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標題: Xanthomonas campestris pv. campestris polyketide合成蛋白XC5357的大量表達與結構解析
Expression and structural analysis of polyketide synthesis protein XC5357 in Xanthomonas campestris pv. campestris
作者: 朱巧莉
Chu, Chiao-Li
關鍵字: 核磁共振儀;Xanthomonas campestris pv. campestris;十字花科;核磁共振;高解析度;抗生素;protein structure;tetracenomycin polyketide synthesis protein;taiwan
出版社: 生物化學研究所
Xanthomonas campestris pv. campestris屬於格蘭氏陰性菌。為兼具學術性及應用性之菌種。X. campestris pv. campestris能分泌多種胞外蛋白,為研究格蘭氏陰性菌蛋白分泌之模式系統,並為一株感染十字花科而造成十字花科黑腐病之植物病原菌,易生長於高溫多濕之環境,屬世界性之病害。此外,X. campestris pv. campestris所產生之多醣體Xanthan為重要之工業原料。X. campestris pv. campestris str. ATCC 33913之基因體定序已由巴西團隊完成且發表;本土菌株X. campestris pv. campestris str. 17也由台灣團隊完成定序及基因註解。
X. campestris pv. campestris str.17的ORF XC5357由生物資訊的方法得知為一tetracenomycin polyketide synthesis protein。Tetracenomycin (Tcm) C 是芳香族polyketide的一種,為一種抗生素並兼具有抗癌(antitumor)的活性,它的合成是由 Streptomyces glaucescens 的 tcm gene cluster (tcmGHIJKLMNOP operon) 所調控。其中Tcm J蛋白估計是催化 Tcm C 形成環狀物以及增加Tcm C前驅物Tcm F2產量的重要蛋白之ㄧ,但其確實功能目前為止仍然不太清楚。
XC5357序列在同種的植物病原菌之間有超過90%以上的相似性,如:Xanthomonas axonopodis pv. citri與Xylella fastidiosa。並且與S. glaucescens 的 Tcm J 蛋白有30%的相似性。因此我們希望藉由解析此蛋白的結構,以提供結構上的基礎來瞭解Tcm J 蛋白在Tcm C合成過程中所扮演的功能。本研究利用X-ray晶體繞射與高磁場核磁共振儀(NMR)異核核磁共振技術來解析此一蛋白的三度空間結構。首先我們先將 XC5357 構築在含有 thioredoxin的pET-32a(+)載體上以取得大量的可溶性融合蛋白,再以蛋白酶切下目標蛋白。在NMR實驗上,以15N標定蛋白後,進行初步2D 15N-1H HSQC 的NMR 實驗,可得到強度相似且分散的交叉峰。再進一步製備15N、13C標定之目標蛋白,以收集多種結構運算所必須之三維光譜。利用所收集的2D 15N-1H HSQC、3D HNCACB、3D CACB (CO)NH、HNCO、3D H(CC-CO)NH-TOCSY、3D (H)C(C-CO)NH-TOCSY等NMR光譜,我們已完成大部分1H、15N以及13C之共振頻率判讀。在X-ray實驗上,在初步結晶篩選時已得到一高解析度的單晶(1.7 Å),並已進一步製備Selenomethionine標定蛋白進行多波長異常繞射數據收集,以解析XC5357之三級結構。目前XC5357的結構已接近完成,得知XC5357單體是由9個β-strands組成一個β barrel的構形,並且2個單體之間以疏水性作用力形成雙聚體。

Xanthomonas campestris pv. campestris is a bacterium that is phytopathogenic to cruciferous plants and causes worldwide agricultural loss. However, it also produces exopolysaccharide (xanthan) that is of great industrial importance. Hence it is an important pathovar both academically and industrially. The genome of X. campestris pv. campestris str. ATCC 33913 was sequenced by the ONSA/FAPBSP/Brazil group in 2002 and that of X. campestris pv. campestris str. 17 was also sequenced and annotated by an integrated structural and functional group in Taiwan in 2002.
One of the ORFs in X. campestris pv. campestris str. 17 was annotated as a tetracenomycin polyketide synthesis protein by a bioinformatics approach. Tetracenomycin (Tcm) C is one of the aromatic polyketide antibiotic that also exhibits antitumor activity. The biosynthesis of the Tcm C is produced in Streptomyces glaucescens and regulated by the tcm gene cluster (tcmGHIJKLMNOP operon). Tcm J is one of the proteins presumably to cyclize Tcm C and greatly increases the yield of TCM F2, the precursor of Tcm C. Until now, however, its function is still unknown.
XC5357 shares a larger than 90 % identity with other proteins from phytopathogens of the same genus including Xanthomonas axonopodis pv. citri and Xylella fastidiosa, and approximately 30% with the Tcm J protein in S. glaucescens. Here we describe the near complete structure determination of XC5357, which can serve as a basis for further structural and functional characterization of this protein for the synthesis of TCM F2. In the present thesis, we aim to identify and characterize the three-dimensional structures of XC5357 using X-ray crystallography and high-resolution NMR techniques. In order to obtain an over-expressed and soluble protein, we have cloned the DNA fragment of XC5357 into a modified pET-32a(+) vector. The final construct encodes a fused thioredoxin-XC5357 protein, which was then cleaved by thrombin to produce the final target protein. In the NMR experiment, we have assigned nearly complete resonances of the 1H, 15N, 13C nuclei of XC5357 using 2D 15N -1H HSQC and a bunch of triple resonance experiments including 3D-HNCACB, 3D-CACB(CO)NH, HNCO, 3D-H(CC-CO)NH-TOCSY and 3D-(H)C(C-CO)-NH-TOCSY. Also, XC 5357 crystals were obtained from purified recombinant protein and exhibit a variety of forms diffracted to at least 1.7 Å resolution by using X-ray crystallography. We have solved the structure of XC5357 by multiwavelength anomalous diffraction (MAD) method using selenomethionine-substituted protein. Now, the structure of XC5357 was near complete. XC5357 composed of 9 β-strands and formed a β barrel.
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