野生株與突變株仙人掌桿菌幾丁質酵素之結構與功能研究

Crystal structure and functional study of wild type and mutated Bacillus cereus NCTU2 chitinase

郭爵源(Chueh-Yuan Kuo)

指導教授 ： 陳俊榮

國立清華大學/生命科學院/生物資訊與結構生物研究所/碩士(2006年)

https://doi.org/10.6843/NTHU.2006.00309

摘要

幾丁質,一種由N-acetyl-D-glucosamine (GlcNAc) 經β-(1,4)鍵結而成的聚合務,廣泛地分佈在自然界中,是構成多數黴菌細胞壁、昆蟲外骨骼、甲殼素動物的主要成份,也存在於軟體動物類、腔腸動物類、線蟲類及原生動物類中。在大多數的生物有機體中,包括病毒、細菌、真菌、昆蟲、高等植物及動物都利用幾丁質酵素分解幾丁質後來取得碳和氮。屬於醣類水解型酵素18家族之仙人掌桿菌幾丁質酵素NCTU2,其基因內含一個27個胺基酸的訊息胜肽 (signal peptide) 與一個333個胺基酸的成熟蛋白。經過胺基酸多重比對後,發現Glu145及Tyr227為ChiNCTU2中參與催化反應的重要胺基酸。ChiNCTU2與其突變株E145Q之分子量為36 KDa,利用氣相擴散法的hanging drop method以含有zinc acetate、polyethene glycerol 8000及solium cacodylate的母液,將蛋白成功地長成晶體。經X-ray繞射後得到ChiNCTU2晶體解析度為1.2 Å, 晶格之空間群 (space group) 為P21,其參數為a = 50.789 Å, b = 48.788 Å , c = 66.867 Å。E145Q之晶體解析度為1.49 Å, 晶格之空間群 (space group) 為P1, 其參數為a = 61.306 50.820 Å, b =72.888 Å and c = 76.343 Å。ChiNCTU2與E145Q分別以多波長非尋常散射法(multiwavelength anomalous dispersion method ) 及分子置換法 (molecular replacement method) 解出其結構。ChiNCTU2之結構為單元體,而E145Q 結構之晶體堆積為四元體。 ChiNCTU2之蛋白質結構由12個α-helices 與 10 個 β-sheets所組成,在其活性區內,有五個胺基酸Asp143、Glu145、Glu190、Gln225 及 Tyr227與鋅原子鍵結。經由DNS (3,5-Dinitrosalicylic acid) 測試酵素產物生成量後,證明鋅原子鍵結在活性區內,會造成酵素活性降低。將二元體ChiNCTU2與E145Q分子結構做重疊(superimpose),發現殘基E145與Q145在角度上有明顯的差別, 而殘基Q145深埋進催化區內的現像,能輔助說明E145Q活性喪失的原因。我們也利用DNS (3,5-Dinitrosalicylic acid)與幾丁二醣反應後, 測量產物之OD540吸收值,證明S.marcescens ChiA的chitin_binding domain 能幫助酵素催化水解膠狀幾丁質,並且提高酵素的水解速率。在與其他屬於醣類水解型酵素18家族之幾丁質酵素,做序列及結構比對後,發現ChiNCTU2為微生物幾丁質酵素中分子量最小的一種。

關鍵字

仙人掌桿菌幾丁質酵素

並列摘要

Chitin, a β-(1,4)-linked polymer of N-acetyl D-glucosamine (GlcNAc), is widely distributed in nature, particularly as a structural polysaccharide in fungal cell walls in the exoskeleton of arthropods, the outer shell of crustaceans, nematodes, etc. Chitinases which hydrolyze chitin as carbon and nitrogen nutrient, occur in a wide range of organisms include in viruses, bacteria, fungi, insects, higher plants, and animals. A gene of family 18 chitinase from Bacillus cereus NCTU2 encodes a signal peptide (27 amino acids) and a mature protein (333 amino acids), The gene of family 18 chitinase from Bacillus cereus NCTU2 was constructed in pET-22b(+) and over-expressed by E. coli BL21 (DE3) strain.. Amino acid multi-alignment reveals that E145 and Y227 are the potential residues mediating the catalytic function of ChiNCTU2. ChiNCTU2 and mutant E145Q of MW 36 kDa have been crystallized using the hanging-drop vapor diffusion method with solution consisted of polyethene glycerol 8000、sodium cacodylate and zinc acetate dihydrate. According to diffraction of ChiNCTU2 crystals at resolution 1.20 Å, the unit cell belongs to space group P21 and has parameters a = 50.789 Å, b = 48.788 Å and c = 66.867 Å. And E145Q crystal at resolution 1.49, the unit cell belongs to space group P1 and has parameters a = 61.306 50.820 Å, b =72.888 Å and c = 76.343 Å. The protein structure of ChiNCTU2 is monomer by using multiwavelength anomalous dispersion method and the crystal packing of E145Q is tetramer by using molecular replacement method. The structure of ChiNCTU2 comprises 12 α-helices and 10 β-sheets . Five residues Asp143、Glu145、Glu190、Gln225 and Tyr227 bind with zinc atoms in the catalytic domain of ChiNCTU2 protein structure. We proved that zinc atoms will cause declined activity of ChiNCTU2 by detecting the amount of chitobioside using DNS (3,5-Dinitrosalicylic acid) .We find an angular difference between residue E145 and Q145 when doing superimposition with dimer form ChiNCTU2 and E145Q protein structures reveals the declined activity of mutant E145Q. We also find that S.marcescens ChiA has greater catalytic velocity than Bacillus cereus chitinase when interact with colloidal chitin after doing DNS (3,5-Dinitrosalicylic acid) experiment .After comparing sequences and structures with other 18 family chitinase, we discover that ChiNCTU2 is the smallest protein among microbe chitinases.

參考文獻

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