica enzymes is often in the range of 6.0-8.0, with activity profiles typically bell-shaped across pH 4.0-9.0 .

Ion requirements:
Like many R. baltica enzymes, CysN/CysC would likely show strict dependence on divalent cations in the following order of efficiency: Mg²⁺ > Ni²⁺ > Co²⁺. Maximum activity would be expected with approximately 10 mM Mg²⁺. Monovalent cations like K⁺ and Na⁺ may also activate the enzyme .

Stability:
Half-lives for R. baltica enzymes have been determined to be approximately 5.3 hours at 40°C and 9.6 hours at 25°C , suggesting moderate thermostability consistent with a mesophilic marine organism.

What crystallization strategies have proven successful for R. baltica enzymes similar to CysN/CysC?

Successful crystallization of R. baltica enzymes has been reported in the literature, providing valuable insights for CysN/CysC crystallization:

• Protein preparation considerations:

  • High purity (>95% by SDS-PAGE) is essential

  • Monodisperse samples confirmed by dynamic light scattering

  • Concentration typically between 5-20 mg/mL

• Crystallization conditions:

  • Inclusion of substrates or substrate analogs to stabilize active conformations

  • Addition of divalent metal ions (particularly Mg²⁺)

  • PEG-based precipitants with pH range 6.0-8.0

  • Sitting or hanging drop vapor diffusion methods

• Optimization techniques:

  • Microseeding to improve crystal quality

  • Additive screening to reduce nucleation and promote ordered growth

  • Truncation constructs if full-length protein proves recalcitrant to crystallization

As reported for other R. baltica enzymes: "The 2Fo-Fc electron density maps depict the cofactors at atomic resolution and illustrate similar interactions with protein residues" , suggesting high-resolution structures can be achieved.

What structural features likely facilitate substrate channeling between the CysN and CysC domains?

While specific structural data for R. baltica CysN/CysC is not available in the search results, the bifunctional nature of the enzyme suggests the presence of substrate channeling mechanisms:

• The physical fusion of CysN and CysC domains would position the active sites in proximity to facilitate direct transfer of the APS intermediate.

• Electrostatic guidance likely plays a key role, with positive charge distribution creating a pathway for the negatively charged APS molecule.

• Conformational changes upon substrate binding to CysN may create or expose a tunnel leading to the CysC active site.

• The interdomain linker region (identifiable through sequence analysis) would be crucial for maintaining proper domain orientation while allowing necessary conformational flexibility.

Mutagenesis studies targeting residues at the domain interface would be valuable for experimentally confirming these mechanisms, similar to the approach described: "Structure-activity relationship analysis identified residue 18 as a gatekeeper" .

How is the expression of cysNC regulated during different growth phases and environmental conditions in R. baltica?

Transcriptomic studies of R. baltica have revealed important insights into gene regulation patterns during growth:

"Comparison of the transition phase and stationary growth phase (82 h) with the mid-exponential phase (62 h) revealed that more genes were regulated in the stationary phase than the mid-exponential. It could be speculated that genes necessary for exponential growth under favourable conditions were already expressed in early log and mid-log phase" .

While the search results don't specifically detail cysNC regulation, evidence from other bacteria suggests that sulfur metabolism genes are often regulated in response to:

• Nutrient limitation, particularly sulfur availability

• Oxidative stress conditions

• Changes in growth phase

In M. tuberculosis, "cysD and cysNC are also induced during macrophage infection, underscoring the importance of sulfur metabolism to intracellular survival" , suggesting these genes respond to environmental stressors.

Further experimental approaches to characterize cysNC regulation would include:

• Quantitative RT-PCR across growth phases

• Reporter gene assays with the cysNC promoter

• Chromatin immunoprecipitation to identify potential regulators

How does R. baltica's sulfur metabolism integrate with its unique cell biology and compartmentalization?

R. baltica possesses a complex cellular organization with distinct compartments, which may significantly impact its sulfur metabolism:

"The cell is surrounded by the intracytoplasmic membrane that defines the pirellulosome and contains the riboplasm with ribosome-like particles and the condensed nucleoid. The region between the intracytoplasmic and cytoplasmic membranes contains the paryphoplasm that harbors some RNA but no ribosome-like particles" .

This compartmentalization raises important questions about the localization of sulfur metabolism enzymes like CysN/CysC. The search results suggest that:

• Proteins involved in core metabolic functions are likely localized to the "riboplasm" compartment: "Proteins, which were most abundant in 2-DE gels and the coding genes of which were also predicted to be highly expressed, could be linked mainly to housekeeping functions in glycolysis, tricarboxic acid cycle, amino acid biosynthesis, protein quality control and translation. Absence of predictable signal peptides indicated a localization of these proteins in the intracellular compartment, the pirellulosome" .

• The CysN/CysC enzyme, being involved in essential sulfur metabolism, would likely be located in the riboplasm compartment, where protein synthesis and primary metabolism occur.

• The compartmentalization may facilitate the maintenance of optimal conditions for sulfur reduction: "Reduction of sulfate to sulfide requires a complex set of enzymes, as well as the maintenance of a very low oxido-reduction potential, which seems difficult with the simultaneous presence of oxygen molecules. Cell compartmentalization is therefore a prerequisite" .

How can site-directed mutagenesis be used to probe the catalytic mechanism of R. baltica CysN/CysC?

A systematic mutagenesis approach would provide valuable insights into the structure-function relationships of this bifunctional enzyme:

This approach has been successfully applied to other enzymes: "Molecular analysis indicates that a flexible N-terminus in both enzymes likely plays a key role in substrate binding. Furthermore, phenylalanine and tyrosine act as gatekeepers and can either adopt an open or closed conformation important for catalysis" .

What computational approaches can help model the complex catalytic mechanism of R. baltica CysN/CysC?

Several complementary computational methods would be valuable for studying this bifunctional enzyme:

• Homology modeling:

  • Using crystal structures of homologous enzymes (e.g., M. tuberculosis CysN/CysC)

  • Molecular dynamics refinement to improve model quality

  • Validation using experimental data from mutagenesis studies

• Molecular docking:

  • "Using the AutoDock Vina tool" to analyze substrate binding modes

  • Screening potential inhibitors for structure-based drug design

  • Modeling substrate channeling pathways between domains

• Quantum mechanics/molecular mechanics (QM/MM):

  • Hybrid methods to model the electronic structure of the active sites

  • Calculation of reaction energy profiles and transition states

  • Investigation of metal ion roles in catalysis

• Network analysis:

  • Integration with genome-scale metabolic models of R. baltica

  • Flux balance analysis to predict metabolic impacts of enzyme modulation

  • Identification of key control points in sulfur metabolism

These methods would provide a comprehensive understanding of the enzyme's mechanism and its role in R. baltica's metabolism.

What does R. baltica's sulfur metabolism reveal about its ecological adaptations to marine environments?

R. baltica's sulfur metabolism, including the bifunctional CysN/CysC enzyme, reflects important adaptations to its marine habitat:

• Efficient sulfate utilization:
  The fusion of CysN and CysC functions may represent an adaptation for efficient utilization of marine sulfate, which is abundant but requires significant energy investment to assimilate.

• Specialized sulfur chemistry:
  "R. baltica has a set of unique sulfatases and C1-metabolism genes" , suggesting specialized capabilities for processing diverse sulfur compounds found in marine environments.

• Salt adaptation mechanisms:
  The search results indicate that R. baltica produces compatible solutes to deal with salt stress: "The biosynthetic pathway for the rare compatible solute mannosylglucosylglycerate (MGG) accumulated by Rhodopirellula baltica, a marine member of the phylum Planctomycetes, has been elucidated" . This suggests adaptations to fluctuating salinity that may also influence sulfur metabolism.

• Environmental sensing:
  "Rhodopirellula baltica distinctly stands out from this trend with only 2.4% (174) of all genes predicted to encode transcriptional regulators... revealed a clear shift towards high numbers of two-component systems (66) as well as high numbers of sigma factors (49)" . This extensive environmental sensing capability likely allows precise regulation of sulfur metabolism in response to changing conditions.

How does the bifunctional CysN/CysC in R. baltica compare evolutionarily to similar enzymes across bacterial phyla?

The bifunctional organization of CysN/CysC in R. baltica represents an interesting evolutionary case that provides insights into metabolic adaptation:

• Distribution pattern:
  The fusion of cysN and cysC genes occurs in several bacterial lineages but is not universal. In many bacteria (including E. coli), these functions are performed by separate proteins. The bifunctional arrangement in R. baltica likely represents either convergent evolution or an ancient fusion that has been maintained due to selective advantage.

• Planctomycetes uniqueness:
  The Planctomycetes phylum shows several unusual features including compartmentalized cells and unique metabolic pathways. The CysN/CysC enzyme may reflect these distinctive evolutionary characteristics: "This is the first characterization of genes and enzymes for the synthesis of compatible solutes in the phylum Planctomycetes" .

• Functional adaptation:
  The fusion likely provides catalytic advantages through substrate channeling, potentially an adaptation to environments where efficient sulfur utilization provides a competitive advantage.

• Regulatory implications:
  The fusion may also allow coordinated regulation of both enzymatic activities, ensuring stoichiometric production of the two enzymes and efficient use of transcriptional/translational resources.

Comprehensive phylogenetic analysis comparing CysN/CysC sequences across bacterial phyla would provide further insights into the evolutionary history and selective pressures that shaped this bifunctional enzyme.

How might recombinant R. baltica CysN/CysC be utilized in biotechnological applications?

The bifunctional nature and unique properties of R. baltica CysN/CysC present several potential biotechnological applications:

• Biocatalysis:

  • Production of activated sulfate compounds (APS, PAPS) for use as sulfur donors in enzymatic sulfation reactions

  • Generation of sulfated biomolecules with potential pharmaceutical applications

  • Integration into enzyme cascades for complex biotransformations

• Biosensors:

  • Development of sulfate detection systems using the ATP-consuming activity of CysN

  • Creation of coupled enzyme assays for environmental monitoring of sulfur compounds

• Metabolic engineering:

  • Enhancement of cysteine production in industrial microorganisms

  • Improvement of sulfur-containing pharmaceutical precursor biosynthesis

  • Engineering of sulfur metabolism in crop plants for improved nutrition or stress tolerance

• Structural biology tools:

  • Use as a model system for studying substrate channeling in bifunctional enzymes

  • Platform for investigating domain communication and allosteric regulation

These applications would leverage R. baltica's adaptations that make it "biotechnologically promising" with "a set of unique sulfatases and C1-metabolism genes" .

What experimental approaches could help engineer improved variants of R. baltica CysN/CysC for specific applications?

Several protein engineering strategies could be employed to enhance or modify the properties of R. baltica CysN/CysC:

• Directed evolution:

  • Error-prone PCR to generate variant libraries

  • Selection or screening for improved thermostability, activity, or substrate specificity

  • DNA shuffling with homologous enzymes from other organisms

• Rational design:

  • Structure-guided mutations to improve catalytic efficiency

  • Modification of metal binding sites to alter cofactor preferences

  • Engineering of substrate binding pockets for novel substrates

• Domain engineering:

  • Optimization of the interdomain linker to enhance substrate channeling

  • Fusion with additional domains for multi-functionality

  • Creation of chimeric enzymes with domains from other sulfur metabolism enzymes

• High-throughput approaches:

  • Deep mutational scanning to comprehensively map sequence-function relationships

  • Microfluidic droplet screening for activity-based sorting of variants

  • Computational design followed by experimental validation

Success metrics would include improved kinetic parameters, enhanced thermostability, altered substrate specificity, or increased expression levels, depending on the specific application targeted.