The Crystal Structure Analysis Of Pyrethroid Hydrolase From Sphingobium Wenxiniae Strain JZ-1

Pyrethroid pesticides are a class of synthetic analogues of the natural pyrethrins. Previous studies showed that most pyrethroid pesticides are highly toxic to some nontarget organisms, such as fish, bees, and aquatic invertebrates. Moreover, they may damage the mammalian reproductive, nervous, immune, endocrine and cardiovascular systems. PytH is a kind of highly efficient and broad-spectrum pyrethroid hydrolase which is discovered in strain Sphingobium wenxiniae JZ-1. Analysis of its three-dimensional structure allows us to understand how PytH degrade permethrin in molecular level.In this study, pytH gene was amplified by PCR using plamid pET29a-pytH as template, and cloned into the expression vector (pET24b). The recombinant PytH was successfully overexpressed and 20mg protein could be purified from 1 liter of culture using immobilized metal affinity chromatography (IMAC). The purified PytH has a high purity and biological activity.The purified protein was concentrated to 20mg/mL and used for crystallization. The PytH crystallization screening was carried out by vapor diffusion method, at 4-8℃. PytH crystals were observed in two conditions (114-36:O.lmol/L HEPES Sodium pH7.5, lmol/L Sodium citrate tribasic dihydrate; 110-16:O.lmol/L HEPES Sodium pH7.5,1.5mol/L Lithium sulfate monohydrate). A 2.81 A diffraction dataset was collected with the wild type PytH crystals after optimizing the 110-16 crystallization conditions, a 2.39A diffraction dataset of the PytH and bifenthrin complex crystal was collect.Sequences alignment showed that PytH shares the highest identity with some α/β hydrolase fold proteins, e.g. a salicylic acid-binding protein SABP2 from Nicotiana tabacum(21% identity). We tried to resolve the PytH crystal structure by molecular replacement but failed. In order to solve the crystal structure of PytH, the selenomethionine crystals were prepared using the same condition as the wild-type PytH. One of the best selenomethionine PytH crystal diffracted to 1.90A and a dataset was collected. PytH crystal structure was solved by using Se-SAD method. The crystal of PytH and bifenthrin complex crystal is belong to space group P4(2)2(1)2, each asymmetric unit contains 6 PytH monomer molecules. The structure showed that PytH is a typical alpha/beta hydrolase. Each PytH monomer molecule consists of 14 alpha helix and 9 beta sheet, between each of the two beta sheet there is at least one alpha helix. A plurality of parallel alpha/beta structures are observed in the PytH molecule.