Xanthomonas campestris pv.campestris 未知功能蛋白XC1692/XC1691複合物的晶體結構及功能分析

Abstract

The bacterial strain studied in this thesis is Xanthomonase campestris pv. campestris, which is a gram-negative bacterium and an important pathovar both academically and industrially. X. campestris can secrete many kinds of extracellular proteins, and is a model system for studying protein secretion of gram-negative bacteria. It is phytopathogenic to cruciferous plants and can cause worldwide agricultural loss. From the sequenced genome, about 4100 genes were predicted. This thesis tries to study 11 of them by using X-ray crystallography technique. XC1692 is one of them containing 141 amino acids with no identified function. However many sequences with high similarity score were detected by a BLAST search. Until now, there is also no similar tertiary protein structure determined. However, although we have obtained high resolution crystals of native XC1692 (diffracted to 1.45 A), we couldn’t solve its phase using multiwavelength anomalous diffraction (MAD) method, due to the poor resolution data of Se-substituted crystals. To solve the problem, we have used mutagenesis method to change Met to Ala to reduce the number of Met to possibly increase the crystal stability. Using this approach, we have successfully solved the phase problem and got a preliminary structure of XC1692 using the MAD approach. XC1692 was then found to adopt a similar architecture with a ribonuclease inhibitor through a structure alignment. Also, a gene (XC1691) annotated as a ribonuclease was found to be upstream of XC1692 and controlled by the same promoter. Altogether, these data imply that XC1692 may serve as a ribonuclease inhibitor, which was further confirmed by a pull-down assay. We are currently crystallizing the XC1691/XC1692 complex to determine its functions from a structure perspective.