Recombinant Rhodopirellula baltica Carboxylate-amine ligase RB4485 (RB4485)

What is Recombinant Rhodopirellula baltica Carboxylate-amine ligase RB4485?

Recombinant Rhodopirellula baltica Carboxylate-amine ligase RB4485 is a full-length protein (372 amino acids) encoded by the RB4485 gene in Rhodopirellula baltica strain SH1. This enzyme is alternatively known as Putative glutamate--cysteine ligase 2 (EC 6.3.2.2), Gamma-glutamylcysteine synthetase 2 (GCS 2), or Gamma-GCS 2 . The protein belongs to the carboxylate-amine ligase family, which catalyzes the formation of carbon-nitrogen bonds between carboxylate and amine groups. In recombinant form, the protein is typically produced with expression tags to facilitate purification and detection, though the specific tag type may vary depending on the production method . The protein is commonly available in lyophilized powder form for research applications.

The complete amino acid sequence of RB4485 is: MSFQVPTVGVEEEYQLVDPRSGALIPNCKEVMRTIRRNGGSEEAHSEIQHELHLNQIEMASDVCSSLEEVRDALTQTRRMLIDAARSNETELASAGTNPLPIPTDDALTP KDRYQAMTDRYQQIARDLFIFGCHVHVAMEDRELGIQVMNRCRRWLPILQAITANSPYWDGVDTGYASYRRELWAQWPMAGPPAHFDSLADYQSCVDDLVACGAIKDESFLYWDIRLPTRVPTIEFRAADVMTRVEETVGYVGMIRAIVMLAISEEEQGKPIVPIRPSVLSYAIWHAARYG MNEQLVDPESREMIPASEL LNRLMTAIDPALKATGEARPVEAFANQLIKSGTGADRQRRGGELSSVVANVVAETVPSAILA . This sequence information is essential for designing experimental approaches targeting specific domains or functional regions of the protein.

How does the structure of RB4485 relate to its putative function?

While the complete three-dimensional structure of RB4485 has not been fully elucidated in the available research, sequence analysis suggests structural similarities to other glutamate-cysteine ligases, which play critical roles in glutathione biosynthesis. The putative functional domains include an ATP-binding site, glutamate-binding regions, and cysteine interaction motifs that collectively contribute to the enzyme's catalytic activity. Based on homology with related enzymes, RB4485 likely adopts a structure with distinct N-terminal and C-terminal domains connected by a flexible linker region.

Structural prediction models suggest the presence of conserved residues that coordinate with metal ions, typically magnesium, which are essential for ATP binding and subsequent catalysis. The aspartate residue at position 93 may be particularly important, as similar residues in homologous enzymes have been shown to be critical for catalytic activity. The protein likely undergoes conformational changes during the catalytic cycle, transitioning between open and closed states to facilitate substrate binding and product release. Understanding these structural features is essential for designing site-directed mutagenesis experiments to probe structure-function relationships and for developing potential inhibitors or activators of the enzyme.

What expression systems are available for producing recombinant RB4485?

The E. coli expression system (product code: CSB-EP756172RDR) provides high protein yields and is suitable for structural studies where glycosylation is not required. For applications requiring eukaryotic post-translational modifications, yeast expression systems (product code: CSB-YP756172RDR) offer an intermediate approach with moderate yields and some post-translational processing capabilities . For research requiring mammalian-like glycosylation patterns, the mammalian cell expression system (product code: CSB-MP756172RDR) provides the most native-like modifications but typically with lower yields. The baculovirus expression system (product code: CSB-BP756172RDR) offers a compromise between protein yield and post-translational modification capabilities .

Additionally, an E. coli-based expression system incorporating Avi-tag biotinylation (product code: CSB-EP756172RDR-B) has been developed specifically for applications requiring oriented immobilization or detection via streptavidin interaction . In this system, E. coli biotin ligase (BirA) catalyzes the covalent attachment of biotin to a specific lysine residue within the 15-amino acid AviTag peptide fused to RB4485. This biotinylation occurs in vivo during protein expression and provides a consistent, site-specific modification for downstream applications.

What analytical methods are most effective for characterizing the purity and activity of recombinant RB4485?

Comprehensive characterization of recombinant RB4485 requires a multi-faceted analytical approach. For purity assessment, SDS-PAGE analysis provides a straightforward method, with commercial preparations typically achieving >85% purity . Higher resolution analysis can be performed using two-dimensional gel electrophoresis, which allows for the detection of post-translational modifications and protein isoforms, as demonstrated in proteomic studies of Rhodopirellula baltica . Mass spectrometry, particularly MALDI-TOF-MS, offers precise molecular weight determination and can verify protein identity through peptide mass fingerprinting.

For activity characterization, spectrophotometric assays measuring the rate of ADP formation coupled to NADH oxidation provide a continuous method for monitoring enzymatic function. Alternatively, radioisotope-based assays using 14C-labeled glutamate can directly quantify product formation. Kinetic parameters (Km, Vmax) should be determined for key substrates: glutamate, cysteine, and ATP. Circular dichroism spectroscopy provides valuable information about secondary structure elements, while thermal shift assays can assess protein stability under various buffer conditions.

Functional characterization should include confirmation of metal ion requirements, typically magnesium or manganese, by assessing activity in the presence of chelating agents (EDTA) and subsequent metal restitution. pH-activity profiling is essential given that related ligases often display bell-shaped pH-dependency curves with optima between pH 7.0-8.5. Additionally, size-exclusion chromatography can determine the oligomeric state of the active enzyme, as many related ligases function as homodimers or higher-order structures.

What are the optimal conditions for expressing and purifying recombinant RB4485 in E. coli?

Optimizing expression and purification of recombinant RB4485 in E. coli requires careful consideration of multiple parameters. The expression construct should contain a codon-optimized RB4485 sequence with appropriate regulatory elements, such as the T7 promoter in pET series vectors, and fusion tags like His6 or GST to facilitate purification. BL21(DE3) or Rosetta(DE3) strains are recommended hosts due to their reduced protease activity and enhanced translation of rare codons found in marine bacterial genes.

Induction conditions significantly impact protein solubility and yield. Lower temperatures (16-18°C) after induction with reduced IPTG concentrations (0.1-0.3 mM) often improve the proportion of soluble protein by slowing expression and allowing proper folding. Growth media supplementation with 1% glucose can reduce basal expression before induction, while the addition of 2.5 mM betaine and 660 mM sorbitol may enhance protein solubility. Post-induction culture periods should be optimized experimentally, typically ranging from 16-20 hours at lower temperatures.

For purification, a sequential approach is most effective. Initial capture via immobilized metal affinity chromatography (IMAC) using a gradient of 20-500 mM imidazole in buffers containing 300 mM NaCl and 50 mM Tris-HCl (pH 8.0) provides primary enrichment. This should be followed by ion-exchange chromatography using Q-Sepharose at pH 8.0 (above the protein's theoretical pI of approximately 5.2) with elution in a 0-500 mM NaCl gradient. Final polishing via size-exclusion chromatography using Superdex 200 equilibrated in 25 mM HEPES (pH 7.5), 150 mM NaCl, and 10% glycerol yields highly purified protein. Throughout purification, reducing agents (5 mM β-mercaptoethanol or 1 mM DTT) should be included to prevent cysteine oxidation, and protease inhibitors should be used in early stages to prevent degradation.

How can researchers effectively assess the enzymatic activity of recombinant RB4485?

Establishing reliable enzyme activity assays for recombinant RB4485 requires a multifaceted approach that addresses the putative glutamate-cysteine ligase function. The primary method employs a coupled spectrophotometric assay measuring ADP formation during ATP hydrolysis. In this system, pyruvate kinase converts the released ADP to ATP while converting phosphoenolpyruvate to pyruvate, which is subsequently reduced to lactate by lactate dehydrogenase with concomitant oxidation of NADH to NAD+. The decrease in NADH absorbance at 340 nm provides a continuous measurement of enzyme activity.

A direct product formation assay can be established using HPLC or LC-MS/MS to quantify γ-glutamylcysteine formation. The reaction mixture typically contains 50 mM HEPES (pH 7.5), 20 mM MgCl2, 5 mM ATP, varying concentrations of L-glutamate (1-20 mM) and L-cysteine (0.1-5 mM), and purified enzyme. Following incubation at 30°C, reactions are terminated with trichloroacetic acid (5% final concentration), and the supernatant is derivatized with o-phthalaldehyde before HPLC analysis using a C18 reverse-phase column. Alternatively, 14C-labeled glutamate can be used with subsequent product separation by thin-layer chromatography.

Enzyme kinetic parameters can be determined through Michaelis-Menten analysis by varying substrate concentrations while maintaining excess concentrations of co-substrates. This approach allows calculation of Km values for glutamate, cysteine, and ATP, as well as Vmax and kcat values. Inhibition studies using buthionine sulfoximine (BSO) or glutathione, known modulators of glutamate-cysteine ligase activity, provide valuable insights into reaction mechanisms. Additionally, metal dependency should be assessed through activity measurements in the presence of various divalent cations (Mg2+, Mn2+, Ca2+) and metal chelators like EDTA to establish cofactor requirements.

What techniques can be used to study protein-protein interactions involving RB4485?

Investigating protein-protein interactions involving RB4485 requires a combination of complementary techniques to establish physiologically relevant binding partners and interaction characteristics. Pull-down assays represent a fundamental approach, utilizing recombinant RB4485 with affinity tags (His, GST, or biotin via AviTag) as bait to capture interacting proteins from Rhodopirellula baltica lysates . Captured proteins can be identified through mass spectrometry analysis, with particular attention to proteins co-regulated during stationary growth phase, as these may represent functional interaction networks .

Co-immunoprecipitation experiments using antibodies against RB4485 can validate interactions in more native conditions. For this approach, crosslinking with formaldehyde (0.1-0.5%) prior to cell disruption may preserve transient interactions. Surface plasmon resonance (SPR) provides quantitative binding kinetics by immobilizing RB4485 on a sensor chip surface and measuring the association and dissociation rates of putative binding partners in real-time. Similarly, isothermal titration calorimetry (ITC) offers label-free quantification of binding thermodynamics, providing values for binding affinity (Kd), stoichiometry, enthalpy, and entropy changes.

For in vivo interaction studies, bacterial two-hybrid systems can be established by fusing RB4485 to the T18 fragment of adenylate cyclase, with potential interacting proteins fused to the T25 fragment. Protein-protein interactions reconstitute adenylate cyclase activity, producing cAMP that activates reporter gene expression. Fluorescence resonance energy transfer (FRET) approaches can visualize interactions in real-time by tagging RB4485 with a donor fluorophore (e.g., CFP) and potential partners with acceptor fluorophores (e.g., YFP). Additionally, chemical crosslinking coupled with mass spectrometry (XL-MS) can identify interaction interfaces by analyzing peptides connected through crosslinks, providing structural insights into the interaction geometry.

How does growth phase affect the expression and activity of native RB4485 in Rhodopirellula baltica?

Comprehensive proteomic studies of Rhodopirellula baltica have revealed significant growth phase-dependent regulation of protein expression, which likely includes RB4485. The number of regulated proteins (showing fold changes greater than 2) increases dramatically from early stationary phase (10 proteins) to late stationary phase (179 proteins), with some proteins showing fold changes as high as 40 . This dynamic regulation suggests that RB4485 expression and activity may vary significantly across growth phases as part of the bacterium's adaptation to changing environmental conditions.

Growth phase transitions are accompanied by major metabolic shifts in Rhodopirellula baltica, including opposing regulation of the tricarboxylic acid cycle and oxidative pentose phosphate pathway . As a putative glutamate-cysteine ligase, RB4485 likely participates in redox homeostasis through glutathione biosynthesis, which would be particularly important during oxidative stress conditions often associated with stationary phase. The enzyme's regulation might be coordinated with the observed downregulation of several enzymes involved in amino acid biosynthesis during stationary phase, reflecting a reallocation of metabolic resources .

The upregulation of the alternative sigma factor sigmaH in stationary phase suggests activation of specific transcriptional programs that could include RB4485 regulation . Given that 26 proteins of unknown function were found to be differentially regulated in stationary phase, RB4485 might participate in protein interaction networks that adapt during growth phase transitions. Additionally, several proteins are specifically regulated during growth on solid surfaces, which correlates with the development of different Rhodopirellula baltica morphotypes, including motile swarmer cells and sessile cell aggregates called rosettes . This pattern suggests potential involvement of RB4485 in morphological differentiation processes, possibly through metabolic adaptations requiring modified glutathione biosynthesis.

What role might RB4485 play in the cellular stress response of Rhodopirellula baltica?

As a putative glutamate-cysteine ligase, RB4485 likely plays a central role in the cellular stress response of Rhodopirellula baltica through its involvement in glutathione biosynthesis, which is crucial for maintaining redox homeostasis under oxidative stress conditions. The enzyme catalyzes the first and rate-limiting step in glutathione synthesis, forming γ-glutamylcysteine from glutamate and cysteine with ATP consumption. This reaction represents a critical control point for cellular antioxidant capacity, directly influencing the organism's ability to detoxify reactive oxygen species (ROS) generated during aerobic metabolism or environmental stress.

Proteomic studies have demonstrated significant remodeling of the Rhodopirellula baltica proteome during the transition to stationary phase, including opposing regulation of central metabolic pathways such as the tricarboxylic acid cycle and oxidative pentose phosphate pathway . This metabolic shift likely coincides with increased oxidative stress, requiring enhanced glutathione production through enzymes like RB4485. The coordinated downregulation of various amino acid biosynthetic enzymes in stationary phase suggests a redirection of amino acid pools, potentially increasing substrate availability for stress-responsive pathways including glutathione synthesis .

The upregulation of alternative sigma factor sigmaH observed in stationary phase Rhodopirellula baltica is particularly noteworthy, as homologous sigma factors in other bacteria often control stress response genes . This transcriptional regulator might directly or indirectly influence RB4485 expression under stress conditions. Furthermore, the specific protein regulation observed during growth on solid surfaces suggests that RB4485 might participate in stress responses associated with biofilm formation or the development of sessile cell aggregates (rosettes), potentially through modulating the redox environment required for cell differentiation and extracellular matrix formation .

How can researchers use Avi-tag biotinylated RB4485 for advanced protein interaction studies?

The availability of Avi-tag biotinylated recombinant RB4485 (product code: CSB-EP756172RDR-B) provides researchers with a powerful tool for protein interaction studies with precise orientation control . The biotinylation occurs in vivo during protein expression through the BirA enzyme, which catalyzes the formation of an amide linkage between biotin and a specific lysine residue within the 15-amino acid AviTag sequence. This site-specific modification ensures uniform orientation of immobilized RB4485, in contrast to chemical biotinylation methods that often result in heterogeneous modification.

For protein interaction screening, Avi-tag biotinylated RB4485 can be immobilized on streptavidin-coated magnetic beads with exceptionally high affinity (Kd ≈ 10^-15 M), creating a stable platform for pull-down assays. Cellular lysates from Rhodopirellula baltica harvested at different growth phases can then be incubated with the immobilized protein to capture physiologically relevant interaction partners. The extremely stable biotin-streptavidin interaction allows for stringent washing conditions that reduce non-specific binding, while maintaining specific interactions for subsequent MS identification of binding partners.

Surface plasmon resonance (SPR) applications benefit particularly from the oriented immobilization provided by Avi-tag biotinylation. By capturing biotinylated RB4485 on streptavidin-functionalized sensor chips, researchers can measure the binding kinetics of potential interacting proteins with high reproducibility. This system allows determination of association rates (kon), dissociation rates (koff), and equilibrium dissociation constants (Kd) under various buffer conditions. The oriented nature of the immobilization ensures that all potential binding surfaces of RB4485 remain accessible, unlike random chemical coupling methods that may obscure interaction sites.

For structural studies, biotinylated RB4485 can be used in electron microscopy applications through streptavidin-conjugated gold nanoparticles as specific labels. This approach allows visualization of RB4485 within protein complexes, providing information about its spatial arrangement relative to interaction partners. Additionally, the biotin-streptavidin system can be leveraged for microarray applications, where biotinylated RB4485 is immobilized in a microarray format to simultaneously screen interactions with multiple proteins or cellular lysates under varying conditions of pH, salt concentration, or redox state.

What approaches can be used to determine the three-dimensional structure of RB4485?

Elucidating the three-dimensional structure of RB4485 requires a multi-faceted approach combining complementary structural biology techniques. X-ray crystallography represents the gold standard for high-resolution protein structure determination. For this approach, purified RB4485 (>95% purity) should undergo systematic crystallization screening using commercial sparse matrix kits with varying precipitants, buffers, and additives. Optimization typically requires exploring protein concentrations between 5-15 mg/mL, with and without substrates or substrate analogs. Co-crystallization with glutamate, cysteine, and non-hydrolyzable ATP analogs (AMP-PNP) may stabilize the protein in a catalytically relevant conformation.

Cryo-electron microscopy (cryo-EM) offers an alternative approach, particularly valuable if crystallization proves challenging or to visualize different conformational states. Sample preparation for cryo-EM typically requires protein concentrations of 1-5 mg/mL applied to glow-discharged grids before vitrification in liquid ethane. For higher resolution, Avi-tag biotinylated RB4485 bound to streptavidin provides increased particle mass, improving alignment accuracy during image processing . Both techniques can be complemented with hydrogen-deuterium exchange mass spectrometry (HDX-MS) to identify flexible regions and conformational changes upon substrate binding.

How can researchers investigate the potential role of RB4485 in morphotype development of Rhodopirellula baltica?

Investigating the potential role of RB4485 in Rhodopirellula baltica morphotype development requires a comprehensive experimental approach that connects enzyme function to cellular differentiation. Proteomic studies have identified proteins specifically regulated during growth on solid surfaces that may be involved in the development of different morphotypes, including motile swarmer cells and sessile cell aggregates (rosettes) . To establish if RB4485 participates in this process, researchers should first quantify its expression levels using quantitative PCR and Western blot analysis across different growth conditions and morphological states.

Gene knockout or knockdown studies provide a direct approach to assess functional importance. CRISPR-Cas9 or homologous recombination techniques can be employed to generate RB4485-deficient strains, followed by comprehensive phenotypic characterization including growth curves, microscopic examination of cell morphology, and surface attachment properties. Complementation studies with wild-type or mutated RB4485 variants can confirm phenotype specificity. For temporal control, inducible antisense RNA systems can down-regulate RB4485 expression at specific developmental stages to distinguish between its roles in initiation versus maintenance of morphological differentiation.

Localization studies using fluorescently tagged RB4485 (GFP fusion) can reveal spatial distribution patterns during morphotype development. Co-localization with known morphotype-specific proteins may indicate functional relationships. Time-lapse microscopy of the tagged protein during the transition from planktonic to surface-attached growth can identify critical time points for RB4485 recruitment or redistribution. These studies should be complemented with metabolomic analysis focusing on glutathione and related thiol compounds across different morphotypes, as altered redox homeostasis might be a mechanism through which RB4485 influences cellular differentiation.

Additionally, transcriptomic analysis comparing wild-type and RB4485-deficient strains during morphotype transitions can identify downstream genes affected by RB4485 activity, potentially revealing regulatory networks. Chemical complementation experiments using exogenous glutathione or its precursors might rescue morphological defects in RB4485-deficient strains, confirming the mechanistic link between enzyme activity and cellular differentiation. This integrated approach would establish whether RB4485 represents a critical node connecting metabolic state to morphological development in Rhodopirellula baltica.

What strategies can be employed to optimize RB4485 for biotechnological applications?

Directed evolution offers a complementary approach when structural information is limited. Error-prone PCR can generate libraries of RB4485 variants with random mutations, which are then screened for desired properties such as increased thermostability, broader pH tolerance, or altered substrate preference. This process can be accelerated using high-throughput screening methods based on colorimetric activity assays in microtiter plate format. DNA shuffling between RB4485 and homologous carboxylate-amine ligases can create chimeric enzymes with hybrid properties, potentially combining the most favorable characteristics from multiple parent enzymes.

Computational design represents an increasingly powerful strategy for enzyme optimization. Rosetta-based computational protocols can predict stabilizing mutations by analyzing energy landscapes and identifying destabilizing residue interactions. Similarly, molecular dynamics simulations can identify flexible regions that may compromise stability, guiding the introduction of disulfide bonds or salt bridges to rigidify these regions without impacting catalytic activity. These approaches can be particularly valuable for enhancing RB4485 stability under industrial conditions involving elevated temperatures or organic solvents.

Finally, enzyme immobilization strategies can significantly enhance RB4485 stability and reusability for industrial applications. The available Avi-tag biotinylated variant (CSB-EP756172RDR-B) provides an elegant approach for oriented immobilization on streptavidin-functionalized supports . Alternative immobilization methods include covalent attachment to activated resins through surface lysine residues, entrapment in sol-gel matrices, or cross-linked enzyme aggregates (CLEAs). Each approach offers different advantages in terms of enzyme loading, activity retention, and operational stability under application-specific conditions.