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.
(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.
(ADH3 Gel Filtration Chromatogram)
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.
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).
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:
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.
We resolved three sets of high-resolution structures of ADH3 using single-particle cryo-electron microscopy:
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.
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.
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.
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.
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.
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.
