Recombinant Pseudomonas putida Methionine import ATP-binding protein MetN 2 (metN2)

What is the MetN2 protein in Pseudomonas putida and how does it function in methionine transport?

MetN2 in P. putida is an ATP-binding protein component of the MetNI methionine ABC transporter system. Similar to the well-characterized E. coli system, it likely works in conjunction with transmembrane domains (MetI) and a substrate-binding protein (MetQ) to facilitate the ATP-dependent uptake of methionine and its derivatives into the cell. The MetN component contains nucleotide binding domains (NBDs) that bind and hydrolyze ATP to power the transport process .

How does the P. putida MetNI transporter system compare structurally to other bacterial methionine transporters?

While specific structural data for P. putida MetNI is limited, insights can be drawn from the E. coli system. The E. coli MetNI transporter functions as a complex with distinct conformational states: an inward-facing conformation where the translocation pathway is open to the cytoplasm, and an outward-facing conformation where it opens to the periplasm when complexed with MetQ. The system undergoes significant conformational changes during the transport cycle, with the MetN subunits rotating approximately 18° toward the molecular twofold axis when transitioning from inward-facing to outward-facing conformations .

What is the genetic organization of the methionine transport system in P. putida?

In P. putida, genes encoding the methionine transport system are typically organized in an operon structure. While the specific organization may vary between strains, these genes are generally co-regulated to ensure coordinated expression in response to methionine availability. The system includes genes encoding the ATP-binding protein (MetN), transmembrane components (MetI), and the periplasmic binding protein (MetQ). Regulatory elements, including promoters and potential binding sites for transcriptional regulators, are typically located upstream of these structural genes .

What are the most effective methods for expressing and purifying recombinant P. putida MetN2 protein?

For optimal expression and purification of recombinant P. putida MetN2:

• Expression system selection: E. coli BL21(DE3) is commonly used, though P. putida itself can serve as an expression host for homologous proteins.

• Vector design: Incorporate a T7 promoter system for controlled expression and a 6×His-tag for purification purposes.

• Culture conditions: Grow cultures at 30°C (rather than 37°C) to enhance protein folding and solubility.

• Purification protocol:

  • Lyse cells using sonication in buffer containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10% glycerol

  • Perform immobilized metal affinity chromatography (IMAC) using Ni-NTA resin

  • Further purify via size exclusion chromatography

• Stability considerations: Include 1-5 mM ATP or non-hydrolyzable ATP analog (ATPγS) in purification buffers to stabilize the protein .

How can researchers effectively measure the ATP hydrolysis activity of recombinant MetN2?

ATP hydrolysis activity can be quantified using the following methodological approach:

• Malachite green assay: Measures released inorganic phosphate with the following specifications:

  • Reaction buffer: 50 mM HEPES (pH 7.5), 150 mM NaCl, 5 mM MgCl₂

  • ATP concentration: 1-5 mM

  • Protein concentration: 50-200 nM

  • Temperature: 30°C (optimal for P. putida proteins)

  • Time points: Measurements at 0, 5, 10, 20, and 30 minutes

• Coupled enzyme assay: Using pyruvate kinase and lactate dehydrogenase to couple ATP hydrolysis to NADH oxidation:

  • Monitor absorbance decrease at 340 nm

  • Reaction components include phosphoenolpyruvate, NADH, pyruvate kinase, and lactate dehydrogenase

• Data analysis: Calculate specific activity as μmol ATP hydrolyzed/min/mg protein.

Table 1: Typical ATP Hydrolysis Parameters for MetN2 Variants

Note: Values based on related studies of ATP binding cassette transporters .

What transport assay systems can be used to study methionine uptake mediated by the MetNI system in P. putida?

Researchers can employ several complementary approaches to study MetNI-mediated transport:

• Radioactive substrate uptake:

  • Use ¹⁴C or ³H-labeled methionine (1-50 μM)

  • Measure accumulation in cells over time (30-120 seconds)

  • Filter cells and quantify radioactivity by scintillation counting

  • Calculate transport rates in nmol·min⁻¹·mg⁻¹ of protein

• Reconstituted proteoliposome assays:

  • Purify MetNI complex and reconstitute into liposomes

  • Initiate transport by adding ATP and methionine

  • Monitor substrate accumulation inside vesicles

  • Particularly useful for measuring kinetic parameters (K_m, V_max)

• Growth complementation:

  • Use methionine auxotrophic strains expressing various MetNI variants

  • Measure growth rates in minimal media supplemented with limiting methionine

  • Particularly valuable for in vivo functional analysis

For selenomethionine transport studies, specific protocols have been developed that can detect uptake rates of 0.2-15.4 nmol·min⁻¹·mg⁻¹ of transporter, depending on the MetQ variant used .

How do mutations in the C2 domain of MetN2 affect transinhibition by intracellular methionine?

Mutations in the C2 domain of MetN2 can significantly alter the regulatory mechanism of transinhibition:

• Mechanism of transinhibition: The C2 domains, located at the C-terminal end of MetN subunits, bind intracellular L-methionine. This binding triggers conformational changes that increase the distance between catalytic residues (like H199), preventing the formation of the catalytically competent NBD dimer required for ATP hydrolysis .

• Key residues: The N295 residue is particularly critical, as it forms hydrogen bonds with bound L-methionine from the adjacent C2 domain. In E. coli MetN, the N295A mutation significantly reduces transinhibition .

• Conformational effects: Mutations can disrupt the β-sheet hydrogen-bonding network between C2 domains, preventing the conformational shift that normally occurs upon methionine binding.

• Functional consequences: MetN N295A variants show enhanced transport activity (V_max = 10 ± 0.5 nmol·min⁻¹·mg⁻¹) compared to wild-type transporters (V_max = 6.3 ± 0.4 nmol·min⁻¹·mg⁻¹) due to reduced inhibition by intracellular methionine .

This structure-function relationship provides valuable targets for engineering MetN2 variants with altered regulatory properties for research applications.

What role do the nucleotide binding domains (NBDs) of MetN2 play in the transport mechanism?

The nucleotide binding domains of MetN2 serve critical functions in the transport cycle:

• ATP binding and hydrolysis: NBDs contain conserved motifs (Walker A, Walker B, H-motif) that coordinate ATP binding and catalyze hydrolysis. The H199 residue in the H-motif is particularly important for catalysis .

• Conformational coupling: ATP binding induces NBD dimerization, which triggers the conformational changes in the transmembrane domains necessary for substrate translocation. During the transition from inward-facing to outward-facing conformations, the NBDs rotate approximately 18° toward the molecular twofold axis .

• Regulatory integration: The NBDs are structurally coupled to the C2 domains, allowing intracellular methionine levels to regulate transport activity through transinhibition. When methionine binds to the C2 domains, the separation between catalytic H-motif residues increases, preventing NBD dimerization .

• Interaction with MetI: The NBDs transmit conformational changes to the transmembrane MetI subunits, which rotate approximately 31° toward the molecular twofold axis during the transition to the outward-facing state, opening the translocation pathway toward the periplasm .

What are the optimal genetic tools and strategies for engineering the metN2 gene in Pseudomonas putida?

P. putida offers several effective genetic manipulation approaches for metN2 engineering:

• Homologous recombination systems:

  • Two-step recombination using suicide plasmids like pEMG (containing I-SceI recognition sites)

  • Counter-selection with I-SceI endonuclease expression from a helper plasmid

  • Recently improved systems with self-curing helper plasmids reduce processing time

• Recombineering methods:

  • RecET-based markerless recombineering for large gene manipulations

  • Thermoinducible single-stranded recombineering systems

  • CRISPR/Cas9 technologies for efficient editing and counterselection

• Vector systems:

  • Broad host range origins like RK2 (low-copy, stable in P. putida)

  • RSF1010 (high-copy) origins for higher expression levels

  • pBBR1 origins requiring only a single gene for replication

• Expression control:

  • Inducible promoters like XylS/Pm (induced by 3-methylbenzoate)

  • T7 RNA polymerase-dependent systems for high-level expression

  • Constitutive promoters of varying strengths for stable expression

These tools enable precise genetic modifications ranging from point mutations to complete gene replacements and expression level optimization.

How can researchers optimize heterologous expression of metN2 in P. putida for functional studies?

Optimizing heterologous expression of metN2 requires careful consideration of several factors:

• Genomic integration sites: rRNA-encoding rrn operons have been identified as especially favorable sites for heterologous gene integration in P. putida. Studies have shown that all seven rrn operons can support functional expression, though the specific operon and distance between the rrn promoter and integrated genes significantly affect expression levels .

• Codon optimization: While not always necessary for genes from related species, codon optimization for P. putida's preferred codon usage can enhance expression of genes with substantially different GC content.

• Expression systems:

  • For stable, moderate expression: chromosomal integration using Tn5-based TREX system

  • For high-level expression: T7 RNA polymerase-dependent promoters

  • For controlled expression: XylS/Pm system with 3-methylbenzoate induction

• Host strain selection: Genome-reduced strains like P. putida EM42 or SEM10 offer enhanced heterologous gene expression capabilities due to reduced metabolic burden and elimination of competing pathways .

• Growth conditions: Cultivation at 30°C rather than 37°C typically provides optimal protein folding and activity for P. putida recombinant proteins.

What are the key considerations when designing mutational studies of P. putida MetN2?

When designing mutation studies of MetN2 in P. putida, researchers should consider:

How is the MetNI transport system integrated with methionine biosynthesis and degradation pathways in P. putida?

P. putida has evolved sophisticated integration between methionine transport, biosynthesis, and degradation:

• Methionine biosynthesis pathway:

  • P. putida primarily uses direct sulfhydrylation of O-succinylhomoserine for methionine synthesis

  • The transsulfuration pathway (involving cystathionine) is expressed at low levels under normal conditions

  • Both pathways are potentially regulated in coordination with transport activity

• Methionine degradation:

  • P. putida converts methionine to methanethiol via methionine γ-lyase (encoded by mdeA)

  • The mdeB gene encodes α-ketobutyrate dehydrogenase E1 component, which metabolizes α-ketobutyrate produced from methionine

  • This degradation is upregulated when methionine is the sole sulfur source

• Coordinated regulation:

  • Transinhibition mechanism where intracellular methionine inhibits the MetNI transporter

  • The mdeR gene, encoding a protein in the leucine-responsive regulatory protein (Lrp) family, acts as a positive regulator for methionine degradation genes

  • Global regulatory systems likely coordinate transport with biosynthetic and catabolic pathways

• Sulfur metabolism connection:

  • In P. putida, methionine can serve as a sulfur source through conversion to methanethiol, oxidation to methanesulfonate, and desulfonation by alkanesulfonatase

  • This pathway connects methionine utilization to the broader sulfur metabolism network

Table 2: Key Enzymes in P. putida Methionine Metabolism

How does the availability of different sulfur sources affect MetN2 expression and function in P. putida?

The availability of different sulfur sources significantly influences MetN2 expression and function in P. putida:

• Sulfate as primary sulfur source:

  • Under these conditions, methionine biosynthesis is active

  • MetNI expression is typically at basal levels

  • Transinhibition regulates transport activity based on intracellular methionine concentration

• Methionine as sole sulfur source:

  • P. putida can grow well with methionine as the sole sulfur source

  • Unlike P. aeruginosa (which uses the reverse transsulfuration pathway), P. putida desulfurizes methionine through the methionine γ-lyase pathway

  • Methionine γ-lyase (MdeA) is upregulated approximately 10-fold under these conditions

  • MetNI transport likely increases to support enhanced methionine uptake

• Response to selenate:

  • Growth with methionine is somewhat inhibited but not halted by 1 mM selenate

  • Selenate completely inhibits growth with methanesulfonate as the sulfur source

  • Alkanesulfonate sulfonatase is inhibited ~50% by selenate

  • This suggests complex interactions between selenium compounds and the methionine utilization pathway

• Coordination with other sulfur acquisition systems:

  • An ssuD (alkanesulfonatase) mutant cannot grow with either methanesulfonate or methionine as sulfur sources

  • This indicates that P. putida converts methionine to methanethiol, then to methanesulfonate, which is desulfonated by alkanesulfonatase

What is the relationship between methionine transport and branched-chain amino acid metabolism in P. putida?

The relationship between methionine transport and branched-chain amino acid (BCAA) metabolism in P. putida involves several interconnected pathways:

• Regulatory connections:

  • The mdeR gene product belongs to the Lrp (leucine-responsive regulatory protein) family

  • This suggests coordinate regulation between methionine and branched-chain amino acid metabolism

  • Lrp-type regulators often respond to multiple amino acids, creating a regulatory network

• Metabolic intersections:

  • α-Ketobutyrate (produced from methionine by MdeA) can feed into the same pathways that process α-ketoacids derived from branched-chain amino acids

  • MdeB (α-ketobutyrate dehydrogenase E1 component) shows high specificity for α-ketobutyrate rather than pyruvate

  • This enzyme likely participates in a specialized α-keto acid dehydrogenase complex

• Enzyme similarities:

  • The α-keto acid dehydrogenase complex components involved in BCAA metabolism share structural and functional similarities with those processing methionine-derived intermediates

  • This suggests evolutionary relationships and potential overlapping functions

• Coordination during growth on specific carbon sources:

  • When P. putida uses aromatic compounds as carbon sources, both methionine and BCAA metabolism may be coordinately regulated

  • The metabolic versatility of P. putida allows it to integrate these pathways efficiently based on nutrient availability

How can the P. putida MetNI system be engineered for improved D-methionine or selenomethionine transport for biotechnology applications?

Engineering the P. putida MetNI system for enhanced D-methionine or selenomethionine transport involves several promising strategies:

• MetQ binding protein modifications:

  • The MetQ N229A variant has been shown to support higher D-selenomethionine uptake rates (10.5 ± 0.9 nmol·min⁻¹·mg⁻¹) than wild-type MetQ (6.3 ± 0.4 nmol·min⁻¹·mg⁻¹)

  • This counterintuitive finding suggests that impaired substrate binding by MetQ can facilitate a noncanonical transport mechanism for certain substrates

  • Structure-guided mutations at the MetQ-MetNI interface could further enhance this noncanonical pathway

• Transinhibition attenuation:

  • Mutations in the C2 domain (e.g., N295A) reduce transinhibition by intracellular methionine

  • Combining MetN N295A with MetQ N229A yields further increased transport rates (15.4 ± 1.3 nmol·min⁻¹·mg⁻¹)

  • Complete removal of the C2 domain could maximize transport capacity but might affect protein stability

• Pathway integration:

  • Co-expression with enzymes that rapidly metabolize intracellular methionine (like MdeA) could reduce transinhibition

  • Integration with selenomethionine utilization pathways could enhance selenoprotein production

• Expression optimization:

  • Integration into highly active genomic loci such as rrn operons

  • Use of strong, constitutive promoters for overexpression

  • Selection of optimal RBS sequences for balanced expression of all components

These approaches could develop P. putida strains with enhanced capabilities for selenoprotein production, D-amino acid metabolism, or other biotechnological applications.

What insights can comparative studies of MetN2 from different Pseudomonas species provide about its evolution and functional specialization?

Comparative studies of MetN2 across Pseudomonas species offer valuable insights:

• Evolutionary adaptation to ecological niches:

  • Soil-dwelling species like P. putida have evolved efficient methionine uptake systems to compete in nutrient-limited environments

  • Pathogenic species such as P. aeruginosa show adaptations for host-associated environments where methionine availability differs

  • These adaptations are reflected in sequence variations in key functional domains

• Pathway diversity:

  • P. aeruginosa contains the reverse transsulfuration pathway (with cystathionine γ-lyase) for methionine-to-cysteine conversion

  • P. putida lacks this pathway, instead using methionine γ-lyase to convert methionine to methanethiol

  • These differences suggest that methionine transport systems may be optimized for different metabolic fates of the imported substrate

• Regulatory variations:

  • Species-specific transinhibition mechanisms may reflect adaptations to different methionine utilization strategies

  • Lrp-family regulators like MdeR show diversification across species, potentially allowing specialized responses to environmental conditions

• Structural conservation and variation:

  • Core ATP-binding and hydrolysis domains are highly conserved

  • Substrate specificity determinants and regulatory domains show greater variation

  • These patterns provide insights into the balance between conserved function and adaptive specialization

How might systems biology approaches help elucidate the role of MetN2 in the broader metabolic network of P. putida?

Systems biology approaches offer powerful tools for understanding MetN2's role in P. putida metabolism:

These systems approaches can help position MetN2 within the broader metabolic and regulatory networks of P. putida, providing a foundation for both fundamental understanding and biotechnological applications.