Parts Summary

1. ADH3

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.

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.

2. ADH3-S88E

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.

3. ADH3-D344N

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.

4. LlADH

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.

5. LlADH-I326A

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℃.