Recombinant Arabidopsis thaliana Rhodanese-like domain-containing protein 10 (STR10)

What is the biochemical function of rhodanese-like domain-containing proteins in Arabidopsis thaliana?

Rhodanese-like domain (RLD) containing proteins in Arabidopsis thaliana function primarily as sulfurtransferases, mediating the transfer of sulfur atoms between various donor and acceptor molecules. These enzymes catalyze transsulfuration reactions through a transient persulfide intermediate formed at a conserved cysteine residue in their active sites . The canonical rhodanese reaction involves the transfer of sulfur from thiosulfate to cyanide, forming thiocyanate (SCN-) and sulfite.

In Arabidopsis, RLDs participate in several aspects of sulfur metabolism, including:

• Sulfide oxidation in mitochondria

• Iron-sulfur cluster biogenesis

• Thio-cofactor biosynthesis

• Detoxification processes

Importantly, experimental evidence from characterized Arabidopsis sulfurtransferases indicates that some RLDs prefer 3-mercaptopyruvate to thiosulfate as a sulfur donor, suggesting specialized functions beyond classical rhodanese activity . This substrate preference diversity likely reflects adaptations to specific physiological roles within plant metabolism.

Where are rhodanese-like proteins localized in Arabidopsis thaliana cells?

Subcellular localization studies provide important insights into the potential physiological functions of rhodanese-like proteins in Arabidopsis. Research on a characterized Arabidopsis sulfurtransferase revealed that the protein contains a putative transit peptide sequence of approximately 7.1 kDa, suggesting targeted subcellular localization .

Experimental evidence using monospecific antibodies against the mature recombinant protein detected a single band equating to the size of the mature protein in purified Arabidopsis mitochondria, while no antigenic reaction was observed with proteins from chloroplasts . This indicates mitochondrial localization for at least some Arabidopsis rhodanese-like proteins.

For definitive determination of STR10 localization, researchers should employ multiple complementary approaches:

• Sequence analysis for targeting signals using prediction algorithms

• Fluorescent protein fusion experiments with confocal microscopy

• Subcellular fractionation followed by western blot analysis

• Immunogold electron microscopy for high-resolution localization

The mitochondrial localization of some RLDs correlates with potential roles in sulfide oxidation pathways or iron-sulfur cluster assembly processes that occur within this organelle.

What is the protein structure of rhodanese-like domains in Arabidopsis?

Rhodanese-like domains in Arabidopsis exhibit an α-β-repeat fold typical of the PF00581 rhodanese domain family . Structural studies of Ab-RLD (an example RLD) reveal that it forms a homodimer where the two active sites are located at opposite sides of the protein complex .

Key structural features include:

• An α-β-repeat fold characteristic of the rhodanese superfamily

• A conserved catalytic cysteine residue in the active site

• Homodimeric quaternary structure in some cases

• N-terminal extensions that may contribute to protein stability or interactions

• N-terminal α-helix (α1) not typically found in standard PF00581 rhodanese domains

For recombinant expression studies, researchers should note that some Arabidopsis RLDs contain putative transit peptides (approximately 7.1 kDa in the characterized example), which would be cleaved in the mature protein . Expression constructs may need to be designed with or without this sequence depending on research objectives.

What are the optimal expression conditions for recombinant Arabidopsis rhodanese-like proteins in E. coli systems?

Based on successful expression strategies documented for Arabidopsis rhodanese-like proteins, researchers can optimize recombinant protein expression using the following methodological approach:

Expression system selection:

• E. coli has been successfully used for Arabidopsis RLD expression

• Consider strains that address codon bias issues for plant proteins

• For proteins with disulfide bonds, specialized strains may be beneficial

Vector design considerations:

• Include an N-terminal His6-tag for affinity purification

• Express both full-length protein and mature protein (without transit peptide)

• Consider codon optimization for E. coli expression

Purification strategy:

• Affinity chromatography using Ni-NTA for His-tagged proteins

• Include reducing agents in buffers to protect the active site cysteine

• Consider additional purification steps like ion exchange or size exclusion chromatography

Protein quality assessment:

• SDS-PAGE and western blotting to confirm size and purity

• Activity assays using thiosulfate and cyanide to confirm functional expression

• Circular dichroism to verify proper folding

How can enzyme kinetics of rhodanese-like proteins be accurately measured?

Accurate enzyme kinetic measurements for rhodanese-like proteins require careful experimental design to account for the unique properties of sulfur transfer reactions. Based on published studies, researchers can implement the following methodology:

Standard rhodanese activity assay:

• The canonical assay measures thiocyanate (SCN-) formation from thiosulfate and cyanide

• Formation of SCN- can be detected spectrophotometrically at 460 nm using the ferric nitrate method

• Control reactions without enzyme are essential to account for non-enzymatic reactions

Kinetic parameters determination:

• For Ab-RLD, reported kinetic parameters include:

  • kcat values of 0.608 s-1 and 0.975 s-1 for thiosulfate and cyanide, respectively

  • Km values of 114.9 μM for thiosulfate and 41.6 mM for cyanide

• Vary substrate concentrations to determine Km and Vmax using Michaelis-Menten kinetics

Alternative substrate testing:

• Evidence suggests that some Arabidopsis RLDs prefer 3-mercaptopyruvate to thiosulfate

• Screen multiple potential sulfur donors including:

  • Thiosulfate

  • 3-mercaptopyruvate

  • Lipoic acid

  • Cysteine persulfide

Table 1: Comparison of kinetic parameters for different rhodanese-like proteins

What experimental approaches can be used to determine the in vivo substrates of rhodanese-like proteins?

Determining the physiological substrates of rhodanese-like proteins is challenging due to the reactivity of sulfur compounds and the complexity of sulfur metabolism. Researchers can employ the following complementary approaches:

Genetic approaches:

• Generate knockout/knockdown mutants in Arabidopsis

• Assess changes in metabolite profiles using metabolomics

• Perform genetic complementation experiments

• Create conditional expression systems to study essential genes

Metabolomics profiling:

• Targeted liquid chromatography-mass spectrometry (LC-MS) analysis of sulfur-containing metabolites

• Stable isotope labeling with 34S or 35S to track sulfur transfer reactions

• Sample preparation under anaerobic conditions to prevent oxidative artifacts

Protein-substrate interaction studies:

• Substrate trapping using active site mutants (e.g., C→S mutations)

• Chemical crosslinking followed by mass spectrometry

• Thermal shift assays to identify ligands that stabilize the protein

Structural biology approaches:

• Co-crystallization with putative substrates or substrate analogs

• Molecular docking studies combined with site-directed mutagenesis

• NMR-based screening of metabolite libraries

How does the 3D structure of rhodanese-like domains relate to their catalytic function?

The structure-function relationship in rhodanese-like domains provides insights into their catalytic mechanism and substrate specificity:

Active site architecture:

• The conserved catalytic cysteine residue is positioned at the end of a nucleophilic elbow

• The active site environment tunes the pKa of the catalytic cysteine to enhance its nucleophilicity

• Positively charged residues often surround the active site to interact with negatively charged substrates like thiosulfate

Mechanistic implications from structural studies:

• The α-β-repeat fold positions the catalytic cysteine optimally for nucleophilic attack on sulfur donors

• The formation of the persulfide intermediate involves significant conformational changes

• The homodimeric arrangement of Ab-RLD places active sites on opposite sides, potentially allowing for coordinated catalysis or regulation

Experimental approaches to probe structure-function relationships:

• Site-directed mutagenesis of active site and substrate-binding residues

• Hydrogen-deuterium exchange mass spectrometry to identify flexible regions

• Small-angle X-ray scattering (SAXS) to study conformational states in solution

How conserved are rhodanese-like domains across different species and within Arabidopsis?

Bioinformatic analyses indicate that rhodanese-like domains are highly conserved across diverse organisms from bacteria to plants and mammals, while showing important variations that may reflect functional specialization:

Evolutionary conservation:

• RLDs belong to a single protein family (PF00581) with multiple clusters at 29% sequence identity threshold

• Sequence similarity network (SSN) analysis reveals distinct clusters with conserved genome neighborhoods and gene synteny

• Highly conserved catalytic residues, particularly the active site cysteine

• Variable N-terminal regions, often containing targeting sequences in eukaryotes

Conservation within Arabidopsis:

• Southern blot analysis with genomic Arabidopsis DNA showed the occurrence of at least two sulfurtransferase-like isozymes

• Specialized clusters of encapsulin-associated RLDs with distinct evolutionary relationships have been identified

• The existence of multiple isoforms suggests functional diversification within the plant

For comparative studies, researchers should perform multiple sequence alignments with rhodanese-like proteins from diverse organisms to identify both universally conserved residues and plant-specific features that might indicate specialized functions in plant metabolism.

What are the potential effects of rhodanese-like protein knockout or overexpression on Arabidopsis phenotypes?

Understanding the phenotypic consequences of altering rhodanese-like protein expression requires systematic genetic studies:

Knockout/knockdown approaches:

• T-DNA insertion mutants from available Arabidopsis collections

• CRISPR/Cas9 gene editing for precise mutations

• RNAi or artificial microRNA for conditional knockdown

• Careful genotyping to confirm complete loss of function

Overexpression strategies:

• Constitutive expression using the CaMV 35S promoter as demonstrated in other Arabidopsis studies

• Tissue-specific or inducible expression systems

• Expression with and without transit peptides for altered localization

Potential phenotypes based on RLD functions:

• Altered sulfur metabolism

• Changed tolerance to cyanide or heavy metals

• Mitochondrial dysfunction

• Oxidative stress sensitivity

• Altered iron homeostasis due to impacts on Fe-S cluster assembly

Molecular mechanisms investigation:

• Transcriptome analysis to identify compensatory responses

• Protein-protein interaction studies to identify affected complexes

• Enzyme activity measurements in plant extracts

• In vivo imaging of reactive sulfur species using specific probes

How can rhodanese-like protein research contribute to understanding plant stress responses?

Research on rhodanese-like domain-containing proteins has significant implications for understanding plant stress responses:

Oxidative stress connections:

• RLDs may function in detoxification of reactive oxygen species

• Mitochondrial localization places certain RLDs at a major site of ROS production

• Persulfide chemistry intersects with cellular redox systems

Heavy metal stress responses:

• Rhodaneses may contribute to heavy metal detoxification via formation of metal-thiocyanate complexes

• Potential involvement in sulfide homeostasis, which affects metal chelation

• Possible roles in iron-sulfur cluster repair or protection from metal-induced damage

Translational research potential:

• Insights from Arabidopsis could be applied to crops, similar to other successful examples where Arabidopsis genes improved stress tolerance in crops

• The SALT- AND DROUGHT-INDUCED RING-FINGER1 (SDIR1) gene from Arabidopsis has been shown to increase drought tolerance when expressed in tobacco and rice

• Understanding sulfur metabolism networks could lead to improved nutrient use efficiency

How can protein-protein interactions of rhodanese-like proteins be identified and characterized?

Identifying the interaction partners of rhodanese-like proteins is crucial for understanding their biological context and regulation:

In vitro interaction studies:

• Pull-down assays with recombinant protein as bait

• Surface plasmon resonance (SPR) for quantitative binding measurements

• Analytical size exclusion chromatography to detect complex formation

In vivo interaction discovery:

• Co-immunoprecipitation using specific antibodies

• Tandem affinity purification coupled with mass spectrometry (TAP-MS)

• Proximity-dependent biotin identification (BioID) or APEX2 proximity labeling

• Yeast two-hybrid screening with appropriate controls for autoactivation

Validation of interactions:

• Bimolecular fluorescence complementation (BiFC) in planta

• Förster resonance energy transfer (FRET) or fluorescence-lifetime imaging microscopy (FLIM)

• Co-localization studies by confocal microscopy

• Genetic interaction studies (e.g., synthetic lethality, suppressor screens)

Functional characterization of interactions:

• Activity assays in the presence of interaction partners

• Structural studies of protein complexes

• Mutagenesis to map interaction interfaces

What are the emerging research directions for rhodanese-like domain-containing proteins in plants?

Several exciting research directions are emerging for rhodanese-like domain-containing proteins in plants:

Integration with sulfur signaling networks:

• Investigation of RLDs as potential sensors or transducers of sulfur availability

• Connections between sulfur metabolism and plant hormone signaling

• Roles in systemic sulfur distribution and allocation

Persulfidation (protein S-sulfhydration) biology:

• RLDs as potential catalysts of protein persulfidation

• Regulatory roles of persulfidation in modulating enzyme activities

• Development of methodologies to detect and quantify protein persulfidation in vivo

Cellular compartmentalization of sulfur metabolism:

• Organelle-specific functions of different RLD family members

• Inter-organelle communication mediated by sulfur metabolites

• Evolutionary adaptation of RLDs to specific subcellular compartments

Translational research opportunities:

• Biofortification of crops with enhanced sulfur-containing nutrients

• Engineering of sulfur metabolism for improved crop stress tolerance

• Potential applications in translational research similar to other Arabidopsis genes that have been successfully transferred to crops