Enzyme Cofactor Engineering Service

Enzyme Cofactor Engineering is a specialized service focused on modifying an enzyme's interaction with its non-protein chemical helper molecule (cofactor), aiming to enhance catalytic efficiency, switch cofactor specificity, or improve cofactor recycling. Many industrially relevant enzymes, such as dehydrogenases and monooxygenases, rely on expensive cofactors like NADH, NADPH, or ATP. By rationally engineering the cofactor binding pocket, we can reduce the reliance on costly cofactors, improve the enzyme's turnover rate, or introduce non-native cofactors to facilitate novel chemical transformations.

CD Biosynsis offers expert CRO services in Cofactor Engineering, combining structural bioinformatics, molecular docking, and targeted mutagenesis. Our platform precisely identifies key residues responsible for cofactor recognition and binding affinity. We specialize in designing mutations to switch cofactor preference (e.g., from NADPH-dependent to the cheaper NADH-dependent), increasing the efficiency of cofactor regeneration systems, and stabilizing the bound cofactor structure. This engineering approach is critical for reducing the operating costs of large-scale biocatalytic processes and expanding the scope of enzymatic reactions in green chemistry.

Highlights

We provide methods to overcome the limitations of expensive cofactors and inefficient cofactor utilization.

• Cofactor Specificity Switching: Rationally redesign the binding pocket to switch the enzyme's preference (e.g., from NADPH to the cheaper NADH or vice versa) for cost-effective bioprocesses.
• Enhanced Cofactor Binding Affinity: Introduce mutations that optimize interactions (e.g., H-bonds) with the cofactor, leading to lower Km and higher catalytic efficiency.
• Improved Turnover Rate: Engineer the active site residues to optimize the geometric positioning of the cofactor relative to the substrate for faster hydride transfer or electron transfer.
• Integration with Regeneration Systems: Design the enzyme surface or structure to be compatible with a co-expressed, regenerating enzyme, maximizing cofactor utility.

Applications

Cofactor engineering drives innovation in industrial biocatalysis where cost is a major factor:

Cost-Reduced Bioprocesses

Reducing the industrial cost of synthesis by engineering enzymes to use cheaper or easier-to-regenerate cofactors (e.g., NADH over NADPH).

Metabolic Engineering Optimization

Balancing the flux and redox state within engineered microorganisms by tuning the consumption of cofactors like NAD+/NADH or ATP/ADP.

Expanded Enzyme Scope

Enabling enzymes to utilize non-native cofactors, which may allow for new reaction chemistries or improved tolerance to harsh industrial conditions.

Biosensor Specificity

Engineering detection enzymes to exclusively rely on a specific cofactor, enhancing the orthogonality and reliability of biosensing devices.

Platform

Our platform employs rational design to precisely manipulate the cofactor binding environment.

Structural Comparison & Docking

Modeling the target enzyme with its native cofactor and the desired non-native cofactor to identify steric and electrostatic clashes/gaps.

Specificity Residue Targeting

Focusing on conserved residues that specifically interact with the unique chemical moieties (e.g., 2’-phosphate group in NADPH) to mediate the switch.

Charge and Hydrogen Bond Optimization

Designing mutations that introduce or remove charged groups (e.g., Lys/Arg to Asp/Glu) to create favorable electrostatic interactions with the new cofactor.

Combined Active Site & Cofactor Tunneling

Modeling the coupled movement of the cofactor and the substrate to ensure the engineering enhances both binding and the catalytic transfer step.

Kinetic Validation (Km and kcat)

High-resolution kinetic assays to quantify the improvement in Km (affinity) and kcat (turnover) for both the native and the engineered cofactor.

Workflow

Our Cofactor Engineering process employs a structural and kinetic-driven design cycle:

• Structural Analysis and Comparison: Model the enzyme with both the native cofactor and the target cofactor (e.g., NADH vs. NADPH) in the binding pocket to identify structural differences.
• Mutation Design Strategy: Rationally select residues for mutation, targeting those that clash with the native cofactor but could favorably interact with the target cofactor (e.g., targeting the 2’-phosphate recognition loop).
• In Silico Screening and Validation: Model single and multiple mutants, using docking and binding energy calculation (MM/GBSA) to predict which variants will have the desired affinity switch.
• Gene Synthesis and Cloning: Synthesize the gene constructs encoding the most promising engineered variants and clone them into the expression vector.
• Expression, Purification, and Kinetic Characterization: Express and purify the mutant enzymes. Measure the Km, Vmax, and kcat/Km for both the native and the target cofactor to quantify the engineered switch.
• Optimization and Iteration: Analyze the kinetic data to guide the next round of design, focusing on further improving the catalytic performance with the new cofactor.

CD Biosynsis delivers functionally switched enzymes with validated kinetic profiles for industrial use. Every project includes:

• Design Rationale: A report detailing the structural basis for the engineered cofactor switch, including key residue interactions.
• Engineered Clone: The plasmid containing the optimized enzyme sequence, verified by sequencing.
• Comparative Kinetic Data: Full Km and kcat values for both the native and the target cofactor for the engineered variant, clearly demonstrating the ratio change.
• Structural Models: PDB files of the predicted mutant enzyme bound to the desired non-native cofactor.