Recombinant Uncharacterized oxidoreductase dhs-5 (dhs-5)

What is the basic structural information available for oxidoreductase dhs-5?

Oxidoreductase dhs-5 from Caenorhabditis elegans is a full-length protein consisting of 378 amino acids (1-378aa). It corresponds to UniProt ID Q10130 and is also known by the synonyms F56D1.5 and short-chain dehydrogenase 5. The complete amino acid sequence is available and includes the primary structure: MAPTPPPPLSSTLIPEESSTSFFASLHSLLPTFHVENGAQLAVTMLLLIPVVYVGYRLYR TFFAFLKAIFIYTIAPLFYKPNLEQYQHRWTVVSGGTDGIGKAYTLELAKRGLRKFVLIG RNPKKLDSVKSEIEEKHSDAQIKTFVFDFGSGDFSSLRDYISDIDVGFVVNSVGTGRDNL ERYGDNPDEDTQILRVNGMGAAEFLSCVLPPMEKSGGGQIVVLSSSQGVRPIPMLAAYCA TKALMTFLCESIDREYSTINVQTLIPALVATKMTYYTKGSTFVVTPENFCHQAVGSIGLT KKTAGCLNHELQMLGFHLFPWTILKYLIMPIYYHQRKRVTELHNTSNNPEQEISLQELNE ETVVPTTKKTVTRDSATA . As a member of the short-chain dehydrogenase family, it likely shares the characteristic Rossmann fold typical of oxidoreductases that utilize NAD(P)H as a cofactor.

How should recombinant dhs-5 be stored and reconstituted for experimental use?

For optimal stability and activity, recombinant dhs-5 should be stored at -20°C/-80°C upon receipt, with aliquoting necessary for multiple use to avoid repeated freeze-thaw cycles. The lyophilized protein powder should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL. For long-term storage, it is recommended to add glycerol to a final concentration of 5-50% (with 50% being the standard recommendation) and then aliquot before storing at -20°C/-80°C . Working aliquots can be maintained at 4°C for up to one week, but repeated freezing and thawing is not recommended as it may compromise protein integrity and activity. The protein is typically supplied in a Tris/PBS-based buffer containing 6% trehalose at pH 8.0 .

What expression systems are most effective for producing recombinant dhs-5?

E. coli is the primary expression system used for recombinant production of dhs-5, as demonstrated in the commercially available preparations . E. coli is particularly advantageous for heterologous protein expression due to its genetic tractability, well-established growth conditions, and versatility in various applications . For dhs-5 specifically, an N-terminal His-tagged construct has been successfully expressed in E. coli systems, facilitating purification through affinity chromatography. When designing expression strategies, researchers should consider codon optimization for E. coli, as C. elegans genes often contain codons rarely used in E. coli. Additionally, expression conditions such as temperature, IPTG concentration, and induction time should be optimized to maximize soluble protein yield while minimizing inclusion body formation.

What are the predicted cofactor requirements for dhs-5 based on sequence homology?

Based on sequence analysis and classification as a short-chain dehydrogenase, dhs-5 likely requires NAD+ or NADP+ as a cofactor for its oxidoreductase activity. The protein contains the characteristic sequence motif TGXXXGXG that is typically associated with the Rossmann fold, which binds nicotinamide cofactors . Additionally, analysis of the amino acid sequence reveals potential binding sites for these cofactors in the N-terminal region of the protein. The specificity for either NAD+ or NADP+ would need to be determined experimentally through cofactor preference assays.

Researchers investigating cofactor requirements should consider the following experimental approach:

Similar to other oxidoreductases, altering cofactor supply and availability in experimental systems may significantly impact the enzymatic activity of dhs-5 .

How might the redox sensitivity of dhs-5 compare to other characterized dehydrogenases?

To investigate redox sensitivity, researchers should consider:

• Conducting activity assays under varying ratios of reduced/oxidized cofactors

• Examining potential allosteric regulation by measuring enzyme kinetics at different redox potentials

• Exploring whether mutations similar to the PDH E353K variant, which decreases sensitivity to NADH in E. coli, might alter the redox sensitivity of dhs-5

• Investigating potential oxidative stress effects on enzyme stability and function

Understanding the redox sensitivity would provide insights into the physiological conditions under which dhs-5 functions optimally and its potential role in metabolic pathways influenced by cellular redox status.

What approaches can be used to determine the physiological substrate and function of dhs-5 in C. elegans?

Determining the physiological substrate and function of an uncharacterized enzyme like dhs-5 requires a multi-faceted approach:

• Metabolomics analysis: Compare metabolite profiles between wild-type and dhs-5 knockout C. elegans strains using LC-MS/MS to identify accumulated substrates or depleted products.

• In vitro substrate screening: Test a library of potential substrates using purified recombinant dhs-5, monitoring NAD(P)H consumption or production spectrophotometrically.

• Phenotypic analysis: Characterize phenotypes of dhs-5 mutants under various growth conditions and stressors to identify functional pathways.

• Co-expression analysis: Identify genes with expression patterns that correlate with dhs-5 in C. elegans, suggesting functional relationships.

• Protein-protein interaction studies: Use immunoprecipitation coupled with mass spectrometry to identify interaction partners, potentially revealing functional pathways.

The combination of these approaches can provide complementary evidence for the physiological role of dhs-5. When performing substrate screening, it is crucial to monitor both forward and reverse reactions since oxidoreductases can catalyze both oxidation and reduction reactions depending on the cellular environment and substrate availability.

What are the optimal conditions for assaying dhs-5 enzymatic activity?

Without specific literature on dhs-5 activity assays, the following general approach for oxidoreductases can be applied:

When developing the assay, researchers should consider that short-chain dehydrogenases typically show activity towards a range of substrates including alcohols, steroids, sugars, and aromatic compounds. A systematic approach testing different substrate classes would be advisable for initial characterization. Additionally, assay conditions should be optimized through factorial design experiments varying pH, temperature, ionic strength, and potential activators or inhibitors.

How can researchers effectively utilize site-directed mutagenesis to explore the catalytic mechanism of dhs-5?

Site-directed mutagenesis is a powerful approach for elucidating catalytic mechanisms of enzymes like dhs-5. Based on sequence analysis and structural predictions, researchers can target the following residues:

• Catalytic triad residues: Short-chain dehydrogenases typically contain a conserved catalytic triad (Ser-Tyr-Lys). Identify these residues in dhs-5 sequence and create single and double mutants.

• Cofactor binding residues: Target glycine-rich regions (e.g., TGXXXGXG motifs) involved in cofactor binding.

• Substrate binding pocket residues: Identify and mutate hydrophobic or charged residues likely involved in substrate recognition.

The experimental workflow should include:

• Design primers for QuikChange or other site-directed mutagenesis methods

• Express and purify mutant proteins following the same protocol as wild-type

• Compare biochemical properties (Km, kcat, substrate specificity) between wild-type and mutant proteins

• Perform structural analysis (if possible) to confirm the effects of mutations

Researchers should consider using conservative mutations (e.g., Ser→Ala, Lys→Arg) to specifically probe the role of functional groups. Additionally, creating a library of multiple mutants using techniques like alanine scanning can provide comprehensive insights into structure-function relationships.

What protein crystallization conditions should be considered for structural studies of dhs-5?

For protein crystallization of dhs-5, researchers should consider:

• Protein preparation:

  • Ensure high purity (>95% by SDS-PAGE)

  • Remove His-tag if it interferes with crystallization

  • Use size-exclusion chromatography to ensure monodispersity

  • Concentrate to 5-15 mg/mL in a stabilizing buffer

• Initial screening:

  • Use commercial sparse matrix screens (Hampton Research, Molecular Dimensions)

  • Include conditions for other oxidoreductases (typically 15-25% PEG, pH 6.5-8.0)

  • Screen with and without cofactors (NAD+/NADP+) and potential substrates

  • Try both vapor diffusion and batch crystallization methods

• Optimization:

  • Fine-tune promising conditions by varying precipitant concentration, pH, and additives

  • Test seeding techniques for crystal improvement

  • Consider surface entropy reduction mutations if initial screening fails

• Alternative approaches:

  • If crystallization proves challenging, consider cryo-EM as an alternative structural approach, especially if dhs-5 forms larger oligomeric complexes

  • Nuclear Magnetic Resonance (NMR) may be suitable for structural studies of specific domains

Researchers should note that the presence of cofactors or substrate analogs often stabilizes oxidoreductases and improves crystallization success rates. Additionally, including reducing agents like DTT or β-mercaptoethanol may prevent oxidation of catalytic cysteine residues and improve homogeneity.

What are common challenges in achieving soluble expression of dhs-5 and how can they be addressed?

Researchers may encounter several challenges when expressing dhs-5 in E. coli:

When troubleshooting expression issues, a systematic approach testing multiple variables is recommended. Small-scale expression trials followed by solubility analysis can rapidly identify optimal conditions before scaling up. Additionally, researchers should be aware that as an oxidoreductase, dhs-5 might be sensitive to oxidizing conditions, so maintaining a reducing environment during purification may improve yield and activity.

How can researchers distinguish between specific and non-specific activities when characterizing dhs-5?

Distinguishing between specific and non-specific activities is crucial when characterizing uncharacterized enzymes like dhs-5:

• Perform comprehensive controls:

  • Use heat-inactivated enzyme preparations

  • Include samples lacking substrate or cofactor

  • Test activity with structurally similar but non-substrate compounds

  • Use denatured protein controls to check for non-enzymatic reactions

• Analyze enzyme kinetics:

  • True enzymatic activities follow Michaelis-Menten kinetics

  • Determine Km and kcat values for potential substrates

  • Compare catalytic efficiency (kcat/Km) across substrates to identify preferred ones

  • Verify substrate depletion and product formation by complementary analytical methods (HPLC, LC-MS)

• Employ inhibition studies:

  • Test known inhibitors of related oxidoreductases

  • Perform product inhibition analysis

  • Use site-directed mutants of catalytic residues as negative controls

• Validate with orthogonal methods:

  • Confirm product formation using mass spectrometry

  • Perform isotope labeling studies to track atom transfer

  • Use binding studies (ITC, SPR) to confirm substrate interactions

When evaluating potential substrates, researchers should be cautious about artifactual activities that may arise from protein impurities or non-enzymatic reactions between assay components. Validation across multiple experimental approaches provides the strongest evidence for physiologically relevant activities.

What bioinformatic approaches can help predict the function of dhs-5 prior to experimental characterization?

Several bioinformatic approaches can provide valuable insights into the potential function of dhs-5:

• Sequence-based analysis:

  • BLAST and PSI-BLAST searches against characterized proteins

  • Multiple sequence alignment with known oxidoreductases

  • Identification of conserved motifs using MEME or PROSITE

  • Phylogenetic analysis to identify evolutionary relationships

• Structural prediction:

  • Homology modeling using related oxidoreductase structures as templates

  • Prediction of binding pockets using CASTp or similar tools

  • Molecular docking of potential substrates and cofactors

  • Molecular dynamics simulations to evaluate substrate-enzyme interactions

• Systems biology approaches:

  • Gene co-expression network analysis in C. elegans datasets

  • Pathway enrichment analysis of co-expressed genes

  • Metabolic network reconstruction to identify potential reaction nodes

  • Genetic interaction networks from high-throughput C. elegans studies

• Function prediction tools:

  • COFACTOR and COACH for enzyme function prediction

  • EFICAz for enzyme function inference

  • Gene Ontology term prediction using tools like PANNZER or DeepGOPlus

When applying these approaches, researchers should integrate results from multiple methods to build a consensus prediction. The predictions should guide experimental design but not substitute for biochemical characterization. Particular attention should be paid to residues conserved across related oxidoreductases, as these often represent functionally important sites for catalysis or substrate binding.

How might high-throughput screening approaches be optimized for identifying dhs-5 substrates?

High-throughput screening for dhs-5 substrates can be optimized through:

• Library design and preparation:

  • Create focused libraries based on metabolomics data from C. elegans

  • Include metabolites from pathways where short-chain dehydrogenases typically function

  • Organize compounds into chemical similarity clusters to identify structure-activity relationships

  • Prepare libraries in microplate format compatible with liquid handling systems

• Assay development:

  • Optimize a NAD(P)H-coupled fluorescence or absorbance-based assay in 384-well format

  • Develop assays for both oxidation and reduction directions

  • Include internal standards and positive controls in each plate

  • Validate assay robustness using Z'-factor determination

• Screening execution and data analysis:

  • Screen at multiple substrate concentrations to avoid missing high-Km substrates

  • Implement machine learning algorithms to identify patterns in substrate preference

  • Use hierarchical clustering to group active compounds by structural features

  • Perform validation assays with hit compounds including dose-response curves

• Follow-up characterization:

  • Confirm hits using orthogonal assays (e.g., HPLC product detection)

  • Determine detailed kinetic parameters for the most promising substrates

  • Investigate structure-activity relationships to define the substrate recognition pattern

  • Test physiological relevance using in vivo approaches in C. elegans

This comprehensive approach increases the likelihood of identifying physiologically relevant substrates among the potentially numerous compounds that may show some activity with dhs-5 in vitro.

What is the potential significance of studying dhs-5 in relation to metabolic engineering applications?

Given the oxidoreductase activity of dhs-5, studying this enzyme has several potential applications in metabolic engineering:

• Novel biocatalyst development:

  • If dhs-5 shows unique substrate specificity or stereo/regioselectivity, it could be valuable for chemical synthesis

  • Engineered variants might catalyze reactions not efficiently performed by currently available enzymes

  • Integration into multi-enzyme cascades for complex transformations

• Pathway engineering in microbial hosts:

  • Expression in E. coli or other production hosts could create new metabolic routes

  • Potential role in redox balance control in engineered pathways

  • May enable production of novel secondary metabolites or fine chemicals

• Cofactor regeneration systems:

  • If dhs-5 shows high activity with specific substrates, it might be useful in NAD(P)H regeneration systems

  • Could be paired with other enzymes requiring reduced cofactors in biotransformation processes

  • Similar to how formate dehydrogenase is used to regenerate NADH in biocatalytic applications

• Synthetic biology applications:

  • Integration into designer metabolic pathways

  • Potential orthogonal components for synthetic circuits if the enzyme has unique properties

  • Engineered variants with altered cofactor specificity (NAD+ vs. NADP+) for specific applications

Understanding the catalytic mechanism and substrate profile of dhs-5 is the first step toward these applications. Subsequently, protein engineering through rational design or directed evolution could optimize the enzyme for specific biotechnological purposes.

How does the study of uncharacterized oxidoreductases like dhs-5 contribute to our understanding of C. elegans metabolism?

Characterizing uncharacterized oxidoreductases like dhs-5 significantly advances our understanding of C. elegans metabolism through:

• Filling knowledge gaps in metabolic networks:

  • Many predicted metabolic reactions in C. elegans lack assigned enzymes

  • Characterizing dhs-5 may connect orphan metabolic activities to specific genes

  • Improves the accuracy of metabolic modeling and flux balance analysis

• Understanding unique metabolic adaptations:

  • C. elegans has several unique metabolic pathways not found in other model organisms

  • Characterizing dhs-5 may reveal nematode-specific metabolic processes

  • Could identify novel metabolites with signaling or regulatory functions

• Developmental and aging metabolism insights:

  • Oxidoreductases often play crucial roles in developmental transitions and stress responses

  • dhs-5 function may reveal metabolic shifts associated with C. elegans life stages

  • Could provide insights into metabolic aspects of aging, as C. elegans is a key model for longevity studies

• Comparative biochemistry perspectives:

  • Comparing dhs-5 function to homologs in other organisms provides evolutionary insights

  • May reveal how metabolic pathways have adapted in different niches

  • Contributes to understanding the conservation and divergence of core metabolic functions

• Potential biomedical relevance:

  • C. elegans is a model for studying human disease mechanisms

  • Characterizing dhs-5 may provide insights into functions of human homologs

  • Could reveal potential drug targets for parasitic nematodes if the enzyme is conserved

The systematic characterization of unassigned enzymes like dhs-5 represents a crucial step toward a complete functional annotation of the C. elegans genome and a comprehensive understanding of nematode metabolism.