Rational Design And Molecular Engineering Of Zearalenone Hydrolase From Clonostachys Rosea

Zearalenone(ZEN) is a kind of mycotoxin with estrogenic activity and is widespread in forages contaminated by molds.The contamination of ZEN causes huge loss to feed industry and livestock husbandry and even brings serious public health problems.The enzyme ZHD101 from Clonostachys rosea which can degrade ZEN and its derivatives effectively is being widely studied.However,ZHD101 prefers to hydrolyze ZEN rather than ZOL with higher toxicity.We thus conducted structure-based rational design to modify several lactone-surrounding residues in an attempt to enhance ZHD101 activity towards ZOLs.Since its application in feed industry was limited by low thermostability,in this study,we attempted to improve the thermostability of ZHD101 through structure-based rational design and molecular engineering of disulfide bridges.(1)By analysis of ZHD101-S102A/ZOL complexes,we found that the major structural difference lies in the flexible lactone ring of the substrate and the H242 side chain of the catalytic triad.By designing site-directed mutagenesis,V153 H showed a 3.7-fold increase in specific activity against ?-ZOL while maintaining activity against ZEN.Kinetic studies showed that V153 H has about 2.5-fold reduction in substrate affinity but a 5.2-fold higher turn-over rate.To analyze the structural basis of improved substrate specificity,we then solved two ZHD101-S102A/V153H/ZOL complex structures.Here the substrate lactone ring is hydrogen bonded to the mutated side chain of H153.As a consequence,the position of ?-ZOL lactone ring is shifted to yield a space to accommodate H242,which turns back to the position of forming catalytic triad.(2)7 mutants(A110C/P196 C,S136C/R189 C,D143C/P181 C,S147C/P181 C,D199C/A202 C,L200C/A231 C,and R204C/G205C)were constructed for disulfide bridge formation.Two mutants,S136C/R189 C and D143C/P181 C,showed enhanced thermostability.Notably,D143C/P181 C mutant,which exhibits similar specific activity as wild type enzyme,showed two-fold increase in thermostability after heated at 50? for 2 min.A quadruplet mutant(S136C/D143C/P181C/R189C)was subsequently constnnnructed and examined,but it showed lower thermostability comparing to D143C/P181 C mutant.In conclusion,structure-based engineering was successfully employed to improve the enzyme activity towards ZOL and the thermostability,which provides significant benefit for the application of ZHD101 in further commercial utilizations.