Our iGEM team has made significant contributions through a systematic and iterative research effort aimed at addressing Ochratoxin A (OTA) contamination in food products. Below are the key experimental achievements:
We not only resolved the high-resolution structure of ADH3 via cryo-EM---including its apo-form, product-bound complex, and world's first substrate-bound complex with OTA---but also comprehensively deciphered its catalytic mechanism. By combining structural insights with functional validation through targeted mutagenesis (e.g., catalytic dead mutant D344N/A and alanine scanning), we demonstrated the critical roles of key residues (H163, L218, H251, H253, etc.) in substrate binding and catalysis. This work provides an atomic-level understanding of OTA hydrolysis by ADH3, setting a solid foundation for future enzyme engineering and rational design.
Through three complete cycles of Design-Build-Test-Learn, we implemented a recursive engineering strategy that led to continuous improvement in enzyme performance:
These contributions not only advance the scientific understanding of OTA-degrading enzymes but also establish a robust engineering framework for future research in enzyme design and toxic compound biodegradation.
ADH3 was first uploaded to the iGEM Parts Registry in 2023 (BBa_K4613014). This year, based on this part, we supplemented and expanded its content, updating the structural analysis and catalytic mechanism of ADH3.
We determined the crystal structure of ADH3.
The monomer adopts the canonical fold of the ADH family: an (α/β)8 triosephosphate isomerase (TIM)-barrel catalytic domain (residues 23–386) flanked by a β-roll subdomain (residues 22–77 and 387–427).
The OTA-binding pocket, visualized in the D344N/OTA complex, is defined by residues S88, H163, V217, L218, H253, I325 and V347, together with six Zn2+-coordinating ligands (H83, H85, H251, H271, K210 and N344).
OTA is anchored through hydrogen bonds to H163, L218, H251, H253 and the β-metal ion. Additional π-stacking is provided by H85 against the OTα moiety, whereas H253 and H271 engage the L-β-phenylalanine portion in T-shaped π-interactions.
The dinuclear metal centre is arranged as follows:Metal-α is ligated by H83, H85, D344 and the carboxylated K210；Metal-β is ligated by H251, H271 and the same carboxylated K210, which thereby bridges the two Zn2+ ions.
ADH3 (wild type) is a highly efficient amidohydrolase derived from Stenotrophomonas acidaminiphila CW117, specifically designed to degrade Ochratoxin A (OTA). OTA is a mycotoxin with strong carcinogenic and nephrotoxic properties, contaminating agricultural products such as grains, coffee, and wine. ADH3 hydrolyzes the amide bond of OTA to produce non-toxic Ochratoxin α (OTα) and L-β-phenylalanine (Phe). Its catalytic efficiency is 67 times higher than that of OTase, the previously most optimal enzyme. The optimal reaction conditions for the enzyme are pH 8.5 and a temperature range of 40-50°C. Under standard conditions (1.2 μg/mL enzyme, 50 μg/L OTA), it can completely degrade OTA within 90 seconds. ADH3 exists as a homotetramer with a molecular weight of approximately 360 kDa and utilizes a binuclear metal center for catalysis.
ADH3-S88E is a rationally designed, high-efficiency amide hydrolase mutant derived from the wild-type ADH3 enzyme of Stenotrophomonas acidaminiphila CW117. This enzyme is specifically used to degrade Ochratoxin A (OTA), a mycotoxin with strong carcinogenicity and nephrotoxicity that is commonly found in cereals, coffee, and wine. By mutating the serine at position 88 to glutamic acid (S88E), the catalytic activity of ADH3-S88E is 3.7 times higher than that of the wild-type ADH3. The mutation introduces additional hydrogen bond interactions, which stabilize the binding of the OTA substrate, thereby accelerating the hydrolysis of the amide bond and generating non-toxic ochratoxin α (OTα) and L-β-phenylalanine (Phe). The optimal reaction conditions for the enzyme are pH 8.5 and a temperature range of 40-50°C; under standard conditions, it can completely degrade 50 μg/L of OTA within 90 seconds.
ADH3-D344N is a rationally designed mutant of ochratoxin A (OTA)-degrading amidohydrolase ADH3 derived from Stenotrophomonas acidaminiphila strain CW117, with lost catalytic activity. This mutant retains substrate-binding ability but lacks hydrolytic activity due to the replacement of the key catalytic residue aspartic acid 344 (Asp344) with asparagine (Asn). It is an indispensable tool in structural studies, capable of capturing enzyme-substrate complexes and elucidating the key residues involved in the recognition and catalytic process of ochratoxin A.
LlADH is a wild-type amide hydrolase extracted from Lysobacter luteus. Its core function is to specifically degrade ochratoxin A (OTA), which is highly nephrotoxic and carcinogenic (classified as a Group 2B carcinogen by the International Agency for Research on Cancer). OTA widely contaminates foods such as wheat, corn, wine, and coffee, as well as feed. LlADH can catalyze the hydrolysis of the lactam bond within the OTA molecule, converting it into non-toxic ochratoxin α (OTα) and phenylalanine (Phe). It is currently the most efficient OTA hydrolase discovered, with a catalytic activity towards OTA that is twice that of the previously reported high-efficiency enzyme ADH3.
LlADH-I326A is a rationally designed, high-efficiency amide hydrolase mutant derived from the wild-type LlADH enzyme of Lysobacter luteus. This enzyme specifically degrades ochratoxin A (OTA), a potent carcinogen and nephrotoxin commonly found in grains, coffee, and wine. By mutating the 326th positions isoleucine to alanine (I326A), LlADH-I326A demonstrates 75% higher catalytic activity than the wild-type enzyme. The mutation reduces the side chain length, enabling OTA to better access the active pocket. The LlADH molecule has a molecular weight of approximately 45 kDa, while structural analysis confirms its existence as a homologous octamer with a molecular weight of about 360 kDa. Optimal reaction conditions are 45℃ and pH 8.5, with the highest thermal stability achieved at 20℃.
In this project, the modeling component established a closed-loop research paradigm of "experimentation-driven modeling and modeling-guided experimentation," achieving multi-scale integration from macroscopic condition optimization to microscopic mechanism elucidation. At the macroscopic level, principal component analysis (PCA) and response surface modeling were employed to reveal the synergistic effect between temperature and pH. Multiple hypothetical models were used to predict the optimal enzymatic reaction conditions, with experimental validation subsequently confirming the reliability of the minimum model. At the microscopic level, computational methods including homology modeling, molecular dynamics (MD) simulations, and MM/PBSA binding free energy calculations were systematically applied to investigate the interaction mechanism between ADH and OTA. Key residues, such as HIS-217, were identified, guiding the experimental team to successfully obtain the I326A mutant with 75% enhanced activity.
The methodological framework developed in this modeling work offers significant reference value for future research: First, it establishes a bidirectional macro-micro research framework enabling cross-scale integration from reaction conditions to atomic-level mechanisms. Second, it implements an iterative "hypothesis-validation-optimization" modeling workflow that continuously refines model predictive capability through experimental feedback. Third, it demonstrates an efficient collaborative mechanism based on cross-validation between computation and experiment. Future teams engaged in enzyme rational design or studies of biocatalytic mechanisms can adopt this closed-loop research paradigm and multi-scale modeling strategy. Particularly under limited experimental resources, leveraging modeling for preliminary prediction and direction screening can optimize experimental design, reduce trial-and-error costs, and advance protein engineering towards more precise development.
Guided by "synthetic biology serving human society" and rooted in solving food security and public health issues, our team takes Responsible Research and Innovation (RRI) as the criterion to advance Human Practice and Education, turning scientific exploration into sustainable development actions.
We conducted multi-region research: collaborated with Xinjiang's agricultural academies and granaries to grasp grain (corn) storage contamination; surveyed toxin (AFB1, DON) pollution in Southwest (dried chili), Central China (peanut) products; and surveyed 330 people, finding only 15.15% clearly understood aflatoxin hazards. This confirmed governance urgency and laid a foundation for targeted project design.
We also joined industry conferences: received "single enzyme degrading multiple toxins" (OTA, AFB1, ZEN) suggestions; collaborated with Tsinghua to introduce AI-assisted enzyme design; and obtained 50 molecular dynamics calculation systems for research support.
In Lushan, we tested soil/tea OTA and gave popular science lectures---public OTA cognition rose from 35.1 to 82.5 points. We collaborated with Xinjiang's granaries to verify enzyme effectiveness in real contaminated samples, laying groundwork for industrial application.
With "stimulating exploration and closing education loops" as core, we built an "Observation-Inquiry-Creation-Sharing" ecosystem covering all ages.
Using "Design-Build-Test-Learn", we turned students into active explorers (e.g., junior students designing enzyme experiments). We integrated iGEM spirit: responsibility via biosafety training, innovation via independent exploration, and cultural inheritance via Chinese painting-science integration.
Our work aligns with iGEM's core: Human Practice connects technology to society; Education builds closed-loop literacy cultivation. We will continue upholding "synthetic biology serving humanity" to safeguard food security, public health, and ecology.
We are HUBU-China!