Enzyme Allosteric Regulation Design Service

Enzyme Allosteric Regulation Design is a sophisticated protein engineering service focused on introducing or modifying remote binding sites (allosteric sites) to regulate enzyme activity through the binding of a non-substrate molecule (allosteric effector). Unlike active site engineering, this approach exploits the enzyme's natural dynamic properties, allowing precise control over catalysis, affinity, or stability without directly interfering with the catalytic pocket. Designing allosteric sites is crucial for creating synthetic biological circuits, developing highly specific regulatory drugs, and achieving conditional control over industrial biocatalysis.

CD Biosynsis utilizes advanced computational techniques, including Molecular Dynamics (MD) simulations and sequence co-evolution analysis, to identify potential allosteric pockets and the transmission pathways connecting them to the active site. Our service involves designing specific mutations to tune the enzyme's response to an effector molecule, converting it into a potent inhibitor or activator. We offer a full design-to-validation pipeline, providing rationally engineered enzymes that can be reversibly controlled by temperature, pH, light, or small-molecule ligands, opening new avenues for controllable biological systems.

Highlights

We provide methods to install novel, controllable regulatory mechanisms into target enzymes.

• Computational Pocket Prediction: Utilize algorithms to search for new, functional binding pockets remote from the active site suitable for effector binding.
• Allosteric Transmission Pathway Mapping: Identify the dynamic communication network (residue-residue contacts) that links the allosteric site to the active site.
• Introduction of Novel Regulation: Design mutations that create a high-affinity binding site for a chosen synthetic effector (e.g., small drug molecule, peptide).
• Tuning Regulatory Response: Engineer the enzyme to exhibit specific regulatory characteristics (e.g., increasing fold-activation, switching from inhibition to activation).

Applications

Allosteric engineering provides sophisticated control for applications ranging from therapeutics to synthetic biology:

Controllable Biologics

Designing therapeutic enzymes that can be activated or inhibited in vivo by an orally available small molecule, improving safety and dosing control.

Synthetic Biosensors

Engineering enzymes to act as detectors that change activity upon binding a target environmental signal molecule, creating novel diagnostic tools.

Industrial Process Shutoff

Developing industrial biocatalysts that can be instantly deactivated (or activated) by a chemical signal, enabling precise control over reaction timing and stopping product formation.

Optogenetic Control

Fusing enzymes with photo-switchable domains to achieve control via light exposure, which requires understanding and engineering the transmission interface.

Platform

Our platform combines dynamic simulation and rational design to exploit the principles of allostery.

Dynamic Correlated Motion Analysis

Using Molecular Dynamics (MD) to analyze correlated motions between the predicted allosteric site and the active site, revealing the communication pathway.

Evolutionary Coupling (Co-Evolution)

Identifying pairs of residues that co-evolve, suggesting they are structurally or functionally linked, often revealing allosteric networks.

Computational Hotspot Mutagenesis

Designing mutations at the allosteric site to maximize binding affinity for the effector molecule while optimizing the transmission signal to the active site.

Binding Affinity Prediction (FEP/MMGBSA)

Using rigorous free energy calculation methods to accurately predict the binding affinity of the effector to the engineered allosteric site.

Gene Synthesis and Validation

Accurate synthesis of engineered DNA and expression/purification of mutant enzymes for experimental validation of regulatory function.

Workflow

Our Allosteric Regulation Design workflow is a comprehensive loop integrating computation, design, and experimental proof-of-concept:

• Target and Effector Selection: Define the enzyme target and the desired small-molecule effector/regulator. Obtain the enzyme's 3D structure.
• Allosteric Pocket Identification: Use structural and dynamic analysis (e.g., MD) to identify cryptic or accessible binding pockets suitable for the effector.
• Effector Docking and Design: Model the effector binding to the predicted pocket. Design mutations at the pocket to maximize binding affinity (Kd).
• Transmission Pathway Modulation: Design secondary mutations along the allosteric pathway to enhance or attenuate the signal transmission to the active site.
• Gene Synthesis and Cloning: Synthesize the engineered enzyme genes (containing allosteric pocket and pathway mutations) and clone them for expression.
• Functional Characterization: Validate the regulatory function by measuring enzyme activity (Km, kcat) in the presence and absence of the effector, quantifying the fold-change in activity.

CD Biosynsis delivers functionally validated enzymes with integrated regulatory control. Every project includes:

• Design Rationale: A report detailing the predicted allosteric site coordinates and the mechanistic link between the effector binding and active site modulation.
• Engineered Clone: The plasmid containing the optimized, validated allosterically regulated enzyme sequence.
• Quantitative Regulation Data: Full kinetic data (Km, Vmax, IC50/EC50) characterizing the enzyme's response to the allosteric effector.
• Structural Visualization: 3D models of the enzyme in both the 'On' (effector-bound) and 'Off' (apo) regulatory states.