DERA Human

What is human DERA and what is its primary function?

Human DERA (deoxyribose-phosphate aldolase) is an enzyme that belongs to the deoC/fbaB aldolase protein family involved in carbohydrate degradation pathways. Its primary function is to catalyze the conversion of 2-deoxy-D-ribose 5-phosphate to D-glyceraldehyde 3-phosphate and acetaldehyde . This aldolase plays a significant role in nucleoside catabolism and the pentose phosphate pathway, which is critical for cellular metabolism and energy production. The enzyme functions by breaking carbon-carbon bonds in aldose sugars, particularly those found in nucleic acid components. This catalytic activity positions DERA as an important metabolic enzyme involved in recycling nucleoside components in human cells.

What are the structural characteristics of human DERA?

Human DERA is a protein with 318 amino acids in its native form. The recombinant version often includes a histidine tag, bringing the total to 338 amino acids with a molecular weight of approximately 37.3 kDa, as confirmed by MALDI-TOF analysis . The protein's amino acid sequence includes conserved regions typical of the aldolase family, with specific domains responsible for substrate binding and catalytic activity. The tertiary structure enables the protein to bind to deoxyribose derivatives and perform its aldolase function. The full sequence begins with MSAHNRGTEL and contains domains essential for substrate recognition and enzymatic activity throughout its structure .

What is the gene information for human DERA?

The human DERA gene is officially designated as "DERA" with the description "deoxyribose-phosphate aldolase (putative)" in Homo sapiens. It has the Gene ID 51071 and its mRNA is represented by RefSeq NM_015954 . The gene has multiple synonyms including DEOC, CGI-26, deoxyriboaldolase, and phosphodeoxyriboaldolase. The enzyme is classified as EC 4.1.2.4 (2-deoxyribose-5-phosphate aldolase) in the enzyme classification system. The gene encodes the protein that catalyzes a specific aldol cleavage reaction in nucleotide metabolism pathways and has been identified across various species, indicating evolutionary conservation of this metabolic function.

How can recombinant human DERA be expressed and purified for research?

Recombinant human DERA is typically expressed in E. coli expression systems with an N-terminal His-tag for facilitated purification . A standard expression protocol includes:

• Cloning the human DERA gene (amino acids 1-318) into an appropriate expression vector with a His-tag sequence.

• Transforming the construct into a compatible E. coli strain optimized for protein expression.

• Inducing protein expression under controlled conditions (temperature, time, inducer concentration).

• Cell lysis under native conditions to preserve protein activity.

• Purification using affinity chromatography (Ni-NTA columns) to bind the His-tagged protein.

• Additional purification steps such as size exclusion or ion-exchange chromatography to achieve higher purity.

This approach typically yields DERA protein with >90% purity as confirmed by SDS-PAGE analysis . The recombinant protein retains its enzymatic activity and can be used for various biochemical and structural studies.

What are the optimal storage conditions for maintaining DERA activity?

For short-term storage (1-2 weeks), recombinant human DERA can be stored at +4°C in an appropriate buffer system . For long-term storage, the protein should be aliquoted to avoid repeated freeze-thaw cycles and stored at -20°C or preferably -70°C . The optimal buffer composition for DERA storage includes:

• 20mM Tris-HCl buffer (pH 8.0)

• 20% glycerol (cryoprotectant)

• 0.1M NaCl (ionic strength maintenance)

• 1mM DTT (reducing agent to prevent oxidation of cysteine residues)

This formulation maintains protein stability and enzymatic activity during storage. It's essential to avoid repeated freezing and thawing as this can lead to protein denaturation and loss of activity. When conducting experiments, working aliquots should be thawed quickly and kept on ice during handling to preserve enzymatic function.

How can enzymatic activity of human DERA be measured?

The enzymatic activity of human DERA can be measured using several approaches:

• Spectrophotometric assays: Monitoring the formation of D-glyceraldehyde 3-phosphate by coupling with NADH-dependent glyceraldehyde-3-phosphate dehydrogenase and measuring the decrease in NADH absorbance at 340 nm.

• Coupled enzyme assays: Using auxiliary enzymes to convert the products of the DERA reaction into measurable signals.

• Direct product quantification: Using HPLC or LC-MS to quantify the formation of D-glyceraldehyde 3-phosphate and acetaldehyde from 2-deoxy-D-ribose 5-phosphate.

Standard reaction conditions include:

• Buffer: 50 mM phosphate or Tris-HCl, pH 7.5-8.0

• Temperature: 25-37°C

• Substrate concentration: 0.1-5 mM 2-deoxy-D-ribose 5-phosphate

• Enzyme concentration: 0.1-1 μg/mL purified DERA

Kinetic parameters (Km, Vmax, kcat) can be determined by varying substrate concentrations and analyzing data using Michaelis-Menten or Lineweaver-Burk plots.

What role does DERA play in the pentose phosphate pathway?

DERA is a key enzyme in the pentose phosphate pathway, specifically in the non-oxidative branch . It catalyzes a reversible aldol reaction that connects the pentose phosphate pathway with glycolysis through the formation of D-glyceraldehyde 3-phosphate. In research contexts, DERA can be used as a tool to study:

• Metabolic flux through the pentose phosphate pathway

• Nucleotide salvage pathways

• Cellular responses to altered nucleoside metabolism

• Connections between carbohydrate metabolism and nucleic acid turnover

Studies involving DERA can employ techniques such as metabolic flux analysis, isotope labeling, and metabolomics to track the flow of carbon through these interconnected pathways. Inhibition or overexpression of DERA in cell models can reveal how cells adapt to perturbations in these metabolic networks, providing insights into cellular metabolism regulation under normal and disease conditions.

How does human DERA differ from DERA in other organisms?

Human DERA shares structural and functional similarities with DERA enzymes from other organisms but exhibits species-specific characteristics. Comparative studies reveal:

These differences can be exploited in research to:

• Develop species-specific inhibitors for antimicrobial applications

• Study evolutionary conservation of metabolic pathways

• Identify structural determinants of substrate specificity

• Engineer enzymes with altered catalytic properties for biotechnological applications

Comparing the crystal structures, catalytic mechanisms, and regulatory properties of DERA across species can provide valuable insights into the evolution of metabolic enzymes and pathway specialization.

What experimental techniques are useful for studying DERA's structure-function relationships?

Several advanced techniques can be employed to investigate DERA's structure-function relationships:

• X-ray crystallography: Determining the three-dimensional structure of DERA in various states (apo-enzyme, substrate-bound, product-bound) to understand catalytic mechanism.

• Site-directed mutagenesis: Systematically altering amino acids in the active site or other functional domains to assess their contribution to enzyme activity, substrate binding, and structural stability.

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): Probing protein dynamics and conformational changes associated with substrate binding and catalysis.

• Molecular dynamics simulations: In silico analysis of protein motion, substrate binding, and catalytic mechanisms based on structural data.

• Isothermal titration calorimetry (ITC): Quantifying thermodynamic parameters of substrate binding and enzyme-inhibitor interactions.

These approaches, when combined, provide comprehensive insights into how DERA's structure determines its function, enabling rational design of inhibitors or engineered variants with altered properties for research applications.

What are potential pitfalls when working with recombinant human DERA?

Researchers working with recombinant human DERA should be aware of several potential challenges:

• Protein aggregation: DERA may form aggregates during purification or storage, particularly at high concentrations. This can be mitigated by including appropriate stabilizing agents in the buffer and avoiding freeze-thaw cycles.

• Loss of activity during purification: Some purification methods may cause partial denaturation or loss of cofactors. Activity assays should be performed at each purification step to track recovery of enzymatic function.

• Substrate stability: The substrate 2-deoxy-D-ribose 5-phosphate can degrade during storage. Fresh preparation or proper storage conditions are essential for reliable activity measurements.

• Interference in coupled assays: When using coupled enzyme assays, components of the reaction mixture may interfere with auxiliary enzymes. Appropriate controls should be included to account for such effects.

• Tag interference: The His-tag used for purification may influence enzyme activity or structural properties. Comparison with tag-cleaved versions or alternative tag positions should be considered for critical experiments.

Addressing these challenges requires careful experimental design and appropriate controls to ensure reliable and reproducible results when studying DERA function and properties.

How can DERA be used in metabolic engineering research?

DERA has significant potential in metabolic engineering research due to its ability to catalyze aldol reactions with high stereospecificity. Applications include:

• Pathway reconstruction: Incorporating DERA into artificial metabolic pathways to produce value-added compounds from simple precursors.

• Metabolic flux analysis: Using DERA as a probe to understand carbon flow through interconnected metabolic pathways.

• Enzyme evolution studies: Engineering DERA variants with altered substrate specificity or enhanced catalytic efficiency through directed evolution or rational design.

• Synthetic biology applications: Utilizing DERA in cell-free enzymatic cascades for the production of complex molecules.

Methodological approaches include:

• Expression of DERA in heterologous systems

• Assessing DERA activity in complex cellular extracts

• Combining DERA with other enzymes in multi-step reactions

• Comparing wild-type and engineered DERA variants for specific applications

These applications demonstrate how fundamental understanding of DERA can be translated into practical research tools and biotechnological innovations.

What considerations should be made when designing inhibitor studies for DERA?

When designing inhibitor studies for human DERA, researchers should consider:

• Inhibitor specificity: Ensure that potential inhibitors specifically target DERA rather than related aldolases or other enzymes in the pentose phosphate pathway.

• Mechanism-based approach: Design inhibitors based on the transition state of the DERA-catalyzed reaction or substrate analogues that bind competitively to the active site.

• Assay compatibility: Confirm that the inhibitor does not interfere with the detection method used in the enzymatic assay.

• Physiological relevance: Consider the cellular environment (pH, ionic strength, presence of metabolites) when evaluating inhibitor efficacy in vitro versus in vivo.

• Structure-activity relationships: Systematically vary inhibitor structures to identify key functional groups responsible for binding and inhibition.

Experimental design should include:

• Determination of IC50 values under standardized conditions

• Kinetic analysis to determine the mode of inhibition (competitive, non-competitive, uncompetitive)

• Direct binding studies using biophysical methods (ITC, SPR, MST)

• Cellular studies to assess inhibitor uptake, stability, and target engagement

These considerations will help develop reliable inhibitor studies that advance understanding of DERA function and potential therapeutic applications.

What are emerging research areas involving human DERA?

Several promising research directions are emerging in the field of human DERA:

• Role in cancer metabolism: Investigating how alterations in DERA expression or activity contribute to metabolic reprogramming in cancer cells, particularly in relation to nucleoside metabolism and the pentose phosphate pathway.

• DERA in neurodegenerative diseases: Exploring connections between DERA function and neuronal metabolism, as the pentose phosphate pathway plays a critical role in maintaining redox homeostasis in neurons.

• Development of specific inhibitors: Designing selective DERA inhibitors as chemical probes to dissect metabolic networks or as potential therapeutic agents.

• Structural biology advancements: Utilizing cryo-EM and time-resolved crystallography to capture transient catalytic states of DERA, providing deeper insights into its reaction mechanism.

• Systems biology integration: Incorporating DERA into comprehensive metabolic models to predict the effects of genetic or environmental perturbations on cellular metabolism.

These emerging areas represent opportunities for researchers to make significant contributions to understanding the fundamental biology of DERA and its potential applications in biomedicine and biotechnology.

How can contradictory findings about DERA function be resolved experimentally?

When faced with contradictory findings about DERA function in the literature, researchers can employ several experimental strategies to resolve discrepancies:

• Standardized assay conditions: Develop and adopt standardized protocols for DERA activity measurements to enable direct comparison of results across laboratories.

• Multi-technique validation: Apply complementary methods to verify findings, such as combining genetic, biochemical, and structural approaches.

• Biological context consideration: Assess whether contradictions arise from differences in experimental models (e.g., cell types, organisms) or physiological conditions.

• Isoform-specific analysis: Determine whether discrepancies result from studying different DERA isoforms or splice variants with distinct properties.

• Reproducibility initiatives: Conduct collaborative studies across multiple laboratories to independently validate key findings.

• Meta-analysis: Systematically analyze existing data to identify patterns, sources of variation, and consensus findings.

By implementing these approaches, researchers can navigate conflicting reports and establish a more coherent understanding of DERA biology, ultimately advancing the field through resolution of apparent contradictions.