The sequence of Zlh, a novel lactonohydrolase metabolizing zerealenone, an estrogenic mycotoxin produced by Fusarim sp. which is of high importance in the food and feed sector, was identified as promising genetic resource for in planta detoxification of the mycotoxin in important crops [1]. The enzyme was recombinantly produced in yeast and purified to homogeneity and successfully crystallized using sparse matrix screening approach.
Applying various crystallization conditions we observed two different crystal forms of Zlh, tetragonal P43212 in the presence of 1.6M of ammonium sulfate and orthorhombic P21212 in the presence of 20% PEG4000. Successful phasing to 2.8 Å resolution has been achieved using the bromide soaked crystals data and then phases extended to 1.65 Å resolution from the native data set. The Zlh structure resembles the α/β hydrolase fold. A special feature of Zlh, which discriminates it from other homologs is the presence of a long loop in the front of the active site, which is disordered in tetragonal crystal form but forms α–helix in the orthorhombic crystal lattice. Tetragonal crystals of Zlh obtained in the presence of ammonium sulfate were populated by protein monomers, whereas Zlh dimers were found in orthorhombic crystals. The active site is capable to bind a very large, but flexible substrate. To further study of the reaction mechanism and substrate specificity of Zlh, we plan to crystallize native enzyme with uncleavable substrate analogs and, in turn, to use inactive mutant in a complex with zerealenone.