DERA

What is DERA and what are its primary functions in biochemical research?

DERA (2-Deoxy-d-ribose-5-phosphate aldolase) is a class I aldolase that catalyzes stereoselective C–C bond formation between acetaldehyde and glyceraldehyde-3-phosphate to generate deoxyribose-5-phosphate . This enzyme was first reported by Racker in 1952 and has since gained significant attention for its ability to catalyze aldol reactions with high stereoselectivity .

DERA enzymes have been found across all kingdoms of life and share a common TIM barrel fold structure despite relatively low sequence identity . This structural conservation highlights the fundamental importance of this enzymatic activity in biological systems.

What is the Delaware Education Research Alliance (DERA) and how does it support academic research?

The Delaware Education Research Alliance (DERA) represents a collaborative research framework designed to facilitate research using teacher, student, and school administrative data . The alliance is a partnership between the Delaware Department of Education (DDOE), Delaware State University (DSU), and the University of Delaware's College of Education and Human Development (CEHD) .

DERA's primary function is to create systematic and ongoing collaboration between researchers and education stakeholders to address pressing education issues in Delaware . The alliance supports research by:

• Matching faculty researchers with DDOE staff

• Facilitating data-sharing across partner organizations

• Supporting project proposals through formal RFP processes and ad hoc opportunities

• Contributing to evidence-based decision-making for Delaware's education system

What catalytic mechanisms underlie DERA enzyme function?

DERA operates via a class I aldolase mechanism involving the formation of a covalent enzyme-substrate intermediate . The catalytic process follows these key steps:

• The catalytic lysine residue in the active site forms a Schiff base (imine) with the carbonyl group of the aldehyde substrate

• This intermediate undergoes deprotonation to form an enamine nucleophile

• The activated enamine attacks the carbonyl carbon of the acceptor aldehyde

• The resulting intermediate forms a new carbon-carbon bond with precise stereochemical control

• Hydrolysis of the Schiff base releases the aldol product with an (S)-configured stereogenic center

This mechanistic pathway enables DERA to catalyze not only single aldol additions but also sequential reactions. The sequential aldol reaction continues until a stable intramolecular hemiacetal is formed, providing a natural termination point for the reaction .

How can researchers effectively immobilize DERA for continuous flow biocatalysis?

Immobilization of DERA represents a critical advancement for implementing continuous enzymatic processes. A particularly effective methodology involves spray-coating DERA onto membrane supports .

The protocol developed through Design-of-Experiment (DoE) optimization includes:

• Preparation of a functional, water-soluble copolymer containing addressable units for covalent enzyme binding

• Mixing purified DERA with the prepared polymer solution

• Spray-coating the mixture onto polyacrylonitrile/polyethylene imine (PAN/PEI) membrane supports

• Post-processing to stabilize the coating through appropriate crosslinking methods

Successful immobilization confirmation requires multiple analytical approaches:

• Atomic force microscopy (AFM) imaging to visualize the enzyme coating morphology

• Activity assays to quantify retained enzyme functionality

• Stability testing under continuous operation conditions

The first fractional factorial design in optimization studies yielded significant performance improvements and critical insights into parameter interactions, while a second full factorial design validated these results . This systematic approach to immobilization enables the creation of enzymatically active membranes suitable for continuous flow reactors for synthesizing enantiomerically pure β-hydroxyaldehydes.

What protein engineering strategies have successfully enhanced DERA catalytic properties?

Protein engineering has dramatically expanded DERA's capabilities beyond its natural substrate range. Several complementary approaches have proven effective:

• Directed evolution: Through 11 rounds of directed evolution, researchers developed DERA-MA with 12 amino acid substitutions, achieving a remarkable 190-fold enhancement in catalytic activity for Michael addition reactions compared to the wildtype enzyme .

• Structure-based design: Utilizing crystallographic data to identify and modify key residues affecting substrate binding, catalysis, and stability.

• Machine learning-guided engineering: Computational approaches are increasingly employed to predict beneficial mutations and accelerate enzyme optimization .

The most striking example of DERA engineering success is its conversion from an aldolase to a "Michaelase" capable of catalyzing enantioselective Michael additions of nitromethane to α,β-unsaturated aldehydes . This transformation represents a fundamental change in catalytic mechanism - from an enamine-based mechanism (activating the substrate as a nucleophile) to an iminium-based mechanism (activating the substrate as an electrophile) .

This engineering achievement demonstrates the remarkable plasticity of enzyme active sites when subjected to appropriate selection pressure and highlights the potential for expanding natural enzyme catalytic repertoires to include entirely new reaction chemistries.

What methodological considerations are crucial for DERA-catalyzed sequential aldol reactions?

DERA's ability to catalyze sequential aldol reactions represents one of its most valuable research applications, but requires careful experimental design :

• Substrate selection and specificity:

  • Not all aldehydes function equally as donors and acceptors

  • Chloroacetaldehyde demonstrated superior performance as an acceptor substrate while being refused as a donor, highlighting the enzyme's excellent selectivity

  • The initial acetaldehyde donor must react with a C2-substituted aldehyde acceptor to form the highly stereospecific first aldol product

• Reaction thermodynamics and control:

  • Sequential aldol reactions with DERA are thermodynamically controlled

  • The reaction typically terminates when a stable intramolecular hemiacetal forms

  • Temperature, concentration, and pH must be carefully optimized

• Stereochemical considerations:

  • DERA provides exceptional stereoselectivity with the enzyme controlling stereochemical outcomes rather than the substrate

  • This results in highly stereospecific polyol systems

• Product analysis:

  • Reaction progress should be monitored using appropriate analytical techniques (HPLC, NMR, etc.)

  • Product structures require thorough verification to confirm stereochemical outcomes

The sequential addition of three achiral aldehydes in DERA-catalyzed reactions can give rise to complex cyclic systems, as demonstrated by Gijsen and Wong in their pioneering studies .

How can researchers utilizing the Delaware Education Research Alliance effectively design their studies?

Researchers interested in collaborating with the Delaware Education Research Alliance should consider several methodological approaches when designing studies :

• Proposal submission process:

  • Formal RFP (Request for Proposal) responses should include concise summaries of research questions, data requirements, design methodology, and potential implications

  • Ad hoc opportunities can be pursued by contacting the appropriate institutional DERA lead

• Research design considerations:

  • Clearly articulate how teacher, student, and school administrative data will be utilized

  • Develop methodologies appropriate to the research questions (quantitative, qualitative, or mixed)

  • Address data privacy and ethical considerations

  • Establish collaborative frameworks with appropriate DDOE staff

• Implementation planning:

  • Articulate how findings will inform educational practice

  • Consider scalability of potential interventions

  • Plan for knowledge translation to practitioners

The ultimate goal of DERA research projects should be to "inform evidence-based decision-making for Delaware students, teachers, and leaders, while simultaneously contributing to the larger body of knowledge guiding education policy" .

How should researchers address stability challenges affecting DERA enzyme function?

DERA enzymes face several stability challenges that require methodological solutions:

• Aldehyde concentration sensitivity:

  • Problem: High aldehyde concentrations can modify lysine residues (including the catalytic lysine), inactivating the enzyme

  • Solution approaches: Continuous substrate feeding strategies, enzyme engineering to reduce surface lysines, implementation of continuous flow systems as demonstrated in membrane immobilization studies

• Thermal stability limitations:

  • Problem: Many native DERA enzymes exhibit moderate thermal stability

  • Solution approaches: Screening thermostable homologs from extremophiles, stability-focused protein engineering, immobilization techniques that enhance thermal stability

• Operational stability:

  • Problem: Activity loss during prolonged reaction times

  • Solution approaches: Immobilization via spray-coating on membrane supports , whole-cell biocatalysis, enzyme stabilization through engineering

A systematic approach combining protein engineering, reaction condition optimization, and process design is typically required to overcome these challenges and develop robust DERA-based biocatalytic processes.

How can researchers reconcile contradictory data in DERA catalytic studies?

When contradictory results emerge in DERA research, several methodological approaches can help resolve discrepancies:

• Systematic condition variation:

  • Create matrices of reaction conditions (pH, temperature, substrate concentrations, buffer compositions)

  • Identify condition-dependent behaviors that might explain contradictory observations

  • Map the complete operational space of the enzyme

• Enzyme quality analysis:

  • Verify enzyme purity and homogeneity

  • Check for different conformational states or oligomeric forms

  • Analyze potential post-translational modifications or degradation products

• Substrate preparation standardization:

  • Ensure consistent substrate quality and preparation methods

  • Address potential substrate stability issues

  • Verify absence of inhibitory contaminants

• Comprehensive kinetic characterization:

  • Determine complete kinetic parameters under various conditions

  • Identify substrate or product inhibition effects

  • Map the full reaction pathway including potential side reactions

By systematically addressing these factors, researchers can often resolve seemingly contradictory results and develop more complete understandings of DERA catalytic behavior.

What are the key considerations for designing DERA Michael addition reactions?

The re-engineered DERA-MA enzyme, which catalyzes enantioselective Michael additions, requires specific experimental design considerations :

• Mechanistic understanding:

  • Unlike natural DERA which operates via an enamine mechanism, DERA-MA functions through an iminium-based mechanism

  • This fundamental change activates the enzyme-bound substrate as an electrophile rather than a nucleophile

• Substrate selection:

  • α,β-unsaturated aldehydes serve as acceptor substrates

  • Nitromethane functions as the nucleophilic donor

  • Substrate structural requirements differ from those of natural DERA substrates

• Reaction optimization:

  • pH, temperature, substrate concentrations, and co-solvents must be optimized specifically for Michael addition chemistry

  • Reaction equilibria differ significantly from aldol reactions

• Product analysis:

  • Enantioselectivity determination requires appropriate analytical methods

  • Product structures and stereochemistry must be thoroughly verified

The successful engineering of DERA for Michael addition chemistry demonstrates the potential for expanding natural enzyme catalytic repertoires to include entirely new reaction types when appropriate directed evolution strategies are applied .

What are the primary research applications of DERA in synthetic biology?

DERA offers diverse research applications in synthetic biology and biocatalysis:

• Pharmaceutical intermediate synthesis:

  • Production of statin side chains and other cholesterol-lowering drug precursors

  • Generation of enantiomerically pure β-hydroxyaldehydes as chiral building blocks

  • Creation of complex polyol systems with multiple stereogenic centers

• Multi-enzyme cascade systems:

  • Integration of DERA into in vitro and in vivo multi-enzyme cascades

  • Combination with other C-C bond-forming enzymes for complex molecule synthesis

  • Development of consolidated bioprocesses

• Methodological innovations:

  • Development of biocatalytic alternatives to traditional organic synthesis

  • Exploration of new reaction types through enzyme engineering

  • Creation of immobilized enzyme systems for continuous biocatalysis

The versatility of DERA stems from its ability to accept various aldehydes as substrates and its high stereoselectivity, making it particularly valuable for asymmetric synthesis applications requiring precise stereochemical control.

How does the Dialog-Enabled Resolving Agents (DERA) framework enhance clinical research?

The Dialog-Enabled Resolving Agents (DERA) framework represents an innovative methodology for improving clinical research through agent-based dialog systems :

• Framework structure:

  • Two complementary agents - a Researcher and a Decider

  • The Decider generates an initial output for the task

  • The agents then collaborate through conversation

  • The Researcher identifies crucial problem components

  • The Decider integrates the Researcher's inputs to improve the final output

• Implementation in clinical settings:

  • In care plan generation, the initial output from the Decider contained "Most" necessary care management steps

  • The Researcher highlighted potential drug interactions and the need for patient education on safe sexual practices

  • These improvements were incorporated into the final care plan

  • The revised plan was rated as containing "All" necessary care management steps by physician-expert evaluators

• Experimental methodology:

  • The DERA framework allows for systematic evaluation of how conversational approaches improve output quality

  • Neither agent has knowledge of the ideal final output, making the process more realistic

  • The approach can be evaluated against non-dialogic methods to assess improvement

This framework demonstrates how dialogic approaches between specialized agents can enhance research quality in domains requiring deep expertise, offering promising directions for clinical research methodology advancement .

What computational approaches are advancing DERA engineering efforts?

Computational methods are increasingly central to DERA engineering, offering several advantages for researchers :

• Machine learning approaches:

  • Predictive models for beneficial mutations based on sequence-function relationships

  • Identification of non-obvious correlations between protein sequence and desired properties

  • Acceleration of the engineering process by reducing experimental burden

• Molecular modeling:

  • Analysis of enzyme-substrate interactions

  • Prediction of how mutations might affect binding pocket geometry

  • Investigation of protein dynamics and flexibility

• Quantum mechanical/molecular mechanical (QM/MM) calculations:

  • Detailed investigation of reaction mechanisms

  • Identification of transition states and energy barriers

  • Elucidation of the electronic factors governing catalysis

These computational approaches are expected to significantly accelerate future DERA engineering efforts by providing rational guidance for experimental work and reducing the resources required for optimization .

What analytical methods are most effective for characterizing DERA-catalyzed reactions?

Comprehensive characterization of DERA-catalyzed reactions requires multiple complementary analytical approaches:

• Activity determination:

  • Spectrophotometric assays tracking aldehyde consumption or product formation

  • Coupled enzyme assays for continuous monitoring

  • Discontinuous sampling with derivatization for improved sensitivity

• Product analysis:

  • HPLC with appropriate columns for separating stereoisomers

  • GC-MS for volatile products after derivatization

  • NMR for structural confirmation and stereochemical assignment

• Enzyme characterization:

  • Circular dichroism spectroscopy for secondary structure analysis

  • Thermal shift assays for stability assessment

  • Size-exclusion chromatography for oligomeric state determination

• Reaction monitoring:

  • In-line spectroscopic methods for continuous flow systems

  • Sampling strategies for batch reactions

  • Quenching protocols to prevent continued reaction during analysis

Selection of appropriate analytical methods should be guided by the specific research questions being addressed and the nature of the substrates and products involved in the DERA-catalyzed reaction.

What tools and resources are available through the Delaware Education Research Alliance?

Researchers collaborating with the Delaware Education Research Alliance have access to several specialized resources :

• Data resources:

  • Teacher, student, and school administrative data

  • Longitudinal educational datasets

  • PreK-16 educational information across Delaware

• Collaborative frameworks:

  • Mechanisms for matching with DDOE staff expertise

  • Facilitated data-sharing across partner organizations

  • Dynamic procedures for joint inquiry

• Proposal support:

  • RFP submission processes several times throughout the year

  • Flexible mechanisms for responding to ad hoc opportunities

  • Clear guidelines for project development

The alliance aims to create a system where "collaboration is systematic and ongoing," providing researchers with both the data and institutional support needed to conduct meaningful educational research with potential for real-world impact .

This comprehensive FAQ collection provides researchers with both fundamental knowledge and advanced methodological guidance for working with DERA in both its biochemical and educational research contexts. The information presented reflects current understanding from diverse scientific sources and emphasizes methodological approaches rather than simple definitions.