Xu_2016_ACS.Catal_6_7657

The enzyme ZHD101 from Clonostachys rosea hydrolyzes and deactivates the mycotoxin zearalenone (ZEN) and its zearalenol (ZOL) derivatives. ZHD101 prefers ZEN to ZOL as its substrate, but ZOL, especially the -form, shows higher estrogenic toxicity than ZEN. To enhance alpha-ZOL selectivity, we solved the complex structures of ZHD101 with both ZOLs and modified several lactone-surrounding residues. Among the mutants, V153H maintained activity for ZEN but showed a 3.7-fold increase in specific activity against alpha-ZOL, with an 2.7-fold reduction in substrate affinity but a 5.2-fold higher turnover rate. We then determined two V153H/ZOL complex structures. Here, the alpha-ZOL lactone ring is hydrogen-bonded to the H153 side chain, yielding a larger space for H242 to reconstitute the catalytic triad. In conclusion, structure-based engineering was successfully employed to improve the ZHD101 activity toward the more toxic alpha-ZOL, with great potential in further industrial applications.