Structural determination and functional analysis of unknown function protein XC847 in Xanthomonas campestris pv. campestris

• Main Authors: yen-yu wu, 吳彥宥
• Other Authors: Shir-Ly Huang
• Format: Others
• Language: zh-TW
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/52026438362383142193

碩士 === 國立中央大學 === 生命科學研究所 === 93 === In this thesis, we have choosen Xanthomonas campestris pv. campestris as our target genome. It is a gram-negative bacterium that is phytopathogenic to cruciferous plants and causes worldwide agricultural loss. However, it also produces exopolysaccharide (xanthan gum) that is of great industrial importance. About 4100 genes are predicted in this genome. Five different vectors are used to construct clones and over-express proteins in the E.coli host to produce enough soluble proteins for X-ray and NMR analysis. Until now, 15 target genes were studied. 4 genes (XC4107 、XC3183 、XC6232、 XC6774) are in the PCR stage, 7 genes (XC2797、XC4109、XC6773、XC6835、XC3953、XC4027、XC5047) are in the overexpression stage, 2 are being analyzed by NMR, 1 being screened for crystallization , and one (XC847) of which the structure has been successfully determined. From sequence alignment, XC847 is predicted as an oligoribonuclease that belongs to the DEDDh family. DEDDh family is one of the six members in the 3' to 5' exonuclease superfamily, and is defined by four conserved acidic residues distributed among three separated sequence motifs. Proteins in this family can hydrolyze both DNA and RNA substrates. From the determined 3D structure, XC847 was found to have conserved residues and active site geometry similar to those in the DEDDh family, ISG20、Pop2, andε186. Moreover, the electrostatic surfaces of these 3 exoribonucleses show similar negative charged profile in their active site regions.From sequence alignment and structural comparison, XC847 is identified as an oligoribonuclease.