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Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/22105
標題: Corynebacterium glutamicum NCHU 87078及其噬菌體P1201之thymidylate synthase, intein及lytic enzyme的生化特性
Biochemical Characterization of Thymidylate Synthases (ThyX) from Corynebacterium glutamicum NCHU 87078, and Inteins and Lytic Enzyme from Corynephage P1201
作者: 甘淑貞
Kan, Shu-Chen
關鍵字: 噬菌體;Corynebacterium glutamicum;corynephage;thymidylate synthase;intein;lytic enzyme
出版社: 分子生物學研究所
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摘要: 
從高產量之味精生產菌株Corynebacterium glutamicum NCHU 87078中選殖出兩個可能參與thymidylate合成的基因，分別命名為CgthyA及CgthyX。將兩基因分別構築在pQE30表現載體上進行大量表現並純化蛋白。CgThyA的thymidylate synthase A (ThyA) 活性為414 mU mg-1 protein，但由酵素活性或功能性互補實驗的結果顯示CgThyX不具活性。典型ThyX之結構為四聚體 (homotetramer)，但由膠體過濾 (gel filtration) 及化學偶合(cross-linking) 分析實驗結果初步解得CgThyX屬於雙聚體 (dimer) 。根據光譜 (spectroscopic) 分析發現，CgThyX未如預期與典型之ThyX在450及380 nm具有最大吸收波峰，顯示CgThyX可能缺乏與FAD結合的能力。蛋白質晶體結構的解析指出CgThyX•FAD此複合物晶體的結構解析度達2.3Å，計算次單位間的作用力 (distance≦3.8 Å) 發現，由於單元體間的作用力較少，導致CgThyX形成四聚體時結構較為鬆散。根據結構所設計的突變實驗結果顯示，位於70~73之胺基酸殘基能幫助FAD上的黃素環正確形成有效的催化位置，達到催化反應的進行。本研究證明C. glutamicum中的ThyX不具ThyX活性， C. glutamicum中是藉由ThyA蛋白進行thymidylate的合成。
從corynephage P1201基因體中，將ORF12、16及56分別選殖出並構築至表現載體pET28b後進行大量表現，得到的蛋白產物大小分別為68kDa、103kDa及97kDa，分別命名為CP1201ThyX、CP1201Ter1及CP1201RIR1。由西方墨點法分析後確知此三個蛋白質均具備intein 的功能，能將自身切割成兩個片段，切割後之片段及大小分別為32kDa (putative thymidylate synthase X) 及36kDa (intein 12)、63kDa (putative terminase) 及40kDa (intein 16)、78kDa (putative ribonucleoside diphosphate reductase α subunit) 及19kDa (intein 56)。即時定量 PCR的分析結果顯示，在噬菌體感染宿主細胞過程中，噬菌體本身所攜帶的thymidylate synthase基因 CP1201ThyX 基因會大量表現，表現量提高為未感染時的2000倍。由西方墨點分析得知，於噬菌體感染初期即可偵測到CP1201ThyX蛋白的表現，且有intein 切割反應的發生。在分析CP1201RIR1的全蛋白產物是否能有效率切割以回收純化目標基因產物的過程中，得到一變異株G392S，能受到溫度的調控以誘導切割反應的進行。由Conserved domain database比對後發現ORF38基因具有chitinase之功能性區域，遂選殖此基因至pQE30載體上並進一步純化回收蛋白。以ethylene glycol chitin (EGC)及chitosan為受質時，其chitinase及chitosanase的活性分別為8.9 mUmg-1及30 mUmg-1protein。

The genome of Corynebacterium glutamicum NCHU 87078 contains two putative thymidylate synthase genes, designated CgthyA and CgthyX. These two genes were expressed in Escherichia coli NovaBlue and the expressed His6-tagged enzymes were purified by nickel-chelate chromatography. The purified CgThyA had a specific activity of 414 mU mg-1 protein, whereas thymidylate synthase activity for CgThyX could not be detected in a functional complementation assay using a 10 days incubation period. Gel filtration chromatography and chemical cross-linking experiments showed that CgThyX may exist as a dimer in solution, unlike a typical ThyX protein with homotetrameric structure for catalytic activity. Spectroscopic analysis indicated that purified CgThyX lacked the cofactor FAD. The 2.3 Å resolution crystal structure of CgThyX-FAD demonstrated a loose tetramer, in which FAD is chelated between the subunits via a manner distinct from that of other flavin-dependent thymidylate synthases. Structure-based mutational studies have identified a non-conserved segment (residues 70~73) of CgThyX protein with crucial role in binding to FAD. Taken together, our biochemical and structural analyses highlight unique features of the C. glutamicum ThyX that distinguish this enzyme from ThyX proteins from other organisms. Our results also suggest that thymidylate synthesis in C. glutamicum requires ThyA but not ThyX.
Open Reading Frames (ORFs) of 12, 16 and 56, designated as CP1201ThyX, CP1201Ter1 and CP1201RIR1, in corynephage P1201 genome were cloned into expression vector pET28b and expressed in Escherichia coli BL21(DE3). Gene products with molecular masses of 68, 103 and 97 kDa were purified by Ni-column chromatography. CP1201ThyX gene product could be spliced into two fragments, putative thymidylate synthase X (ThyX) and intein 12, with sizes of 32 and 36 kDa. CP1201Ter1 gene product was spliced into two fragments, putative terminase and intein 16, with sizes of 63 and 40 kDa. CP1201RIR1 gene product was spliced into two fragments, putative ribonucleoside diphosphate reductase (RNR) α subunit and intein 56, with sizes of 78 and 19 kDa. Expression level of CP1201thyX gene in phage-infected Corynebacterium glutamicum was about 2000-fold higher than that of uninfected cell by real-time PCR. Western blot analysis revealed that CP1201ThyX protein was highly expressed and spliced into two fragments in the course of phage infection. We found that CP1201RIR1 with G392S mutation became a temperature-sensitive intein. Conserved domain database revealed that ORF38 possesses chitinase domain. This ORF was cloned into pQE30 vector and expressed in E. coli NovaBlue. The purified protein had specific activities of 30 mU mg-1 and 8.9 mU mg-1 protein to crab chitosan and ethylene glycol chitin (EGC), respectively.
URI: http://hdl.handle.net/11455/22105
其他識別: U0005-1008201014143700
Appears in Collections:分子生物學研究所

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