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The Structural Study of Validoxylamine 7-Phosphate Synthase ValL

Author: ZhengLiNa
Tutor: ZhangHouJin
School: Huazhong University of Science and Technology
Course: Biomedical Engineering
Keywords: validamycin ValL crystal structure
CLC: Q55
Type: Master's thesis
Year: 2011
Downloads: 24
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Abstract


As a widely-used agricultural antibiotic in Asia and a precursor of various medicines, validamycin A bears extraordinary value for agriculture as well as pharmaceutical industry. Therefore, in recent years, the research and development of validamycin A has drawn significant attention from academia and industry alike. The optimization of fermentation condition has lead to ten-fold increase in yield per liter, which caused large drop in the price of validamycin A. Meanwhile, the study on its biosynthesis pathway has demonstrated the essential enzymes required for the validamycin A production. However, the structures and mechanisms of these enzymes on the pathway remain unclear. In this study, in order to construct the new prokaryotic expression vector pET-28a-GST-TEV we cloned the gene of GST and the cleavage site of TEV protease into pET-28a(+) plasmid. Then the target gene valL was inserted into pET 28a-GST-TEV and the recombinant protein was expressed in BL21(DE3) Rosetta cells. Subsequently, the protein was used to grow crystals and x-rays diffraction was chosen as the method to determine the three-dimensional structure.Using the protocol described above, we construct a novel prokaryotic expression vector pET-28a-GST-TEV featuring His tag and GST Tag. It would have good application in the hetero expression of eukaryotic genes. In addition, we collected the diffraction data of native ValL crystals as well as selenomethionine-labelled ValL crystals with resolutions of 1.9? and 1.7? respectively. Based on the above data, we built the three dimensional models of ValL and ValL/trehalose complex. The structural analysis shows that the overall structure of ValL closely resembles the classic fold of‘‘GT-B’’glycosyltransferases. It harbors two domains each of which contains aβ/α/βRossmann fold and an active site at the interface of them. Also found in the ValL structure is a kink between last two C-terminal helices, which is the common feature of GT-B enzyme family. The kink directs the last C-terminal helix into the vicinity of N-terminal domain and causes the residues in the helix to interact with the N-terminal domain. Furthermore, the electron density at the junction of N-terminal and C-terminal unambiguously indicates the location of trehalose embedded in the active site.In this work, we solved the structure of validoxylamine 7-phosphate synthase encoded by one of the eight essential genes required for validamycin biosynthesis. The results laid the framework for the further study of synthetic mechanism of validamycin and they are instrumental for the advanced application of validamycin in agriculture as well as medical science.

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