Overexpression and structural studies of some functional proteins from plant pathogen Xanthomonas campestris

• Main Authors: Yu-Sheng Fang, 方裕勝
• Other Authors: Der-Hang Chin
• Format: Others
• Language: zh-TW
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/18163571000091321990

碩士 === 中興大學 === 化學系所 === 95 === The functional of a protein is related to its three-dimensional structure. Due to the rapidly growing genomic database, structure genomics is paving a way to understand the function and intricate interaction among proteins in a whole organism. Xanthomonas campestris (Xcc) is a gram-negative bacterium that is phytopathogenic to cruciferous plants. Due to its academic and industrial values, we have endeavored to identify and characterize the structures and functions of proteins encoded in Xcc by using X-ray crystallography. From the sequenced genome, approximately 7000 genes were predicted in Xcc. We have studied 10 of them: XC449 (two-component system sensor protein)、XC743 (twitching motility protein)、XC708 (consvered hypothetical protein)、XC1008 (murein hydrolase D)、XC1313 (RNA-directed DNA polymerase)、XC1618 (type II secretion system protein like protein)、XC2895 (phage-related integrase)、XC3620 (Ham1 like protein)、XC4632 (consvered hypothetical protein)、and XC7201 (3-hydroxyanthranilate 3,4-dioxygenase). Among those, XC743、XC1008、XC1313、XC1618、XC3620、XC4632 are insoluble or exhibit low expression after we have tried many different induction conditions. For other proteins, we have tried changing fusion partners or expressing in different hosts to further increase their solubility. XC708 are considered to be a regulator of pathogenicity pathway in Xcc. It can regulate the synthesis of extracellular enzyme, EPS, pathogenicity and other pathogenic factor. It has been fused to the MBP protein for overexpression, but cannot be completely cleaved by TEV; furthermore, it was found to precipitate during the cleavage reaction by TEV at 16℃. A better condition needs to be found for its overexpression. XC2896 was predicted as a phage-related integrase. Its overexpression is OK. However, we found that it degraded during crystallization screening. XC449 comprises 351 amino acid, and is predicted as a two-component system sensor protein. This type of protein usually locates on the cell membrane. It comprises two domain, one domain of that has no similar (identity more than 30%) sequence be found from Protein Data Bank structure. After expression in E. coil, a crystal screening is being performed to find crystallization condition. XC7201 is an interesting protein comprising 176 amino acids. Many sequences in the prokaryote and eukaryote kingdom with high similarity score were detected by BLAST search. It was predicted to a 3-hydroxyanthranilate 3,4-dioxygenase (3HAO), which is an extradiol dioxygenase that catalyzes the final conversion step of tryptophan to quinolinic acid in the kynurenine pathway. Quinolinic acid is an endogenous neurotoxin, that activates the N-methyl-D-aspartate(NMDA) receptor and the biosynthesis of NAD+ precursor. Chemically speaking, 3HAO is an important enzyme that catalyzes the aromatic ring opening reaction. Since aromatic ring is a very stable compound, it would needs very high activation energy to begin the ring opening reaction. It is therefore interesting to investigate how the micro-organism can catalyze this reaction in room temperature at natural buffer condition. A detailed study of this enzyme structure can help understand the mechanism of this reaction, and help to development of its industrial application. We have used the selenomethionine-labeled protein to get a good crystal that diffracted to at least 1.8 Å X-ray diffraction data at the remote and inflection wavelength have been collected. The phase problem has been solved using the MAD approach. A preliminary structure of XC7201 has been obtained. Further structural refinement is now undergoing! After analyzing the protein structure, we found that XC7201 exists as a dimer in the crystal, and stabilized mainly through hydrophobic interaction. Every monomer contains two metal ions, were found to be Ni2+ by X-ray absorption scpectrum. One of the metal ions locates in the active site, and is coordinated by two histidine and one glutamic acid. The other metal ion is located in the C-terminal region of the protein and coordinated by 4 cysteine. This metal center is possibly unrelated to the enzymatic activity, and mainly contributes to the enzyme stability. We found that the XC7201 structure has high similarity with 3HAO from other two species by structure alignment. The most difference from exists at C-terminal region XC7201 which has more amino acids, and forms a α helix. Because this helix is away from active site; it may not affect the enzyme activity ether. It’s importance needs further characterization.