Enhancing the activity of zearalenone lactone hydrolase toward the more toxic α-zearalanol via a single-point mutation

Abstract

Zearalenone (ZEN) and its derivatives are estrogenic mycotoxins known to pose significant health threats to humans and animals. Especially, the derivative α-zearalanol (α-ZAL) is over 10 times more toxic than ZEN. Simultaneous degradation of ZEN and its derivatives, especially α-ZAL, using ZEN lactone hydrolases (ZHDs) is a promising solution to eliminate their potential hazards to food safety. However, most available ZHDs exhibit limited activity toward the more toxic α-ZAL compared to ZEN. Here, we identified a broad-substrate spectrum ZHD, named ZHDAY3, from Exophiala aquamarina CBS 119918, which could not only efficiently degrade ZEN but also exhibited 73% relative activity toward α-ZAL. Through rational design, we obtained the ZHDAY3(N153H) mutant, which exhibited the highest specific activity (253.3 ± 4.3 U/mg) reported so far for degrading α-ZAL. Molecular docking, structural comparative analysis, and kinetic analysis collectively suggested that the shorter distance between the side chain of the catalytic residue His242 and the lactone bond of α-ZAL and the increased binding affinity to the substrate were mainly responsible for the improved catalytic activity of ZHDAY3(N153H) mutant. This mechanism was further validated through additional molecular docking of 18 mutants and experimental verification of six mutants.IMPORTANCEThe mycotoxins zearalenone (ZEN) and its derivatives pose a significant threat to food safety. Here, we present a highly promising ZEN lactone hydrolase (ZHD), ZHDAY3, which is capable of efficiently degrading both ZEN and the more toxic derivative α-ZAL. Next, the ZHDAY3(N153H) mutant obtained by single-point mutation exhibited the highest specific activity for degrading α-ZAL reported thus far. We further elucidated the molecular mechanisms underlying the enhanced hydrolytic activity of ZHDAY3(N153H) toward α-ZAL. These findings represent the first investigation on the molecular mechanism of ZHDs against α-ZAL and are expected to provide a significant reference for further rational engineering of ZHDs, which will ultimately contribute to addressing the health risks and food safety issues posed by ZEN-like mycotoxins.

Keywords: crystal structure; enzyme activity; substrate specificity; zearalenone lactone hydrolase; α-zearalanol.

Fig 1

Degradation ability of ZHDAY3 and ZHDAY3(N153H) variant on different substrates. The activity of ZHDAY3 and ZHDAY3(N153H) variant were measured at pH 9.5 and 40°C using five substrates (ZEN, α-ZOL, β-ZOL, α-ZAL, and β-ZAL). The ability of the enzyme to degrade ZEN was set to 100%. Results are shown as mean ± SD from three biological replicates for each experimental condition.

Fig 2

Overall crystal structure of ZHDAY3. Structural superimposition (left) of ZHDAY3 (orange cartoon model, center) and ZHD101 structures (blue cartoon model, right). The colors of the cap and core domains of ZHDAY3 and ZHD101 are darker and lighter, respectively. Catalytic residues are shown as magenta sticks.

Fig 3

Interaction network of ZEN with wild type ZHDAY3 (a) and ZHDAY3(N153H) mutant (b). The structures of ZHDAY3 and ZHDAY3(N153H) mutants were modeled based on the solved ZHDAY3(S102A) crystal structure as a template. Proteins are shown in cartoons, ZEN is shown in cyan sticks, catalytic residues are shown as orange lines, and other residues interacting with ZEN are shown in deep green lines. The residue 153 is shown as dark green sticks, nucleophilic interactions as black dashed lines, hydrogen bonds as green dashed lines, and Pi–Pi and CH–π interactions as purple dashed lines.

Fig 4

Structural information of ZHDAY3 and mutant ZHDAY3(N153H), respectively, with the substrate α-ZAL. (a) Stereo view of the interaction network between ZHDAY3 and α-ZAL. (b) Stereo view of the interaction network between ZHDAY3(N153H) and α-ZAL. Distances between the NE2 atoms of His242 and the ester oxygen atom (O10') and carbonyl carbon atom (C12') in ZHDAY3 (c), and ZHDAY3(N153H) (d) are indicated. Proteins are shown as cartoons, α-ZAL is shown as pink sticks, catalytic residues are shown as orange lines, and other residues interacting with α-ZAL are shown as deep green lines. The 153 residue is shown as dark green sticks, hydrogen bonds are shown as green dashed lines, and Pi–Pi and CH–π interactions are shown as purple dashed lines.

Fig 5

Relative position of His242 in six mutants and the distances between the NE2 atoms of His242 and the ester oxygen atom (O10') and carbonyl carbon atom (C12') of α-ZAL, respectively. α-ZAL is shown as pink sticks, and H242 residue is shown as orange sticks.

Fig 6

Relative activity of ZHDAY3 and its mutants toward ZEN and α-ZAL. Substrates (ZEN and α-ZAL) were used to measure the activity of ZHDAY3 and its mutants at pH 9.5 and 40^oC. The ability of the enzyme to degrade ZEN was set to 100%. Values represent the average of three replicates and their error bars.