iGEM 2025 — Team hubu-china

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Contents

# Engineering

## Molecular Experiments（Ⅰ）

#### Week 1: 15-22 January 2025

This week we mainly focused on the selection and discussion of topics (excluding preliminary social research and expert interviews).

#### Week 2: 1-22 February 2025

This week's focus is on gene cloning, or in other words, plasmid construction and validation.

We optimized the codon and cloned the ADH3 gene (removed signal peptide sequence) to the pET46/Ek-LIC vector to construct the recombinant plasmid pET46/ADH3.

#### Week 3-4: 23 February - 8 March 2025

In the past fortnight, we has mainly focused on protein characterization and homogeneity analysis.

* **Gel filtration and chromatography**: Conducted gel filtration chromatography, DEAE anion exchange chromatography, concentration and purity verification
* **Oligomeric state determination**: The ADH3 peak was determined to be between the 158 kDa and 440 kDa markers
* **Electron microscopy analysis**: Negative dyeing electron microscopy analysis showed a homogeneous octamer structure with a diameter of about 10 nm

#### Week 5-6: 10-25 March 2025

In the past half month, we has focused on the structural analysis of cryo-EM, which referred to the sample preparation and data collection, data processing and 3D reconstruction, model construction and space optimization.

#### Week 7: 26 March - 6 April 2025

This week we focused on structural analysis. We conducted a detailed analysis of substrate binding pockets, such as pocket composition and interaction, substrate binding key residues, OTA binding patterns, etc.

#### Week 8: 7-16 April 2025

This week we focused on rational design strategies for conceptualizing mutants.

Based on the ADH3-D344N/OTA complex structure, we designed the amino acids bound to the substrate OTA to inactivate them one by one to verify their catalytic activity against OTA.

* **Design principle 1**: Expand the substrate binding space (reduce the volume of the side chain)
* **Design principle 2**: Enhance substrate-enzyme interaction (introduce hydrogen bonding or hydrophobicity)
* **Design principle 3**: Adjust the substrate orientation (change the chemical properties of the side chain)

#### Week 9: 17-25 April 2025

This week we mainly focused on fixed-point mutation experiments. We successfully constructed mutant S88A,H163A,K210A,V217A,L218A,H251A,H253A,I325A and V347A.

#### Week 10: 26 April - 5 May 2025

In the past fortnight, we has mainly focused on the analysis and comparison of mutant enzyme active substances.

After protein expression and purification, a SDS-PAGE was carried out to verify whether the protein had been expressed and the enzyme activity assay showed the key amino acid function, and we proposed the catalytic mechanism model of ADH3.

#### Week 11: 6-15 May 2025

This week we focused on the rational design strategies for conceptualizing mutants.

Based on the results of the previous round of mutations, we designed mutations for L218, V347, and S88 sites:

* **L218 series**: L218S,L218C,L218T,L218F,L218Y,L218W
* **V347 series**: V347D,V347R,V347H,V347E,V347K
* **S88 series**: S88D,S88R,S88H,S88E,S88K

#### Week 14-15: 16-30 June 2025

In the past fortnight, the focus has been on mutant protein expression and purification.

The mutants were constructed and expressed, and the purity was verified through SDS-PAGE, showing that the protein purity was over 95% and the molecular weight was about 45 kDa.

#### Week 16-17: 3 July - 11 July 2025

In the past fortnight, we has mainly focused on the analysis and comparison of mutant enzyme active substances.

The activities of S88E, S88K and S88H were increased by 3.7, 2.4 and 1.3 times, respectively. The activity of the series L218 and V347 mutants was significantly reduced.

#### Week 18: 12-21 July 2025

In the past fortnight, we has mainly focused on the review of the topic and analyzed the reasons for errors in the experiment as a whole and discussed the follow-up development direction of the topic.

## Molecular Experiments（Ⅱ）

#### Week 1: 14-21 March 2025

This week's focus was on the bioinformatics analysis, i.e. sequence analysis and genetic design.

Based on the sequence analysis in the NCBI database, we compared AHD3 to screen the optimal gene sequence of interest (LlADH). Comprehensive analysis through bioinformatics tools. At the same time, its structural domains are predicted and compared.

#### Week 2: 22-31 March 2025

This week's focus was on the gene cloning, which referred to the plasmid construction and validation.

We optimized the codon and cloned the LlADH gene (removed signal peptide sequence) to the pET46/Ek-LIC vector to construct the recombinant plasmid pET46/LlADH. We obtained 8 sequence-correct clones and saved them as LlADH-WT-1~8. Purification verification was performed at the same time.

#### Week 3-4: April 1-16 2025

In the past fortnight, we has mainly focused on protein characterization and homogeneity analysis.

* **Gel filtration chromatography**: The molecular weight corresponding to the peak position of LlADH was about 350 kDa, consistent with the theoretical molecular weight of octamer (372 kDa)
* **Static light scattering analysis**: Molecular weight was determined to be 351.9 kDa, confirming that LlADH was a homologous octamer
* **Electron microscopy analysis**: Negative dyeing electron microscopy showed that the protein particles were uniformly distributed, with a diameter of about 10 nm, showing a typical octamer morphology

#### Week 5: 17-23 April 2025

This week's focus was on the analysis of LlADH hydrolysates and the acquisition of standard sample data.

* **HPLC testing**: OTA retention time: 12.5 minutes, OTα retention time: 9.8 minutes
* **Mass spectrometry verification**: Confirmed the molecular weight of OTα: [M+H]+ m/z 257.0189 (calculated value 257.0211), in line with the theoretical value

#### Week 6-7: 24 April - 11 May 2025

In the past fortnight, we has mainly focused on the analysis and comparison of enzyme active substances.

* **Temperature effect**: Enzyme activity was highest at 45°C, maintained at more than 60% at 20-50°C
* **pH effect**: Optimal pH was 8.5, activity maintained in the range of pH 7.5-9.0 by more than 50%
* **Metal ions effect**: Fe²⁺, Cu²⁺ and Zn²⁺ significantly inhibit enzyme activity, other metal ions also show varying degrees of inhibition
* **Thermal stability**: Stability was best at 20°C, residual activity dropped below 20% at 40°C

We also compared the activity of LlADH with ADH3, and found that the hydrolytic activity of LlADH against OTA was twice that of ADH3.

#### Week 8-9: 12-30 May 2025

In the past fortnight, the focus has been on the structural analysis of cryo-EM. That is, sample preparation and data collection, data processing and 3D reconstruction, model construction and space optimization.

#### Week 10: 1-8 June 2025

This week we focused on the structural analysis and rational design.

We conducted a detailed analysis of substrate-bound pockets, such as pocket composition and interaction, substrate binding to key residues, OTA binding mode (based on ADH3 superposition), etc. At the same time, the structural differences between LlADH and ADH3 were compared in detail.

#### Week 11: 9-14 June 2025

This week we focused on rational design strategies for conceptualizing mutants.

Based on the ADH3/OTA complex structure, we designed mutants targeting substrate-binding pocket key residues. A total of 15 mutation types were designed.

* **Design principle 1**: Expand the substrate binding space (reduce the volume of the side chain)
* **Design principle 2**: Enhance substrate-enzyme interaction (introduce hydrogen bonding or hydrophobicity)
* **Design principle 3**: Adjust the substrate orientation (change the chemical properties of the side chain)

#### Week 12: 14-21 June 2025

This week we mainly focused on fixed-point mutation experiments.

The mutants we successfully constructed are S89D, S89F, S89H, S89K, and S89R; S91R; G131L; L219H; I326A; V348F,V348H,V348S.

Some mutants failed to be built, such as G131F, L219K, and L219R.

#### Week 13: 22-30 June 2025

This week we focused on small-scale protein expression testing. We selected strains E. coli BL21 (DE3) to optimized the conditions for expression by testing.

#### Week 14-15: 2-19 July 2025

In the past fortnight, we has mainly focused on large-scale protein expression and purification.

The purity verification by SDS-PAGE showed that the protein was over 95% and the molecular weight was about 45 kDa.

* **High expression levels**: LlADH wild-type, S89H, S91R, L219H, I326A, and V348S
* **Medium expression levels**: S89D, S89K, S89R, V348F, and V348H
* **Low expression levels**: S89F and G131L

#### Week 16-17: 20 July - 4 August 2025

In the past fortnight, we has mainly focused on the analysis and comparison of mutant enzyme active substances.

Based on wild type, the relative activity was 100%, and the activity of mutant I326A was significantly increased. V348F activity slightly increased; S89D, S89H, S89K, S89R, S91R, G131L, V348S, V348H activity decreased; L219H was completely inactivated.

#### Week 18-19: 5-21 August 2025

In the past fortnight, we has mainly focused on the review of the topic and analyzed the reasons for errors in the experiment as a whole and discussed the follow-up development direction of the topic.

#### Week 20-21: 22 August - 11 September 2025

In the past fortnight, the main focus has been on the upgrading of the expression system of mutants. We achieved efficient expression in Pichia pastoris, laying the foundation for industrialization and practical application.

## Modeling Experiments

#### Biological Modeling Section

* **Week 1**: Swiss-model homology modeling - Used Swiss-model website for homology modeling, compared key parameters GMQE and QMEAN
* **Week 2**: LlADH docks with ligand OTA - Used PyMol to visualize the protein to find rigid structure for preliminary docking
* **Week 3**: Molecular dynamics simulation - Used Amber force field and GAFF for ligand, 100ns simulation
* **Week 4**: Molecular dynamics simulation results analysis - Analyzed RMSD, RMSF, RG and hydrogen bond analysis, found protein ligand complex was unstable
* **Week 5**: Global docking of LlADH - Used Autodock Vina to screen protein ligand complex with lowest binding free energy
* **Week 6**: Molecular dynamics simulation with new docking results - Processed protein and ligand files separately, conducted solvent return and energy minimization
* **Week 7**: Calculating free energy landscape maps - Used Poisson-Boltzmann surface area method to calculate interaction and free energy
* **Week 8**: Visualization of all results - Plotted results with origin to evaluate stability of protein-ligand reaction

#### Condition Optimization Section

* **Week 9**: Data cleaning and preprocessing - Cleaned reaction condition data, dealt with missing outliers, normalized enzyme activity
* **Week 10**: Single-goal optimization analysis - Visualized data with single objective, expanded sample points by triple spline interpolation
* **Week 11**: Prediction of multi-objective combination conditions - Used different hypothesis models and optimization algorithms, predicted by response surface method

#### Climate Prediction Models Section

* **Week 12**: Read relevant materials, familiarized with research background, supplemented basic concepts in meteorology
* **Week 13**: Analyzed calculation ideas, sorted out possible schemes for combining machine learning and meteorological data
* **Week 14**: Focused on design and implementation of model part, chose appropriate machine learning method
* **Week 15**: Focused on model evaluation and result analysis, wrote code for visualization and verification

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Contents

# PREFACE TO ENGINEERING

Against the backdrop of global food security facing the challenge of Ochratoxin A (OTA) contamination in food products, our team has conducted a systematic study on OTA biodegradative enzymes. The uniqueness of this research lies in the fact that every critical decision and directional shift has incorporated in-depth considerations of Human Practices, particularly through continuous dialogue with scientific experts, which has infused profound real-world concern into the scientific exploration of the project.

## Cycle 1: From Social Needs to Precision Design - Expert Guidance at the Research Inception

In the beginning of the project, we consulted with experts in the field of food safety with the question "How to effectively address OTA contamination in food grains?". A senior researcher clearly stated: "Current mainstream physical and chemical detoxification methods, such as ozone treatment, are effective but tend to cause nutrient loss and chemical residues and even biosorbents like zeolites exhibit limited impact. The development of efficient and specific biological degradation methods is an urgent need of the industry." This statement pointed us in the right direction, leading us to abandon traditional approaches and focus on innovative enzyme-based degradation solutions.

Why choose ADH3 among numerous candidate enzymes? This decision was also validated by both literature and expert input. An enzymology expert advised: "Selecting a high-activity 'chassis' element is half the success of the project". This prompted us to conduct extensive literature mining, ultimately identifying ADH3, the most efficient OTA hydrolase discovered to date, as our research starting point, ensuring a high-level foundation for the project.

## Cycle 2: Homologous Enzyme Mining and Performance Leap - Expert Support in Breaking Bottlenecks

After successfully increasing the activity of ADH3-S88E by 3.7-fold in the first cycle, we once believed that enzyme activity modification was approaching its limit. However, during a mid-term presentation, a structural biology professor raised a critical question: 'From a structural perspective, the binding affinity of ADH3's catalytic pocket to the substrate may have already reached its theoretical limit. To achieve a stepwise improvement in efficiency, it may be necessary to search for homologous enzymes with deeper catalytic pockets.'

This insightful observation became a turning point for the entire project. We immediately adjusted our direction and shifted to bioinformatics mining, eventually discovering the sequence of homologous enzyme LIADH, which shares a 73% similarity with ADH3. After systematic expression, purification, and functional verification, the results were inspiring: the activity of LIADH was twice that of ADH3, perfectly validating the expert's prediction and achieving a leap in performance.

## Cycle 3: Structure-Guided Rational Modification - Iteration through Industry-Academia-Research Collaboration

After obtaining the high-resolution structure of LIADH, we initiated rational design work. At this stage, an engineer from a biotechnology company provided a suggestion: "An excellent industrial enzyme preparation requires not only high activity but also good stability." Therefore, while designing mutants to enhance activity (such as I326A, which increased activity by 75%), we also fully recognized the deficiencies of LIADH in terms of thermal stability and metal ion tolerance---issues that must be addressed for future application.

### Summary and Outlook

Looking back on the entire project, expert interviews and Human Practices (HP) are not isolated components outside scientific research, but are deeply integrated into each research cycle, serving as a "compass" to guide the project's direction. From selecting the research direction, breaking performance bottlenecks to planning application pathways, the valuable insights of experts have ensured that our work always targets real-world needs, embodying the core spirit of iGEM---'Responsible Innovation'. In the future, we will continue to listen to the voices of the industry, persist in exploring enzyme stability, heterologous expression, and practical application testing, striving to translate our research outcomes from the laboratory to farmlands and production workshops, and contributing a solid young force to safeguarding global food security.

## CYCLE 1: RATIONALLY DESIGNED ULTRA-HIGH EFFICIENCY OCHRATOXIN A HYDROLASE ADH3

#### 1. Design - Precision Design Based on Social Needs and Cutting-Edge Bioinformatics

#### Design Inspiration Driven by Human Practices (HP)：

Our design stems from addressing the challenge of Ochratoxin A (OTA) contamination in global food security. Following suggestions from expert interviews, our team took note of OTA's strong carcinogenicity, nephrotoxicity, and its widespread contamination in agricultural products such as grains, coffee, and wine. Traditional physical and chemical methods (e.g., adsorption, ozone treatment) suffer from drawbacks including low efficiency, nutrient loss, and chemical residues. Therefore, we aim to design an efficient, safe, and specific bio-enzymatic degradation method to make a modest and prudent contribution to human food safety.

To also establish a paradigm for future research on similar enzymes, our design phase integrates bioinformatics, structural biology, and rational protein engineering to create both an outstanding enzyme and a comprehensive system.

#### Cutting-Edge Literature Research and Bio-Element Selection：

Through literature mining, we selected the amidohydrolase ADH3 from Stenotrophomonas acidaminiphila CW117 as the core biological element. It has been proven to be the most efficient OTA hydrolase------ADH3(WT) discovered to date (1.2 μg/mL enzyme completely degrades 50 μg/L OTA within 90 seconds), with an efficiency 67 times higher than that of OTase, the previously optimal enzyme. This ensures that our research starts with a high-performance "chassis" element.

#### Rational Design and Hypotheses：

Through bioinformatics analysis, we used tools such as Phyre2 to perform sequence alignment between ADH3 and amidohydrolases with known structures. The results showed that ADH3 contains highly conserved amino acids (H83, H85, H251, H271, K210, D344) that coordinate with the binuclear metal center, classifying it into the binuclear metal amidohydrolase superfamily.

We postulated that its catalytic mechanism is similar to that of other enzymes in this family, and predict that the diversity of substrate-binding pockets is determined by the loop region. This provides clear targets and support for our subsequent mechanism exploration and molecular modification.

#### 2. Build - Precision Molecular Construction and High-Quality Protein Preparation

#### Plasmid Construction：

First, we predicted and removed the 20-amino-acid signal peptide at the N-terminus of ADH3 through bioinformatics. The optimized adh3 gene was fully synthesized and cloned into the pET46/Ek-LIC vector to form a recombinant plasmid with a 6×His tag at the N-terminus. Codon optimization was then performed, and the final recombinant plasmid was named pET46/ADH3. This enabled our constructed biological element (the ADH3 gene) to carry a standardized purification tag.

#### Library Generation and Rational Protein Design (Core of Engineering)：

**Design Objectives：**

* **Enhance activity**: Design for tighter substrate binding and faster conversion rates (e.g., S88E mutation to introduce new hydrogen bonds).
* **Elucidate mechanism**: Create catalytically inactive mutants (D344N/A) for structural studies and hydrolysis mechanism analysis.
* **Explore trade-offs**: Design mutants such as I325A to reduce steric hindrance.

Based on the above design objectives, we systematically constructed three major types of mutants using site-directed mutagenesis technology (names were derived from subsequent experimental results):

* **Ala-scanning Library**: Ala mutants were constructed for each of the 9 key residues (S88, H163, K210, V217, L218, H251, H253, I325, V347) in the substrate-binding pocket for functional verification.
* **Catalytic Dead Mutant**: D344A and D344N mutants were constructed targeting the key catalytic residue D344 for capturing the enzyme-substrate complex structure.
* **Rational Design Library**: Based on structural information obtained later, systematic modification was performed on the L218, V347, and S88 sites (e.g., mutation to S, C, T, F, Y, W, D, R, H, E, K, etc.), aiming to introduce new hydrogen bonds or π-π interactions to enhance enzyme activity.

#### Protein Expression & Purification：

All constructs with optimized codons were transformed into the E. coli BL21(DE3) expression system for induced expression. A well-established three-step purification process was developed:

* **Ni-Affinity Chromatography**: Captures the target protein with the His tag.
* **DEAE Ion-Exchange Chromatography**: Further purifies to obtain high-purity protein.

  ![Exam scores chart](https://static.igem.wiki/teams/5577/pages/engineering/1.webp)

  (Figure : SDS-PAGE Image of DEAE Purification)
* **Size-Exclusion Chromatography**: Finally obtains homogeneous protein samples suitable for structural analysis. Through this method, it was confirmed that ADH3 exists as a homotetramer in solution, with a molecular weight of approximately 360 kDa.

  ![Exam scores chart](https://static.igem.wiki/teams/5577/pages/engineering/2.webp)

  (Figure: ADH3 Size-Exclusion Chromatography Profile)

The above construction process demonstrates our ability to efficiently and reliably prepare large quantities of high-quality protein, which forms the basis for all subsequent tests. The final protein purity achieved was >95% (Figure 3.3), meeting the standards for structural biology research.

#### 3. Test - Multi-Dimensional and Rigorous Functional Verification

#### Enzymatic Assay - Functional Test：

We established a high-performance liquid chromatography (HPLC) method to accurately quantify OTA degradation and the production of the product OTα, thereby evaluating enzyme activity.

![Exam scores chart](https://static.igem.wiki/teams/5577/pages/engineering/3.webp)

(Figure: HPLC Chromatogram of Standard Samples)

#### Test Results：

![Exam scores chart](https://static.igem.wiki/teams/5577/pages/engineering/4.webp)

(Figure : Verification of Inactive Mutants)

* **Verification of Key Catalytic Residues**: The D344N and D344A mutants were completely inactive, confirming our bioinformatics predictions and laying the foundation for complex structure analysis.
* **Functional Residue Screening**: Ala-scanning mutant tests showed that mutants such as H163A, L218A, H251A, and H253A completely lost activity, demonstrating the indispensable role of these residues in substrate binding or catalysis. Meanwhile, the I325A mutant showed a 34% increase in activity, indicating potential for optimization at this site.

  ![Exam scores chart](https://static.igem.wiki/teams/5577/pages/engineering/4.webp)

  (Figure : Functional Verification of Key Sites)
* **Verification of Rational Design**: Final tests showed that the S88E, S88K, and S88H mutants exhibited significantly enhanced activity, reaching 3.7-fold, 2.4-fold, and 1.3-fold that of the wild type, respectively, successfully verifying our design hypotheses.

#### Structural Test - Mechanism Elucidation：

This represents the most prominent technical highlight of this project. Using single-particle cryo-electron microscopy (cryo-EM) technology, we successfully resolved three sets of high-resolution structures:

![Exam scores chart](https://static.igem.wiki/teams/5577/pages/engineering/5.webp)

* **Apo-ADH3 (2.71 Å)**: The ligand-free structure of ADH3, revealing the overall architecture of its homotetramer.
* **ADH3/Phe (2.50 Å)**: The complex structure with the product phenylalanine, initially revealing the product-binding mode.
* **D344N/OTA (2.50 Å)**: The complex structure of the inactive mutant D344N bound to the substrate OTA. This is the world's first reported structure of an OTA hydrolase in complex with its substrate, clearly revealing the OTA binding mode: OTA is precisely anchored in the active pocket through a hydrogen bond network (with H163, L218, H251, H253, and Metal β) and π-stacking interactions (with H85, H253, and H271).

![Exam scores chart](https://static.igem.wiki/teams/5577/pages/engineering/6.webp)

(Figure : Details of OTA-Protein Interactions)

These structural data provide us with atomic-level "visual evidence", greatly enhancing the credibility of all our conclusions and serving as a bridge connecting design and learning.

#### 4. Learn & Iteration - From Atomic Insights to Engineering Optimization

#### Mechanism Learning：

Integrating all test data (mutant enzyme activity + high-resolution structures), we elucidated the complete catalytic mechanism of ADH3 for the first time:

* **Metal Activation**: Metal β interacts with the carbonyl oxygen of the OTA amide bond, polarizing the carbonyl carbon.
* **Proton Transfer**: The conserved D344 abstracts a proton from the bridging water molecule.
* **Nucleophilic Attack**: The deprotonated hydroxyl group performs a nucleophilic attack on the polarized carbonyl carbon, breaking the C-N bond, and D344 then donates a proton to the leaving group.

  ![Exam scores chart](https://static.igem.wiki/teams/5577/pages/engineering/7.webp)

  (Figure : Schematic Diagram of the Catalytic Mechanism)

#### Highlight of Iterative Design：

Based on the D344N/OTA complex structure, we gained a key insight: the side-chain hydroxyl group of S88 points toward the phenol hydroxyl group of the OTα moiety of the OTA molecule, but the distance is too great to form a strong interaction.

**New Hypothesis：**If S88 is mutated to an amino acid with a longer side chain or a charged group (e.g., Glu, Lys), stronger hydrogen bonds or electrostatic interactions may form, thereby stabilizing substrate binding more effectively and improving catalytic efficiency.

**New Design：**Based on this, we constructed a series of mutants including S88D, S88R, S88H, S88E, and S88K.

**Iterative Test Results ：**

The enzyme activity test results have perfectly verified our new hypothesis. The S88E and S88K mutants have achieved disruptive performance improvements (3.7-fold and 2.4-fold respectively), which is most likely due to the fact that the carboxyl group of Glutamic acid (Glu) and the amino group of Lysine (Lys) form a stronger hydrogen bond network with the phenolic hydroxyl group of OTα, thereby stabilizing the reaction transition state.

The test results have been directly fed back into the new design cycle and incorporated into broader project considerations.

#### Iterative Engineering:

The success of S88E has validated the effectiveness of our structure-based design approach. However, the failure to introduce bulkier aromatic amino acids at the L218 site (L218F/Y/W, i.e., Phenylalanine/Tyrosine/Tryptophan mutants) has helped us understand the precise spatial constraints of the active pocket, providing key reference information for subsequent designs.

We have developed and revised a bioinformatics structure prediction and fitting model (using AutoDock Vina software) to predict the binding affinity between Ochratoxin A (OTA) and the mutant enzymes we constructed. The predictions of this model (e.g., the lower binding energy of the S88E mutant) are well-correlated with our experimental results, laying a foundation for the bioinformatics-aided design of the next generation of mutants and creating a powerful tool for this purpose.

## CYCLE-2：Screening,Construction,Testing and Functional Analysis of LlADH

## Design

#### 1.Design Background

In the previous engineering cycle, we learned the mechanism from tests and guided rational design. We successfully iterated an optimized component (S88E) with significantly improved performance, which has an enzyme activity 3.7 times that of the prototype ADH3. However, in our subsequent expert interviews, it was keenly pointed out that the binding degree of the catalytic site between the enzyme and substrate, which we have successfully modified, has reached or is expected to reach the limit of this enzyme. To further improve the efficiency of OTA enzymatic hydrolysis, we can only look for isozymes with deeper catalytic pockets to break through the structural bottleneck of the prototype enzyme itself.

#### 2. Design Objectives

Therefore, we plan to screen through databases to identify enzymes that share a similar structure and identical function to ADH3. We will then conduct structural analysis and functional characterization of these enzymes, with the expectation that they will exhibit higher efficiency and stronger adaptability in the degradation of OTA.

## Build

#### 1.Data Screening

Based on the characteristics of amide metal hydrolases and the structural analysis of ADH3, we conducted data mining in the NCBI database and obtained its isozyme---amide hydrolase LlADH derived from Lysobacter luteus. We analyzed LlADH sequence using different online tools and the websites of these tools are listed. LlADH consists of a total of 442 amino acids, with approximate molecular weight 46.45 kDa and a theoretical isoelectric point of 6.20. There are two Cys residues in its sequence, and it has no transmembrane regions.

Bioinformatics Analysis Website

![Exam scores chart](https://static.igem.wiki/teams/5577/pages/engineering/8.webp)

#### 2. Similarity verification

We performed a Sequence alignment between LlADH and ADH3 showed 73% similarity. Based on Phyre2 structure prediction, 76% structural similarity was observed, confirming that it is the chassis component with the highest comprehensive score for us.

![Exam scores chart](https://static.igem.wiki/teams/5577/pages/engineering/9.webp)

(Figure: Sequence Alignment between ADH3 and LlADH)

The conserved amino acids involved in coordinating the binuclear metal center are marked with green circles, among which the Asp (aspartic acid) that participates in the acid/base-catalyzed protonation of the substrate is marked with blue triangles.

#### 3. Construction of recombinant plasmid

By using online bioinformatics tools, we predicted that the first 19 amino acids of LlADH are signal peptides. W In the constructed vector, the first 19 amino acids of LlADH were removed, and the N-terminus was fused with a 6×His purification tag. Codon optimization was performed, and the entire gene was synthesized and subcloned into the pET46/Ek-LIC vector. The plasmid was named pET46/LlADH.

#### 4.Construction, Expression, and Purification of the Expression Strain

The pET46/LlADH plasmid was transformed into competent cells of the expression strain BL21(DE3) for transformation and expression. Purification was performed using nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography once. The eluate was collected according to the UV curve detected by the AKTA protein purification system, followed by ultrafiltration concentration. After ultrafiltration concentration, the protein concentration was 11.7 mg/mL. The purity of the protein was verified by SDS-PAGE, and the results are shown in the figure. The position indicated by the red arrow corresponds to the concentrated LlADH. The molecular weight of the target protein is approximately at the 45 kDa band, which is consistent with the theoretical molecular weight. With a purity of over 95%, it was confirmed that the LlADH protease with high purity and suitable experimental concentration was obtained.

![Exam scores chart](https://static.igem.wiki/teams/5577/pages/engineering/10.webp)

(Figure: SDS-PAGE analysis of concentrated LlADH
 Lane 1: marker; Lane 2: LlADH)

## Test

#### I.Determination of homogeneity and molecular weight of LlADH

To verify the oligomeric state and molecular weight of the recombinant LIADH protein, we conducted a systematic analysis using various biophysical methods.

#### 1.Gel filtration chromatography

The purified LIADH was analyzed using a Superose™ 6 increase 10/300 GL chromatographic column. The results showed that LIADH presented a single symmetric peak in gel filtration chromatography, and the elution position was between the 158 kDa and 440 kDa standard proteins. Based on the theoretical molecular weight of the LIADH monomer (46.45 kDa), it is speculated that the protein may exist in the form of a homooctamer.

![Exam scores chart](https://static.igem.wiki/teams/5577/pages/engineering/11.webp)

(Figure: Size-exclusion chromatography analysis of LlADH)

#### 2.Static Light Scattering

To further accurately determine the molecular weight of LIADH, we used static light scattering technology for analysis. As shown in the figure, the results indicate that the molecular weight of LIADH is approximately 351.9 kDa, which is highly consistent with the theoretical molecular weight of the octamer (~371.6 kDa), confirming that LIADH exists as a homooctamer in solution.

![Exam scores chart](https://static.igem.wiki/teams/5577/pages/engineering/12.webp)

(Figure: Static light scattering analysis of LlADH)

#### 3.Negative staining electron microscopy

The morphology and distribution of LIADH were observed by negative staining electron microscopy. The results showed that the protein particles were evenly distributed with consistent morphology, with a diameter of approximately 10 nm, and obvious four-fold symmetric structures could be observed, which further supported that LIADH is a highly homogeneous octameric protein.

![Exam scores chart](https://static.igem.wiki/teams/5577/pages/engineering/13.webp)

(Figure: Negative staining analysis of LlADH)

#### II. Enzyme Activity Detection and Analysis of LlADH

#### 1.Analysis of LIADH Hydrolyzed OTA Products1

We analyzed the OTA hydrolysis reaction catalyzed by LIADH using HPLC. The results, as shown in the figure, indicate that as the reaction proceeds, the OTA peak gradually decreases, and a new peak appears at the retention time corresponding to the OTα standard. After 50 minutes of reaction, OTA is completely hydrolyzed into OTα. We further verified through HPLC-MS that the molecular weight and isotope distribution of the product are consistent with those of OTα, as shown in the figure, confirming that LIADH can efficiently hydrolyze OTA to produce non-toxic OTα and phenylalanine.

![Exam scores chart](https://static.igem.wiki/teams/5577/pages/engineering/14.webp)

(Figure: HPLC analysis of OTA and OTα standards and OTA-degrading reaction mixtures catalyzed by LlADH)

#### 2.Analysis of the enzymatic properties of LIADH

* **Optimal temperature**: The enzyme activity of LIADH is the highest at 45℃.
* **Optimal pH**: The enzyme activity is the highest in Tris-HCl buffer at pH 8.5.
* **Influence of metal ions**: Fe²⁺, Cu²⁺, and Zn²⁺ significantly inhibit enzyme activity; Li⁺ slightly promotes it; other metal ions have no significant effect or slightly inhibit it.
* **Thermal stability**: LIADH has the best stability at 20℃ and is rapidly inactivated above 40℃.

![Exam scores chart](https://static.igem.wiki/teams/5577/pages/engineering/15.webp)

(Figure: Enzymatic characterization of purified LlADH)

(A) The temperature evaluation. (B) The pH evaluation.

(C) The metal ions evaluation. (D) The thermalstability evaluation.

All assays are performed in triplicate and the results are presented as average ± SD

#### 3.Comparison of LIADH and ADH3 activities

We tested the enzyme reaction samples of ADH3 and LlADH under exactly the same reaction conditions and systems, with the enzyme activity of ADH3 set as 100% for comparison. The results are shown in the figure. The analysis of the OTA hydrolysis reaction results indicates that the hydrolysis activity of LlADH on OTA is twice that of ADH3, making it a more efficient amide hydrolase for hydrolyzing OTA.

![Exam scores chart](https://static.igem.wiki/teams/5577/pages/engineering/16.webp)

(Figure: Specific activity of ADH3 and LlADH)

#### III. Structural Analysis of LlADH

#### 1.Cryo-electron microscopy data collection and processing

We used a 300kV Titan Krios cryo-electron microscope to collect LIADH data, and finally reconstructed a three-dimensional structure with a resolution of 2.67 Å from 151,233 particles.

![Exam scores chart](https://static.igem.wiki/teams/5577/pages/engineering/17.webp)

Detailed image processing and three-dimensional reconstruction are shown in the figure. Figure A is the cryo-electron microscopy image of LlADH, and the results show that the protein particles have good uniformity. Among the 520,000 protein particles initially collected from the original images, 150,000 particles were selected for two-dimensional classification after performing offset correction and contrast transfer function evaluation using cryoSPARCv3.3.1 software. Some good particles among them were used to construct the initial template. As shown in Figure B, which is part of the two-dimensional classification results of LlADH, the protein presents a C4 symmetric unit. These selected protein particles were subjected to further three-dimensional classification, and after multiple rounds of optimization, a high-resolution protein electron density map with a resolution of 2.67Å was finally reconstructed.

![Exam scores chart](https://static.igem.wiki/teams/5577/pages/engineering/18.webp)

(Figure: Cryo-EM data processing for LlADH)

(A)Cryo-EMmicrograph. (B)2D classification.

(C)Local resolution map. (D)FSC curve.

#### 2.Structural Construction and Correction

Using the ADH3 structure (PDB: 8IHQ) as the initial model, we used UCSF Chimera software to superimpose the LlADH structure with the electron density map obtained from electron microscopy data processing. We then performed model building, refinement, and evaluation using COOT and Phenix, ultimately obtaining an atomic model where all indicators meet the standards. It can be seen from the collated data that all the overall data are within the specified values, and the structure is reliable.

#### 3.Structural Analysis

![Exam scores chart](https://static.igem.wiki/teams/5577/pages/engineering/19.webp)

(Figure: LlADH protomer in cartoon representation)

* LIADH is a homologous octamer with a double-layered double-helix structure.
* Each monomer consists of a TIM barrel catalytic domain and a β-roll domain.
* The active center contains a binuclear metal site, which is coordinated by residues such as H84, H86, H252, H272, K211, and N345.
* It is speculated structural alignment that OTA may bind to the enzyme through residues such as H86, H254, and H272.
* To obtain the complex structure of LlADH and OTA and clarify the substrate binding mode of LlADH, we incubated LlADH with OTA before preparing the frozen samples. However, under the cryo-electron microscope, no electron cloud of OTA was observed in the putative substrate pocket of LlADH.

![Exam scores chart](https://static.igem.wiki/teams/5577/pages/engineering/25.avif)

(Figure: Superposition of LlADH onto ADH3/D344N/OTA)

OTA is shown in green sticks. Residues surrounding OTA in LlADH and ADH3/D344N/OTA are shown in magentas- and green-colored lines, respectively. The carboxylated K210 and K211 functions as a bridging ligand between the two metal ions. The binuclear metal cluster (denoted as α and β) is presented as red spheres. Distances < 3.5 Å are indicated with black dashed lines in the modeled LlADH/OTA.

## Learn

#### I.Shortcomings or areas for improvement of monomers in enzyme activity modification

Although LIADH has exhibited extremely high OTA hydrolytic activity, there are still some aspects that need improvement.

* The LIADH monomer cannot provide sufficient hydrophilic interactions or stacking forces for OTA, which further limits the reduction of reaction activation energy.
* Its stability at higher temperatures is poor (it is prone to inactivation above 40°C), which limits its application in industrial environments. In addition, LIADH is sensitive to certain metal ions (such as Fe²⁺ and Cu²⁺), which may affect its enzymatic activity in actual complex systems. In the future, its thermal stability and metal tolerance can be further improved through rational design or directed evolution.

#### II. Preliminary Understanding of the Relationship Between Structure and Function

Through cryo-electron microscopy structural analysis, we have initially revealed the octameric assembly mode and active center composition of LIADH. Key residues such as H86, H254, and H272 may bind to OTA through π-stacking and hydrogen bond interactions, which provides a structural basis for subsequent enzyme activity modification.

However, the failure to obtain the OTA complex structure has limited the in-depth understanding of its substrate recognition mechanism. In the future, we can try to use substrate analogs or inert mutants (such as D345N) to capture the complex structure.

#### III Enlightenment for the application of biological detoxification

The high catalytic activity and metal independence (except for Zn²⁺) of LIADH give it significant advantages in food and feed detoxification applications. Its optimal pH (8.5) is consistent with many agricultural products processing environments, indicating potential for direct application. Future research can focus on the detoxification effect and process optimization of LIADH in real matrices (such as grains and wine).

## Cycle 3: Site-Directed Mutagenesis Design, Construction, Activity Detection and Optimization Direction of LlADH

## Design

#### Design 1 conducts site analogy through structural stacking.

In the previous cycle of LlADH, we have, for the first time, analyzed the structure of its catalytic pocket and its catalytic mechanism using bioinformatics and structural biology (cryo-electron microscopy). At the protein level, we optimized codons using synthetic biology, thereby increasing the expression level and purity of its protein crystals. We will utilize the effective research paradigm established in the first cycle and rationally modify this protein using synthetic biology techniques.

Through structural comparison, we found that the active site residues of ADH3, such as G88, G130, H16, etc., as well as six metal-iron coordinating residues, correspond to similar sites in LlADH like G89, G131, H164, etc., which also have a similar distribution in the LlADH/OTA structure. This indicates that these residues should also play an important role in LlADH catalysis. This further verifies the high conservation of the ADH family in catalytic mechanisms, especially in terms of coenzyme binding and metal ion stabilization. The spatial arrangement of key residues determines substrate specificity and reaction efficiency.

![Exam scores chart](https://static.igem.wiki/teams/5577/pages/engineering/25.avif)

(Figure: Superposition of LlADH onto ADH3/D344N/OTA)

OTA is shown in green sticks. Residues surrounding OTA in LlADH and ADH3/D344N/OTA are shown in magentas- and green-colored lines, respectively. The carboxylated K210 and K211 functions as a bridging ligand between the two metal ions. The binuclear metal cluster (denoted as α and β) is presented as red spheres. Distances < 3.5 Å are indicated with black dashed lines in the modeled LlADH/OTA.

#### Design 2: Select mutation sites and formulate multi-directional mutations

In the modeling structure, the six amino acid residues L219, G131, V348, G89, G91, and I326 are farther from OTA than other OTA-binding amino acid residues. Therefore, it is speculated that replacing these residues to introduce additional substrate-enzyme interactions may increase the enzymatic activity of LlADH.

* **First**, select the residue L219 adjacent to the Phe moiety of OTA and mutate it to a polar residue, so that the mutant provides additional hydrophilic interactions or stacking forces for the Phe moiety of OTA. Three mutations, L219H, L219K, and L219R, are designed;
* **Compared with the Phe moiety**, the OTα moiety of OTA is less restricted due to the lack of sufficient interacting residues. Therefore, the residue G131 was mutated to a hydrophobic residue to provide additional hydrophobic interactions for the OTα moiety, resulting in the design of two mutations: G131F and G131L.
* **The side chain of residue V348** points to the OTα moiety of OTA. Mutating it to amino acid residues with different charges aims to provide additional hydrophilic interactions for the hydroxyl group of the OTα moiety, and three mutations, namely V348F, V348H, and V348S, are designed;
* **The side chain of residue V348** points to the OTα moiety of OTA. Mutating it to amino acid residues with different charges aims to provide additional hydrophilic interactions for the hydroxyl group of the OTα moiety, and three mutations, namely V348F, V348H, and V348S, are designed;
* **The side chain of residue I326** points to the Phe part of OTA. To allow OTA to enter the active pocket better, it is mutated to I326A with a smaller side chain.

## Build

#### Design site-directed mutagenesis primers and molecular cloning

Using the pET46/LlADH plasmid as a template, site-directed mutagenesis of LlADH was performed via polymerase chain reaction with the primers designed above to construct a series of mutant plasmids.

## Test

#### Test 1 Large-scale Expression and Detection of Target Protein

After correct sequencing, expression and purification are performed. The expression and purification process of the mutant protein is the same as that of the LlADH wild type.

Finally, S89D, S89F, S89H, S89K, S89R, S91R, G131F, L219H, L219K, L219R, I326A, V348F, V348H, and V348S were obtained. The purified proteins were verified by SDS-PAGE. The concentration results of LlADH, S91R, L219H, L219K, L219R, I326A, V348F, V348H, and V348S are shown in the figure, and the concentration results of S89D, S89F, S89H, S89K, and S89R are shown in the figure. The size of all concentrated mutant proteins is around 45 kDa, and the protein purity is suitable for subsequent enzyme activity experiments.

![Exam scores chart](https://static.igem.wiki/teams/5577/pages/engineering/22.webp)

(Figure: SDS-PAGE analysis of LlADH variants)

A：1：marker；2：WT； 3-10：S91R、L219H、L219K、L219R、I326A、V348F、V348H、V348S B：1：marker；2-6：S89D、S89F、S89H、S89K、S89R；7：WT

#### Test 2 Determination of Mutant Enzyme Activity

Based on the above analysis results, using the established HPLC detection system, the enzyme activities of LlADH and its mutants were analyzed, and the results are shown in the figure:

* **The enzyme activities of the L219 series mutants** L219H, L219K, and L219R almost completely disappeared.
* **The G131 series mutants** G131F and G131L were not successfully purified;
* **The catalytic activities of the V348 series mutants** V348F, V348H, and V348S almost disappeared.
* **The catalytic activities of the S89 series mutants** S89D, S89F, S89H, S89K, S89R, and S91R were not improved.
* **The catalytic activity of the mutant I326A** was increased by 75% compared with LlADH.

![Exam scores chart](https://static.igem.wiki/teams/5577/pages/engineering/23.webp)

(Figure: LlADH engineering)

The enzyme activity of variants was conducted in triplicate and the average values with the error bars representing standard deviations are shown. ND, not detectabl

#### Analysis of causes

* **The failure of the L219 series mutations** may be due to the polar side chains or larger side chains of His, Lys, and Arg hindering the binding and entry of OTA;
* **The failure of the V348 series mutations** indicates that the hydrophobic interaction of residue V348 is necessary for the LlADH-catalyzed hydrolysis of OTA;
* **The failure of the S89 series mutations** indicates that the charges carried by residues S89 and S91 are necessary for the LlADH-catalyzed hydrolysis of OTA;
* **The catalytic activity of the mutant I326A** is relatively improved compared to LlADH, which may be because the side chain of Ala is smaller, providing more space for the binding of OTA to LlADH.

## Learn

In this round, based on the structural analysis of LlADH, potential sites were predicted through structural stacking analogy, and multi-directional mutations were formulated. After a complete design-build-test process, mutation sites that can significantly improve enzyme activity were identified, and the enzyme activity test was successfully passed. The next step will focus on combinatorial mutations of key residues to further optimize the spatial configuration and charge distribution of the substrate-binding pocket, aiming to synergistically enhance catalytic efficiency and substrate specificity. Meanwhile, the stability of the mutants under different reaction conditions will be verified to provide data support for subsequent large-scale applications. Virtual experiment verification will combine molecular dynamics simulations and free energy calculations. Additionally, efforts will be made to explore the high-efficiency expression process of LlADH in food-safe industrial strains such as Pichia pastoris and optimize fermentation conditions to improve the soluble expression level and catalytic activity.

Additionally, it is exciting that our attempts to express the engineered protein in Pichia pastoris have shown relatively stable and effective relative enzymatic activity efficiency.The relative enzymatic activity tests demonstrated that our engineered protein could be efficiently expressed in both eukaryotic and prokaryotic model organisms, with no significant differences observed.

![Exam scores chart](https://static.igem.wiki/teams/5577/pages/engineering/24.webp)

(Figure: Relative enzyme activity result)

## From One Enzyme One Effect to One Enzyme Multi Effect: Exploration and Prospect of Fpro

### To achieve a leap from one enzyme one effect to one enzyme multi effect, we continued to mine in the database and found an enzyme that can simultaneously degrade three toxins - Fpro enzyme from Bacillus megaterium HNGD-A6.

### Enzyme activity test

According to literature and team enzyme activity testing, Fpro enzyme has been proven to efficiently degrade AFB1, OTA, and ZEN, three fungal toxins, with relatively low degradation rates. We found in subsequent experiments that the enzyme also has the ability to degrade Patulin, achieving a preliminary functional leap of "one enzyme, four effects".

### Structural Analysis

Through electron microscopy analysis, we obtained the empty structure of Fpro, as shown in the schematic diagram below.

![Exam scores chart](https://static.igem.wiki/teams/5577/pages/engineering/26.avif)

### Rational Transformation and Current Limitations

The team has conducted rational design mutations for Fpro, but the mutant enzyme activity test results have the following issues:

* **The activity of key mutants decreased**: the degradation activity of AFB1 by mutants decreased by about 50%;
* **Insufficient experimental stability**: Some mutants did not complete parallel control due to insufficient protein content, resulting in significant data errors;
* **Substrate stability issue**: Some mutants exhibit significant self degradation within 1-3 days, which interferes with enzyme activity evaluation;
* **PH adaptability limitation**: The wild-type Fpro exhibits the highest activity under alkaline conditions (pH 8.5), but the feed industry requires an acidic environment (pH 2.5-4.0).

### Future research direction: Improving efficiency and industrial expression

1. **Enhance catalytic efficiency and acid resistance**
   Structural analysis and rational design: Further analysis of the complex structure of Fpro with AFB1, OTA, ZEN, and Patulin is needed to clarify the substrate binding domain and catalytic residues, and obtain highly active mutants through iterative mutagenesis.
   Acid resistance modification: The plan is to combine QM/MM calculations to simulate the conformational changes of enzymes in acidic environments, and design pH adaptive mutants (such as introducing acid stable residues or adjusting surface charge distribution).
2. **Development of yeast expression system**
   Codon optimization and signal peptide design: Optimize the Fpro gene codon for yeast expression systems (such as Pichia pastoris) and add alpha factor signal peptides to promote secretion expression.
   Fermentation process optimization: It is necessary to explore high-density fermentation conditions (temperature, pH, inducer concentration) to achieve high yields while avoiding protein misfolding or degradation.
3. **Industrial application bottlenecks and countermeasures**
   Improved thermal stability: Preliminary studies have shown that Fpro remains active at 80 ° C, but needs to withstand short-term high temperatures (~120 ° C) during feed processing. Thermal stability can be enhanced by introducing disulfide bonds or rigid residues.Multi substrate synergistic degradation mechanism: We plan to investigate the degradation priority and product toxicity when multiple toxins coexist (such as the reduced toxicity of AFB1 degradation product AFD1, but the remaining toxin degradation products need to be clarified).

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your project. It is designed to provide sufficient information for other teams to replicate our work.{% endblock %}
{% block page\_content %}

## What Should this Page Contain?

---

Provide comprehensive, step-by-step protocols for all experiments conducted. Include a list of materials, reagents, and equipment used. Specify concentrations, volumes, incubation times, temperatures, and other critical parameters. Document any modifications or optimizations made to standard protocols.

Explain the purpose of each experiment and its relevance to your project goals. Describe the experimental design, including controls and replicates. Provide a clear rationale for the chosen methods and approaches.

Document any troubleshooting steps taken and optimizations made during the experimental process. Share any lessons learned that could benefit future teams.

## Inspirations

---

* [2024 Heidelberg](https://2024.igem.wiki/heidelberg/experiments)
* [2024 Marburg](https://2024.igem.wiki/marburg/experiments)
* [2024 EPFL](https://2024.igem.wiki/epfl/experiments)
* [2023 Estonia-TUIT](https://2023.igem.wiki/estonia-tuit/experiments)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block page\_content %}

Contents

# Results

## Ⅰ、Enzymatic Properties and Activity Analysis

### 1. ADH3 Prototype Enzyme (Benchmark)

**Catalytic Efficiency**: Our core biological component, the amide hydrolase ADH3, has been proven to be the most efficient ochratoxin A (OTA) hydrolase to date. At a concentration of 1.2 μg/mL, it can completely degrade 50 μg/L of OTA within 90 seconds, which is 67 times more efficient than the previously optimal enzyme, OTase.

**Metal Dependence**: Using tools such as Phyre2, we aligned the sequence of ADH3 with amide hydrolases of known structures (e.g., PDB: 2QS8, 4C5Y). The results showed that ADH3 relies on a highly conserved dinuclear metal center (Fe²⁺/Zn²⁺), with key residues including H83, H85, H251, H271, K210, and D344.

![Exam scores chart](https://static.igem.wiki/teams/5577/pages/result/image1.webp)

(Sequence Comparison of Dinuclear Metal Amide Hydrolases)

**Oligomeric State**: Gel filtration chromatography and static light scattering were used to obtain homogeneous protein samples for structural analysis. These analyses confirmed that ADH3 exists as a homotetramer in solution, with a molecular weight of approximately 360 kDa.

![Exam scores chart](https://static.igem.wiki/teams/5577/pages/result/image2.webp)

(ADH3 Gel Filtration Chromatogram)

### 2. LIADH Mining and Preliminary Modification

**Activity Enhancement**: Based on the characteristics of amide metal hydrolases and the structural analysis of ADH3, we mined the NCBI database to identify its isoenzyme, the amide hydrolase LlADH from Lysobacter luteus. Through transformation, expression, purification, and testing, high-purity LIADH was obtained. Subsequently, under identical reaction conditions and systems, the enzyme reaction samples of ADH3 and LlADH were detected, with the enzyme activity of ADH3 set as 100% for comparison. The results of the OTA hydrolysis reaction analysis showed that the hydrolysis activity of LlADH towards OTA is twice that of ADH3, making it a more efficient amide hydrolase for OTA hydrolysis.

![Exam scores chart](https://static.igem.wiki/teams/5577/pages/result/image3.webp)

**Optimal Conditions**: Under the premise of controlling variables, the enzyme activity of LIADH was tested under different conditions. The optimal temperature was determined to be 45°C (Figure A) and the optimal pH was 8.5 (Figure B). Its activity is inhibited by Fe²⁺ and Cu²⁺, while slightly promoted by Li⁺ (Figure C).

**Thermal Stability**: LIADH is prone to inactivation above 40°C and exhibits the best stability at 20°C (Figure D).

![Exam scores chart](https://static.igem.wiki/teams/5577/pages/result/image4.webp)

### 3. Rational Design and Iteration of ADH3

**Mutant Activity**: We established a high-performance liquid chromatography (HPLC) method to accurately quantify the degradation of OTA and the production of the product OTα, thereby evaluating enzyme activity. Through tests such as Ala scanning of mutants, the final results showed that:

![Exam scores chart](https://static.igem.wiki/teams/5577/pages/result/image5.webp)

1. The D344N and D344A mutants were completely inactivated, confirming our bioinformatics predictions and laying the foundation for the structural analysis of the complex for mechanism research.

![Exam scores chart](https://static.igem.wiki/teams/5577/pages/result/image8.webp)

1. Mutants such as H163A, L218A, H251A, and H253A completely lost their activity, demonstrating the indispensability of these residues in substrate binding or catalysis. Meanwhile, it was found that the activity of the I326A mutant was increased by 34%, indicating that there is room for optimization at this site.

![Exam scores chart](https://static.igem.wiki/teams/5577/pages/result/image6.webp)

1. The activities of the S88E, S88K, and S88H mutants were significantly enhanced, reaching 3.7 times, 2.4 times, and 1.3 times that of the wild type, respectively.

![Exam scores chart](https://static.igem.wiki/teams/5577/pages/result/image9.webp)

## Ⅱ、 Structural Biology and Mechanism Elucidation

We systematically resolved the three-dimensional structures of ADH3 and its homologous enzyme LIADH using high-resolution structural biology techniques, revealing their catalytic mechanisms and substrate recognition patterns.

### 1. High-Resolution Structure of ADH3

We resolved three sets of high-resolution structures of ADH3 using single-particle cryo-electron microscopy:

* **Apo-ADH3 (2.71 Å)**: It was revealed that ADH3 exists as a homotetramer, with an overall structure presenting a double-layered double-helix architecture. Each monomer consists of a TIM barrel catalytic domain and a β-roll domain.

![Exam scores chart](https://static.igem.wiki/teams/5577/pages/result/10.avif)

* **ADH3/Phe Complex (2.50 Å)**: The complex structure with the product phenylalanine initially revealed the product binding mode, suggesting that residues such as H163 and L218 may be involved in substrate recognition.

  ![Exam scores chart](https://static.igem.wiki/teams/5577/pages/result/11.avif)
* **D344N/OTA Complex (2.50 Å)**: As the world's first OTA-enzyme complex structure, it clearly showed that OTA is precisely anchored in the active pocket through a hydrogen bond network (with H163, L218, H251, H253, and metal β) and π-stacking interactions (with H85, H253, and H271).

  ![Exam scores chart](https://static.igem.wiki/teams/5577/pages/result/12.avif)

### 2. Structural Resolution of LIADH

We resolved the 2.67 Å structure of LIADH using cryo-electron microscopy, confirming that it also exists as a homotetramer (with a molecular weight of 351.9 kDa as determined by static light scattering). The active center coordinates the dinuclear metal center through residues such as H84, H86, H252, H272, K211, and N345, and its structure is highly conserved with that of ADH3.

![Exam scores chart](https://static.igem.wiki/teams/5577/pages/result/16.avif)

### 3. Elucidation of the Catalytic Mechanism

Comprehensive analysis of mutant enzyme activity and structural data led us to propose the atomic mechanism of ADH3-catalyzed OTA hydrolysis: Metal β interacts with the carbonyl oxygen of the amide bond in OTA, polarizing the carbonyl carbon; the conserved residue D344 extracts a proton from the bridging water molecule; the deprotonated hydroxyl group nucleophilically attacks the polarized carbonyl carbon, breaking the C-N bond; and D344 donates the proton to the leaving group, completing the hydrolysis process.

## Ⅲ、 Functional Verification and Mutant Library Construction

Combining cryo-electron microscopy structural resolution and bioinformatics prediction methods, we systematically constructed and verified multiple mutant libraries through rational design and Ala scanning strategies to validate the functions of key residues and enhance enzyme activity.

### 1. ADH3 Rational Design Mutant Library

**Ala Scanning Library**: Ala mutants were constructed for 9 key residues, including S88, H163, K210, V217, L218, H251, H253, I325, and V347. The results showed that mutants such as H163A, L218A, H251A, and H253A were completely inactivated, confirming the indispensability of these residues in catalysis or substrate binding; the activity of the I326A mutant was increased by 34%, indicating the potential for spatial optimization at this site.

**Rational Design Library**: Based on structural information, systematic mutations (e.g., S88D/E/H/K/R) were performed on sites such as S88, L218, and V347 to introduce hydrogen bonds or π-π interactions. Finally, the activities of the S88E, S88K, and S88H mutants were significantly enhanced, reaching 3.7 times, 2.4 times, and 1.3 times that of the wild type, respectively; mutants with large side chains at the L218 site (e.g., L218F/Y/W) lost their activity, indicating that this site is sensitive to spatial factors.

### 2. LIADH Mutant Screening

Through structural alignment and rational design, the following LIADH mutants were successfully constructed and purified: S89D, S89F, S89H, S89K, S89R, S91R, G131F, L219H, L219K, L219R, I326A, V348F, V348H, and V348S.

Enzyme activity test results showed that the activity of I326A was increased by 75%, confirming that reducing the side chain can reduce steric hindrance; the activities of the L219 series (H/K/R) and V348 series (F/H/S) were completely lost, indicating that these residues are crucial for substrate binding or catalysis; the S89 series mutations did not enhance activity, suggesting that the charge property of this site is essential for catalysis.

![Exam scores chart](https://static.igem.wiki/teams/5577/pages/result/13.avif)

* **LIADH**: It can completely hydrolyze OTA to form non-toxic OTα and phenylalanine within 50 minutes, and its optimal pH (8.5) is consistent with the processing environment of many agricultural products, providing a basis for direct application.

![Exam scores chart](https://static.igem.wiki/teams/5577/pages/result/14.avif)

* The next step will focus on combinatorial mutations to further optimize the spatial and charge distribution of the substrate-binding pocket.

Additionally, it is exciting that our attempts to express the engineered protein in Pichia pastoris have shown relatively stable and effective relative enzymatic activity efficiency.The relative enzymatic activity tests demonstrated that our engineered protein could be efficiently expressed in both eukaryotic and prokaryotic model organisms, with no significant differences observed.

![Exam scores chart](https://static.igem.wiki/teams/5577/pages/result/15.avif)

{% endblock %}